Giulia Oliva

Tobias Sahr

Carmen Buchrieser

Emmanuelle Charpentier

Small RNAs, 5' UTR elements and RNA-binding proteins in intracellular bacteria: impact on metabolism and virulence

Keywords: intracellular bacteria, small RNAs, riboswitch, Hfq, CsrA, virulence intracellular bacteria, small RNAs, riboswitch, Hfq, CsrA, virulence

2

Adaptation to environmental changes

. Bacteria that transit between extracellular environments and host cells to replicate have a dual intracellular/extracellular lifestyle that allows those bacteria also to survive and multiply in a cell-free environment. Classical examples of facultative intracellular pathogens are the bacterial genera Legionella, Chlamydia, Listeria, Coxiella, Mycobacterium, Shigella or Francisella, which regularly switch from intracellular to extracellular variants (M.

!

GENERAL INTRODUCTION !

! grown on buffered charcoal yeast extract (BCYE), a nutrient rich medium where the amino acid L-cystein and iron supply are added (Fields, 1996a) (Figure 1). They utilize mainly organic acids and amino acids as carbon and energy sources (Fields, 1996a;[START_REF] George | Amino acid requirements of Legionella pneumophila[END_REF][START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF].

Different to what was thought previously, it was shown recently that carbohydrate usage is also important for the L. pneumophila life cycle [START_REF] Eisenreich | The life stage-specific pathometabolism of Legionella pneumophila[END_REF][START_REF] Eylert | Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates[END_REF]. Furthermore, L. pneumophila encodes a glycoamylase conferring glycogen-and starch-degrading activity [START_REF] Herrmann | GamA is a eukaryotic-like glucoamylase responsible for glycogen-and starch-degrading activity of Legionella pneumophila[END_REF].

The number of species and serogroups constituting the genus Legionella continuously increases; the genus is currently composed of 58 species and more than 70 serogroups which are colonizing natural aquatic environments like lakes, rivers, ponds and are often found in biofilms [START_REF] Fields | Legionella and Legionnaires' disease: 25 years of investigation[END_REF][START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF][START_REF] Steinert | Legionella pneumophila: an aquatic microbe goes astray[END_REF]). An exception is Legionella longbeachae that is found predominantly in potting soil and is thought to be transmitted by inhalation of dust from contaminated soils [START_REF] Cameron | Epidemiological characteristics of Legionella infection in South Australia: implications for disease control[END_REF][START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF]. Despite the fact that Legionella have not co-evolved with humans but in aquatic habitats, bacteria belonging to the genus Legionella can cause severe, often fatal disease in humans. At least 20 Legionella species have been associated to human disease while others have been isolated only from the environment [START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF]. In addition, a number of so-called Legionella-like amoebal pathogens (LLAPs) have been described. These Legionella-like strains do not grow without amoebae on routine growth ! ! media and even though they show similar genetic composition, they are not associated with disease [START_REF] Marrie | Legionella-like and other amoebal pathogens as agents of community-acquired pneumonia[END_REF][START_REF] Rf | Classification of the genus Legionella[END_REF]. The most-studied Legionella species is L. pneumophila, as it is responsible for most cases of Legionnaires' disease [START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF][START_REF] Yu | Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey[END_REF].

Natural and man-made environmental reservoirs for Legionella

Several studies have analyzed the distribution of legionellae in natural habitats.

L. pneumophila has been isolated from samples at temperature ranging from 10 to 42 °C, however they multiply at temperature between 20 and 42 °C, with an optimal growth temperature of 35 °C. Natural freshwater habitats are rarely associated with legionellosis, but most of the cases of legionellosis are due to man-made aquatic systems where the temperature is higher than the ambient temperature. Although osmolarity and pH appear to be environmental factors related to the prevalence of legionellae, temperature gradients influence the distribution of Legionella species and its multiplication [START_REF] Flannery | Reducing Legionella colonization in water systems with monochloramine[END_REF][START_REF] Fliermans | Ecological distribution of Legionella pneumophila[END_REF][START_REF] Lee | Survival and growth of Legionella species in the environment[END_REF]. In contrast to the wide spread distribution of Legionella in fresh water environments, the limiting nutrient levels of these habitats result mostly in low concentrations of non-replicating planktonic bacteria. Biofilm matrices, composed of heterogeneous communities, are known to provide shelter and nutrients required by this bacterium, thereby serving as an ecological niche for replication and persistence of Legionella outside a host cell (Al-Quadan, [START_REF] Al-Quadan | Exploitation of evolutionarily conserved amoeba and mammalian processes by Legionella[END_REF][START_REF] Fields | Legionella and Legionnaires' disease: 25 years of investigation[END_REF]. Furthermore, biofilm colonization can be influenced by the symbiotic association with other microorganisms and more importantly by the presence of free-living amoebae [START_REF] Declerck | Replication of Legionella pneumophila in biofilms of water distribution pipes[END_REF][START_REF] Murga | Role of biofilms in the survival of Legionella pneumophila in a model potable-water system[END_REF]. Indeed, pioneering studies by Rowbotham showed that this facultative intracellular bacterium replicates and establishes a parasitic life style within freeliving protozoa serving as their natural hosts [START_REF] Rowbotham | Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae[END_REF]. Moreover, it had been reported that Legionella spp have the capacity to replicate within at least 14 different species of fresh water amoebae including Acanthamoeba, Hartmannella, Neaglaria, the ciliate Tetrahymena pyriformis and the slime mold Dictyostelium discoideum (Fields, 1996a;[START_REF] Hägele | Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella[END_REF][START_REF] Solomon | Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions[END_REF][START_REF] Steinert | Dictyostelium as host model for pathogenesis[END_REF]. Taken together, free-living amoebae and biofilms constitute the major environmental reservoirs for L. pneumophila and consequently a potential source of contamination for humans. Indeed, since the discovery that the Legionnaires' disease outbreak in Philadelphia in 1979 was caused by L. pneumophila that was present in the hotel's air conditioning system, the link between natural and man-made

GENERAL INTRODUCTION !

! environmental reservoirs has been clearly shown. Furthermore, due to the elevated temperature, thermally altered aquatic environments can shift the equilibrium between freeliving protozoa and bacteria, which may promote bacterial multiplication and the emergence of the disease. As human infection occurs by inhalation of contaminated aerosols, and more and more such devises are developed, it is generally thought that Legionnaires' disease may have emerged in the last decades as consequence of the industrialization and the alteration of many environments by and for human profit.

Figure 2. From the environment to humans. In the environment Legionella is able to survive as an intracellular parasite of protozoa or persist in biofilms. Upon transmission through man made devices (showers, air conditioning systems, cooling towers etc.) the bacteria may reach the lungs, where they can infect and colonize macrophages, which may lead to a severe pneumonia named Legionnaires' disease.

! A great number of devices has been identified as source of aerosol transmission of Legionella, including potable water sources such as showers and taps, water faucets, and non-potable waters such as cooling towers and evaporative condensers, spas, fountains and humidifiers (Figure 2). The sources of outbreaks of Legionnaire's disease have been traced back to also to diverse locations, including hotels, homes, hospitals and cruise ships. Moreover, the material of the piping system, stagnation and periods of non-use of water pipes have been reported as pivotal factors in the occurrence of Legionella outbreaks [START_REF] Atlas | Legionella: from environmental habitats to disease pathology, detection and control[END_REF][START_REF] Steinert | Regrowth of Legionella pneumophila in a heat-disinfected plumbing system[END_REF].

CHAPTER ONE !

Legionnaires' disease 1.3.1 Symptoms and clinical manifestations

The term legionellosis includes collectively the diseases caused by Legionella that are transmitted by the inhalation of contaminated aerosols. Until recently it was thought that human to human transmission does not happen. However, a first possible evidence of personto-person transmission of Legionnaires' Disease (LD) was recently reported to have occurred during a large outbreak in Portugal [START_REF] Borges | Legionella pneumophila strain associated with the first evidence of person-to-person transmission of Legionnaires' disease: a unique mosaic genetic backbone[END_REF]). Legionnaire's disease is a severe pneumonia with additional multisystem diseases [START_REF] Diederen | Legionella spp. and Legionnaires' disease[END_REF][START_REF] Mcdade | Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease[END_REF].

Symptoms and signs of the disease are variable and range from mild fever, which is usually one of the first signs of illness, to respiratory and organ failure. Furthermore, non-productive cough, anorexia, chills, headache, chest pain, rigors, dyspnea and diarrhea are signs of the Legionnaires' disease. Interestingly, also extra pulmonary manifestations may occur such as confusion, memory loss, hallucinations, neurological manifestations, and metastatic or contiguous infections [START_REF] Marrie | Nosocomial Legionnaires' disease: Clinical and radiographic patterns[END_REF][START_REF] Mcclelland | Pneumonia and osteomyelitis due to Legionella longbeachae in a woman with systemic lupus erythematosus[END_REF]). Legionnaire's disease occurs both as sporadic cases and as outbreaks and is either community or nosocomially acquired. The mild form of legionellosis is the so-called Pontiac fever. This non-pneumonic form is a self-limited influenza-like form of disease associated with exposure to Legionella. Symptoms include fever, headache and myalgia [START_REF] Fields | Virulence of a Legionella anisa strain associated with Pontiac fever: an evaluation using protozoan, cell culture, and guinea pig models[END_REF][START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF].

Diagnosis and treatment

Legionnaires' disease is an atypical pneumonia that might clinically resemble pneumococcal or other bacterial pneumonias, thus a rapid and proper diagnosis is essential for starting the correct treatment since legionellosis has a poor prognosis when treatment starts late. However, the diagnosis of legionellosis is challenging and thus the disease is probably underreported.

Legionnaires' disease can be diagnosed by both non-culture and culture techniques. Diagnosis methods are culturing from sputum, bronchial washing or autopsy/biopsy tissue, however although this was until recently the gold standard for diagnosis of legionellosis, it requires specific growth medium, takes time, and has a poor sensitivity. Since the development of the urinary antigen testing, early diagnosis and rapid initiation of appropriate antibiotic therapy has been achieved, thus it is now the most frequently used diagnostic test (Beauté, Zucs, de Jong, European Legionnaires' Disease Surveillance Network, 2013). Over the last decades the

GENERAL INTRODUCTION !

! number of cases diagnosed has remarkably increased because of the high sensitivity of this test. Nevertheless, this non-culture technique is limited solely to the detection of L.

pneumophila Sg1 [START_REF] Cunha | Legionnaires' disease[END_REF]. Thus, the development of molecular techniques, such as nucleic acid amplification-based methods, can certainly provide diagnosis with specificity for the detection of other serogroups and higher sensitivity than culture methods. Indeed, several PCR based methods that rapidly allow the diagnosis of legionellosis including those due to non serogroup 1 L. pneumophila have been developed in the last years [START_REF] Maze | Enhanced detection of Legionnaires' disease by PCR testing of induced sputum and throat swabs[END_REF][START_REF] Merault | Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples[END_REF]. Legionella are intracellular bacteria and as such antibiotics against the bacterium need to act and accumulate within infected cells. Beta-lactam and aminoglycoside antibiotics do not penetrate the cells and therefore are ineffective against

Legionella. In the original outbreak of Legionnaires' disease the patients treated with the macrolide erythromycin have been reported to show lower mortality, however side effects were also reported. To date, most macrolides, tetracyclines, ketolides and quinolones are effective and particularly azithromycin, doxycycline or levofloxacin can be used as first-line treatment [START_REF] Bruin | Wildtype MIC distribution and epidemiological cut-off values in clinical Legionella pneumophila serogroup 1 isolates[END_REF][START_REF] Roig | Legionnaires' disease: a rational approach to therapy[END_REF].

Risk factors

Legionella spp are opportunistic pathogens thus persons presenting specific risk factors are mainly concerned. Risk factors for developing Legionnaires' disease include age older than 50 years, male gender, chronic lung disease, smoking, immunosuppression, cancer and AIDS [START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF][START_REF] Sandkovsky | Legionella pneumonia and HIV: case reports and review of the literature[END_REF]. Moreover, the likelihood of death is increased in patients with hospital-acquired infections and in recipients of organ transplants [START_REF] Kugler | Nosocomial Legionnaires' disease. Occurrence in recipients of bone marrow transplants[END_REF][START_REF] Yu | Nosocomial legionellosis[END_REF]. However, recently, the infection of neonates from contaminated birthing pools has also gained significant attention since two independent cases and subsequent fatalities were reported [START_REF] Fritschel | Fatal legionellosis after water birth, Texas, USA, 2014[END_REF]Phin, Cresswell, Parry-Ford, Incident Control Team, 2014). Furthermore, repeated exposure to infectious sources represents a risk factor for Legionnaires' disease onset [START_REF] Cunha | Legionnaires' disease[END_REF][START_REF] Fraser | Legionellosis: evidence of airborne transmission[END_REF]; J. T. [START_REF] Johnson | Nosocomial legionellosis in surgical patients with head-and-neck cancer: implications for epidemiological reservoir and mode of transmission[END_REF][START_REF] Marrie | Control of endemic nosocomial legionnaires' disease by using sterile potable water for high risk patients[END_REF][START_REF] Muder | Infection due to Legionella species other than L. pneumophila[END_REF].

Epidemiology and incidence

Since the 1976 epidemic event, many sporadic cases and several outbreaks occurred worldwide. The precise incidence of Legionnaire's disease is not known, mostly due to the worldwide different awareness levels, diagnostic methods and surveillance methods, resulting CHAPTER ONE ! in a under-diagnosed and under-reported disease. In the United States about 5 000 cases of Legionnaires' disease are reported each year [START_REF] Dooling | Active Bacterial Core Surveillance for Legionellosis -United States, 2011-2013[END_REF]. Data from the USA passive surveillance for Legionnaires' disease indicated a 286% increase in reported cases per 100,000 people during 2000-2014. This could be due to a true increase in the frequency of disease caused by a number of reasons such as an expanded susceptibility of the population, increased Legionella concentrations in the environment, or to an increased testing for Legionnaires' disease, (http://www.cdc.gov/legionella/surv-reporting.html). The yearly incidence shows seasonal variations, particularly more illness usually occur in the summer and early fall (http://www.cdc.gov/legionella/health-depts/inv-tools-single/index.html).

In 2013, 5851 cases of Legionnaires' disease were reported to the European Centre for Disease Prevention and Control. Within the years of 2005-2012 a notification rate of 11.4 cases per million people was observed. Most of the cases were community-acquired (73%),

19% were travel-associated, and 8% were linked to healthcare facilities. Most cases (88%) were confirmed by the urinary antigen test (http://ecdc.europa.eu/en/healthtopics/legionnaires_disease/pages/index.aspx). In France 1389 cases of Legionnaire's disease were reported in 2015 with a prevalence of 2.1 cases per 100 000 inhabitants (http://invs.santepubliquefrance.fr/Dossiers-thematiques/Maladiesinfectieuses/Infections-respiratoires/Legionellose), a number of infection that remained quite stable over the last years as seen in Figure 3. Quite interestingly, among the 58 Legionella species described until now, the strains belonging to the species L. pneumophila are responsible for over 90% of the Legionnaires' disease cases worldwide. Moreover, the strains belonging to the species L. longbeachae are

GENERAL INTRODUCTION !

! responsible for about 4% of human cases world [START_REF] Yu | Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey[END_REF], however, their distribution in Australia and New Zealand is different, as L. longbeachae accounts for 30.4% of the human Legionnaires' disease cases in this geographical region. Furthermore, among the strains causing Legionnaires' disease, L. pneumophila serogroup 1 (Lp1) alone is responsible for about 84% of cases although 15 serogroups are described within this species (Gomez-Valero, Rusniok, Cazalet, & Buchrieser, 2011a;[START_REF] Kozak-Muiznieks | Prevalence of sequence types among clinical and environmental isolates of Legionella pneumophila serogroup 1 in the United States from 1982 to 2012[END_REF][START_REF] Yu | Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey[END_REF][START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF]. Moreover, in the last years, molecular typing methods allowed to show, that some of the L. pneumophila Sg1 strains are worldwide distributed like L. pneumophila strain Paris [START_REF] Cazalet | Analysis of the Legionella longbeachae genome and transcriptome uncovers unique strategies to cause Legionnaires' disease[END_REF]2004), and others are emerging clones like the L. pneumophila Sg1 subclones ST47 (Lorraine) [START_REF] Ginevra | Lorraine strain of Legionella pneumophila serogroup 1, France[END_REF] or ST1, ST37, ST62 and ST23 (David et al., 2016).

Association with free-living amoebae

Many clinically relevant pathogens like Listeria, Legionella, Vibrio, Mycobacterium or Burkholderia are reported to survive within protozoa in the environment and to establish a symbiotic or parasitic relationship with these hosts (M. R. [START_REF] Brown | Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms[END_REF][START_REF] Greub | Microorganisms resistant to free-living amoebae[END_REF][START_REF] Hilbi | Environmental predators as models for bacterial pathogenesis[END_REF][START_REF] Molmeret | Amoebae as training grounds for intracellular bacterial pathogens[END_REF].

However, Legionella has a special place among these pathogens, as protozoa are their natural hosts. Thus protozoa play a crucial role for the survival, transmission and virulence and their association with protozoa may explain the constant presence of Legionella in the environment. Within protozoa L. pneumophila displays a remarkable increased resistance to harsh conditions such as fluctuations in temperature, pH, acidity and osmolarity, facilitating their survival in the environment (Abu Kwaik, [START_REF] Abu Kwaik | Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant[END_REF]. In addition this intracellular niche provides Legionella with resistance to chemical disinfection and biocides, such as chloride, UV irradiation, compared to in vitro-grown bacteria, hindering thus the eradication of L. pneumophila from environmental sources of infections [START_REF] Barker | Trojan horses of the microbial world: protozoa and the survival of bacterial pathogens in the environment[END_REF] [START_REF] Barker | Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila[END_REF][START_REF] Cunha | Legionnaires' disease[END_REF]. In addition, protozoa have been reported to release respirable L. pneumophila-containing vesicles, which are resistant to freezing-thawing and sonication [START_REF] Berk | Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp[END_REF]. Other observations derived from the co-cultivation of viable but no-culturable (VBNC) L. pneumophila with amoebae, showed that these provided the adequate stimuli and nutrients necessary for resuscitating the hibernated L. pneumophila [START_REF] García | Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection[END_REF][START_REF] Steinert | Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii[END_REF]. Thus, environmental protozoa represent not only a nutrition CHAPTER ONE ! resource for the survival, replication and distribution of Legionella, but also function as shelter affording protection against adverse environmental conditions ( [START_REF] Barker | Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila[END_REF][START_REF] Berk | Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp[END_REF]. In addition to shelter and nutrient provided by amoebae to Legionella, it has been reported that L. pneumophila released from protozoan hosts display a striking increase in infectivity of mammalian cells in vitro. For instance, bacteria released from H. vermiformis are more infectious and kill host cells more efficiently than in vitro grown Legionella [START_REF] Brieland | The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease[END_REF]. L. pneumophila bacteria grown within amoeba are also highly motile and survive and multiply more efficiently within human monocytes, (J. D. [START_REF] Cirillo | Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila[END_REF][START_REF] Cirillo | Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion[END_REF][START_REF] Garduno | Intracellular growth of Legionella pneumophila gives rise to a differentiated form dissimilar to stationary-phase forms[END_REF].

While protozoa are the natural hosts of L. pneumophila, the infection of human cells remains opportunistic, however the dual host specificity may be due to the fact that protozoa are primordial phagocytes and as such they share many features with mammalian phagocytes including conserved antimicrobial processes [START_REF] Hilbi | Environmental predators as models for bacterial pathogenesis[END_REF][START_REF] Al-Quadan | Exploitation of evolutionarily conserved amoeba and mammalian processes by Legionella[END_REF].

Particularly, the intracellular life cycle of L. pneumophila has been shown to be very similar in both hosts [START_REF] Fields | Legionella and Legionnaires' disease: 25 years of investigation[END_REF]. Thus it has been proposed that the million years of coevolution with amoebae has equipped these bacteria with effective strategies to face antimicrobial effectors and the human immune system (Gao, Harb, & Abu Kwaik, 1997;[START_REF] Molmeret | Amoebae as training grounds for intracellular bacterial pathogens[END_REF]. The analyses of the L. pneumophila genome sequence in 2004 further supported this hypothesis (Cazalet et al., 2004). This sequence analyses was a major breakthrough and key to the further understanding of the strategies employed by Legionella to subvert host functions and the evolution of virulence as it led to the identification of a large number of bacterial proteins with eukaryotic-like properties as witness of the tight coevolution between L. pneumophila and its protozoan hosts (Cazalet et al., 2004). It also led to a new concept in host pathogen interactions: bacteria parasitizing protozoa acquire genes from their hosts to subvert host functions. Later the work of several groups including ours, showed that these eukaryotic-like proteins are indeed secreted effectors that act in the host like their eukaryotic homologues -thus molecular mimicry is a major virulence strategy of Legionella (see below) [START_REF] Nora | Molecular mimicry: an important virulence strategy employed by Legionella pneumophila to subvert host functions[END_REF]. Taken together, the association of Legionella with environmental protozoa is essential for the ecology and pathogenesis of these bacteria.

GENERAL INTRODUCTION ! !

Intracellular life cycle within phagocytic cells

The primary feature of L. pneumophila pathogenesis is related to its capability to multiply intracellularly. The intracellular cycle of legionellae has been characterized in both protozoan and mammalian cells. Microscopically, the two processes are identical, even though differences in entering and exiting the host cell steps have been described [START_REF] Escoll | From amoeba to macrophages: exploring the molecular mechanisms of Legionella pneumophila infection in both hosts[END_REF]. Our current understanding of the L. pneumophila infectious cycle is represented in Figure 4. Following the contact of L. pneumophila with phagocytic cells, such as macrophages or amoebae, the bacteria are internalized through conventional phagocytosis or coiling phagocytosis [START_REF] Bozue | Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion[END_REF]; J. A. [START_REF] Elliott | Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila[END_REF][START_REF] Horwitz | Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil[END_REF]. Conventional phagocytosis in macrophages was shown to be mediated through a complement-dependent mechanism by complement receptor CR1 and CR3. Following the CR3 binding to the major outer membrane protein MOMP, the association MOMP-CR3 is sufficient to induce the bacterial phagocytosis (Bellinger-Kawahara & [START_REF] Bellinger-Kawahara | Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes[END_REF]; N. R. [START_REF] Payne | Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors[END_REF]. By contrast, in the amoebae Hartmannella veriformis, the attachment and the induction of L. pnuemophila uptake are mediated through the lectin receptor in a time-dependent manner [START_REF] Escoll | From amoeba to macrophages: exploring the molecular mechanisms of Legionella pneumophila infection in both hosts[END_REF][START_REF] Venkataraman | Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires' disease bacterium[END_REF]. After uptake in protozoa or macrophages, the bacteria persist within the so-called Legionella-containing vacuole (LCV), which subsequently evades the lysosomal network. Within minutes of uptake, numerous vesicles derived from the mitochondria and the endoplasmic reticulum (ER) are docked onto the LCV membrane. Within this ER-like vacuole, the bacteria replicate and at the end of the replication cycle flagellated bacteria are released. Adapted from [START_REF] Isberg | The Legionella pneumophila replication vacuole: making a cosy niche inside host cells[END_REF].

CHAPTER ONE

!

The non-complement mediated attachment and invasion of L. pneumophila in phagocytic cells depends on several factors such as type IV pili [START_REF] Stone | Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells[END_REF], the 60 Kda heat shock protein Hsp60 (R. A. [START_REF] Garduño | Surface-associated hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model[END_REF] and the enhanced entry protein RtxA (Repeats in structural Toxin) encoded by the enh1 locus and the enhanced entry protein EnhC (S. L. [START_REF] Cirillo | Legionella pneumophila entry gene rtxA is involved in virulence[END_REF][START_REF] Cirillo | Identification of novel loci involved in entry by Legionella pneumophila[END_REF]. Recently it has been shown that EnhC also interferes with immunstimulatory muramyl-peptide production to evade innate immunity (M. Liu et al., 2012). Furthermore, the secreted proteins LaiA, SidE and LpnE [START_REF] Newton | Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry[END_REF] are required for efficient host entry [START_REF] Chang | Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila[END_REF][START_REF] Newton | Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry[END_REF]. However, LpnE like EnhC and LidL also influences trafficking of the L. pneumophila containing vacuole [START_REF] Newton | Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking[END_REF]. In addition, the presence of the flagellum and generally motility also positively affects the establishment of infection. Comparative evidences have suggested that elaborate and diverse uptake processes exist in different hosts [START_REF] Steinert | Legionella pneumophila: an aquatic microbe goes astray[END_REF].

After entry into phagocytes, L. pneumophila manipulates the maturation process of the nascent phagosome and forms a unique replication-permissive compartment that is resistant to acidification (Horwitz, 1983a;1983b). This Legionella-containing vacuole (LCV) is a singlemembrane compartment surrounded by numerous small vesicles on the cytoplasmic face.

Distinct and temporal docking events follow the escape of the LCV from the lysosome, including the recruitment of mitochondria and within 4 hours vesicles derived from rough endoplasmic reticulum (ER) cluster near the LCV membrane [START_REF] Isberg | The Legionella pneumophila replication vacuole: making a cosy niche inside host cells[END_REF][START_REF] Roy | Exploitation of the endoplasmic reticulum by bacterial pathogens[END_REF] (Figure 4). Fluorescence microscopy analysis of either intact cells or isolated LCVs has shown that ER-associated proteins, such as Sec22b and the GTPase Rab1, localize near the vacuole, elucidating the origin and the trafficking network of vesicles surrounding the LCVs (Kagan & Roy, 2002;[START_REF] Swanson | Association of Legionella pneumophila with the macrophage endoplasmic reticulum[END_REF]. Further studies demonstrated that the vesicles derived from the ER fuse with the LCV membrane, leading to the release of their soluble content into the LCV lumen [START_REF] Robinson | Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila[END_REF]. Although the ER association with the LCV has been detected during intracellular replication of Legionella, additional membrane trafficking events may occur and modulate the intracellular life cycle of this bacterium. During the late replicative phase, the LCV was found in association with the late endosomal protein LAMP1 (lysosome-associated membrane protein 1), postulating that the lysosomal compartment may provide Legionella with a nutrient-rich environment necessary

GENERAL INTRODUCTION ! ! 14
for replication [START_REF] Sturgill-Koszycki | Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles[END_REF]. Approximately 20-24 hours postinfection, the host cell is extensively filled with bacteria (Figure 5). Following replication, depletion of nutrients drives the differentiation of L. pneumophila from a replicative form, where the bacteria are not motile and not cytotoxic, to a virulent form, characterized as motile, cytotoxic and flagellated bacteria (Molofsky & Swanson, 2004). This transition ensures that the bacteria activate the infectious traits for the escape and the following transmission into a new host [START_REF] Hammer | LEGIONELLA PNEUMOPHILA PATHOGENESIS: A Fateful Journey from Amoebae to Macrophages[END_REF]. During replication in the host cell L. pneumophila induces the expression of several host antiapoptotic genes via activation of the transcription factor NFκB [START_REF] Abu-Zant | Antiapoptotic signalling by the Dot/Icm secretion system of L. pneumophila[END_REF][START_REF] Losick | NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila[END_REF]. Only at the late stages of infection macrophages show increasingly apoptotic phenotypes, such as nuclear condensation and phosphatidylserine exposure but little is known about how Legionella may induce apoptosis to leave the host cell [START_REF] Speir | Programmed cell death in Legionella infection[END_REF]. Recently it has been proposed that inducing apoptosis of the host cell by inhibiting the host factors BCL-XL may help eliminating L. pneumophila during infection [START_REF] Speir | Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis[END_REF].

Hijacking host cell defenses by the Dot/Icm machinery

Genome sequencing and analysis led to an in depth understanding of genomic and but also pathogen-related features of L. pneumophila, like the presence of multiple pathogenicity islands and mobile genetic elements. This bacterium shows high plasticity and genetic diversity, considering that 7.5-10.5% of its genes are strain specific (Gomez-Valero, Rusniok, CHAPTER ONE Jarraud, Vacherie, Rouy, et al., 2011b). However, the most remarkable genomic feature of L.

!

pneumophila is the presence of a high number and variety of eukaryotic-like proteins or eukaryotic-like domains, which are thought to have evolved during the bacterial co-evolution with fresh-water amoebae (Cazalet et al., 2004). Indeed, phylogenetic analyses of some of these eukaryotic-like proteins has been carried out revealing a clustering of the Legionella eukaryotic like proteins with eukaryotic sequences, further supporting the hypothesis that these were acquired by HGT from eukaryotic organisms like amoeba (Gomez-Valero & Buchrieser, 2013;[START_REF] Lurie-Weinberger | The origins of eukaryotic-like proteins in Legionella pneumophila[END_REF][START_REF] Nora | Molecular mimicry: an important virulence strategy employed by Legionella pneumophila to subvert host functions[END_REF]. These eukaryotic-like proteins and proteins encoding domains normally only or mainly present in eukaryotic proteins are good candidates to manipulate host cell functions to the pathogens advantage. As such, these proteins need to be secreted in the host cells. Among the wide varieties of secretion systems identified in L. pneumophila, the type IVB secretion system (T4BSS) named dot/icm (defective organelle trafficking/intracellular multiplication) is essential for intracellular replication of L. pneumophila [START_REF] Berger | Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila[END_REF][START_REF] Marra | Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages[END_REF]. This macromolecular complex, shown in Figure 6, is encoded by 27 genes, which are located in two distinct genomic regions. [START_REF] Buscher | The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02[END_REF]. Individual letters represent Dot protein names, whereas letters preceded by an "I" indicate Icm protein names. Adapted from [START_REF] Isberg | The Legionella pneumophila replication vacuole: making a cosy niche inside host cells[END_REF].

GENERAL INTRODUCTION ! !

The majority of the dot/icm genes are dedicated to the formation of the secretion complex [START_REF] Vincent | Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system[END_REF]. Besides structural proteins, membrane-spanning proteins are associated and interact with the host cell membrane by forming a pore for the translocations of "effectors" into the cytoplasm [START_REF] Segal | The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii[END_REF]. In addition, the Dot/Icm secretion system includes chaperons, such ad IcmS and IcmW/DotB which guide the effectors into the secretion system or provide the necessary energy for the assembly of the whole apparatus [START_REF] Segal | The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii[END_REF]. The sequences of the dot/icm genes is 96-98% conserved across L. pneumophila strains and 62-79% conserved across Legionella species (Gomez-Valero, Rusniok, Cazalet, & Buchrieser, 2011a;Gomez-Valero et al., 2014;[START_REF] Morozova | Comparative sequence analysis of the icm/dot genes in Legionella[END_REF].

The Dot/Icm system translocates the surprisingly high number of over 300 effectors into the host cytoplasma, allowing this bacterium to manipulate many host signalling and metabolic pathways [START_REF] De Felipe | Legionella eukaryotic-like type IV substrates interfere with organelle trafficking[END_REF][START_REF] Heidtman | Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways[END_REF][START_REF] Ninio | A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles[END_REF][START_REF] Zhu | Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila[END_REF]. This secretion apparatus is essential for the establishment of the LCV, the escape from lysosomal fusion and the multiplication in both, amoeba and macrophages [START_REF] Burstein | Genome-scale identification of Legionella pneumophila effectors using a machine learning approach[END_REF][START_REF] De Felipe | Legionella eukaryotic-like type IV substrates interfere with organelle trafficking[END_REF][START_REF] Escoll | From amoeba to macrophages: exploring the molecular mechanisms of Legionella pneumophila infection in both hosts[END_REF]Heidtman et al., 2009;[START_REF] Shohdy | Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking[END_REF]. The first characterized effector of the Dot/Icm T4SS was RalF, a guanine nucleotide exchange factor (GEF) required for the activation and recruitment of the host GTPase ARF-1 to the LCV. ARF-1 is a highly conserved master regulator of membrane trafficking processes, particularly vesicle trafficking between the ER and Golgi complex and thus important for the intracellular growth of L. pneumophila [START_REF] Nagai | A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes[END_REF]. Moreover, a second bacterial GEF named SidM (also known as DrrA) actives the small GTPase Rab1 by promoting nucleotide exchange and recruits it to the LVC, similarly to ARF1 recruitment [START_REF] Murata | The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor[END_REF]. Despite the recruitment of Rab1 by SidM, it is not essential for intracellular growth, the coordinated activity of ARF-1, Rab 1 and Sar1 is required to guide the trafficking and fusion of ERderived vesicles to the LCV [START_REF] Machner | Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila[END_REF][START_REF] Nagai | Show me the substrates: modulation of host cell function by type IV secretion systems[END_REF]. Another Dot/Icm secreted effector named VipA has been described to act as actin nucleator, polymerizing microfilaments. VipA alters host cell vesicle trafficking, however it is not essential for entry or replication in amoeba or macrophages [START_REF] Franco | The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking[END_REF]. Several, functionally distinct translocated substrates have been identified and characterized such as LidA, involved in the recruitment of early secretory vesicles to the LCV in the integrity maintenance of the Dot/Icm complex, and LepA and LepB, involved in the bacterial non-lytic release from the LCV during the amoeba infection (J. [START_REF] Chen | Legionella effectors that promote nonlytic release from protozoa[END_REF].

CHAPTER ONE

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In recent years, the eukaryotic like proteins or proteins with eukaryotic like domains have been functionally analysed. This has shown that they are indeed Dot/Icm effectors that subvert host functions to the pathogens advantage. For example L. pneumophila subverts ubiquitin signalling by secreting F-box and U-box proteins into the host cell where they ubiquitinate proteins on the Legionella containing vacuole [START_REF] Kubori | Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions[END_REF]Lomma et al., 2010;[START_REF] Price | Indispensable role for the eukaryotic-like ankyrin domains of the ankyrin B effector of Legionella pneumophila within macrophages and amoebae[END_REF]. Thanks to the eukaryotic CaaX motif that allows anchoring into membranes, the F-box protein named AnkB was reported to serve as platform for the docking of polyubiquitinated proteins to the LCV to generate aminoacids for intracellular replication of L. pneumophila [START_REF] Ivanov | Lipidation by the host prenyltransferase machinery facilitates membrane localization of Legionella pneumophila effector proteins[END_REF][START_REF] Price | Indispensable role for the eukaryotic-like ankyrin domains of the ankyrin B effector of Legionella pneumophila within macrophages and amoebae[END_REF]. Furthermore authophagy is subverted by L. pneumophila by secreting different effectors like RavZ or Spl, a protein that shows a high degree of sequence similarity to the eukaryotic enzyme sphingosine-1 phosphate (SPL). RavZ is a protease that deconjugates Atg8 in the host cell [START_REF] Choy | The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation[END_REF], whereas Spl is an enzyme that directly targets the host sphingolipid metabolism (Rolando et al., 2016). Thus Spl inhibits autophagosome formation and RavZ autophagosome maturation, showing that L. pneumophila, like other intracellular pathogens, employs several strategies to counteract autophagy in the host cell (Escoll, Rolando, & Buchrieser, 2016). Furthermore, L.

pneumophila modifies the chromatin landscape of its host by secreting the effector RomA that contains a eukaryotic SET-domain conferring methyltransferase activity to tri-methylate lysine 14 of histone H3. RomA promotes H3K14 methylation of 4870 promoter regions, including genes with roles in innate immunity (Rolando et al., 2013).

These examples show, that indeed, the acquisition of eukaryotic like genes by L. pneumophila from its hosts, has shaped its genome and its virulence strategies and many more functions are still to be discovered. However, not all the characterized Dot/Icm effectors are present in all L. pneumophila strains, suggesting that L. pneumophila has acquired strain-specific sets of effector proteins to face distinct host cell niches, to which they may be confronted during their evolution (Cazalet et al., 2004;Gomez-Valero & Buchrieser, 2013).

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CHAPTER TWO

HFQ AND SMALL REGULATORY RNAs

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INTRODUCTION

Pathogenic bacteria have evolved a variety of strategies to replicate in many different niches in their hosts. They have learned how to counteract the host defences and to replicate in specific, normally sterile regions. There are bacteria that remain extracellular and those that are internalized via active or passive pathways (Cossart and Sansonetti 2004). Intracellular bacterial pathogens co-exist with the infected cell in an obligate intracellular state or transit between the extracellular and intracellular environment. However, all have evolved the ability to survive and grow within eukaryotic cells such as professional phagocytes or other cell types by employing diverse mechanisms to manipulate the host cell for their own benefit. After entering the host cell, the bacteria are surrounded by a membrane-bound vacuole that is targeted to the lysosomal compartments to undergo proteolytic degradation. There, the bacteria are facing a changing and very hostile environment characterized by decreasing pH, elevated concentration of reactive oxygen and nitrogen species and poor nutrient content to which either they have to adapt or they have to avoid in order for infection to succeed. Two main strategies are observed: (i) survival in this CHAPTER TWO ! 21 Microbiology Reviews, 2015, Vol. 39, No. 3 niche either by preventing vacuole-lysosome fusion or by modifying the environment within the phagolysosome (vacuolar pathogens) like Legionella, Coxiella, Brucella, Mycobacterium and Salmonella; or (ii) escape from the vacuole to gain access to and proliferate within the host cell cytosol (cytosolic pathogens) like Listeria, Shigella, Burkholderia, Francisella and Rickettsia (Haas 2007;[START_REF] Ray | Life on the inside: the intracellular lifestyle of cytosolic bacteria[END_REF]Fredlund and Enninga 2014). Adaptation to these intracellular lifestyles is regulated in both space and time. Those bacteria that adapt most rapidly to the hostile conditions encountered in the host are the most successful. The adaptation process is accompanied by major changes in gene transcripts, post-transcriptional regulatory molecules and the protein levels of the bacteria. For intracellular bacteria this includes a temporal and spatial highly co-ordinated regulation of the production of specific effector proteins. These proteins are transferred to the host by dedicated secretory systems and are able to modify the immune response and the metabolism of the infected cell, bringing advantage for the pathogens (Hubber and Roy 2010;Agbor and Mc-Cormick 2011;[START_REF] Steiner | Host-pathogen interactions and immune evasion strategies in Francisella tularensis pathogenicity[END_REF]. The bacteria sense specific stimuli and respond with changes in gene expression, a process that is tightly controlled by various and complex regulatory networks. Involved therein are factors that are highly conserved in the microbial world including the stringent response alarmone (p)ppGpp [START_REF] Dalebroux | ppGpp: magic beyond RNA polymerase[END_REF], two-component systems [START_REF] Salazar | Temporal and evolutionary dynamics of two-component signaling pathways[END_REF], the global regulator CsrA (Romeo, Vakulskas and Babitzke 2013), the RNA chaperone Hfq (Sobrero and Valverde 2012) and the alternative sigma factor RpoS [START_REF] Lange | Identification of a central regulator of stationary-phase gene expression in Escherichia coli[END_REF][START_REF] Schellhorn | Elucidating the function of the RpoS regulon[END_REF]. Key players in these important regulatory networks are small, non-coding RNAs (sRNA). In the last years, genome-wide expression studies using high-density tiling arrays or RNA deep sequencing (RNAseq) have uncovered the wide distribution and large number of non-coding RNAs present in bacterial genomes (Papenfort and Vogel 2010). Studies of their functionality attracted more and more attention, and meanwhile it is evident that they play a crucial role in many biological processes such as in environmental sensing and stress adaptation, virulence and infectivity of intracellular bacteria, as well as development and metabolism (Gottesman and Storz 2011). In this review we will focus on how intracellular pathogens regulate their adaptation to the invaded host, allowing their replication, by discussing the fascinating roles that sRNAs and the two major RNA-binding proteins Hfq and CsrA/RsmA play in these regulatory processes. After describing their mechanisms of function we will illustrate their impact by providing selected examples of their regulatory roles in intracellular bacteria.

332

FEMS

MECHANISMS BY WHICH sRNAs FUNCTION IN BACTERIA

Regulatory RNAs in bacteria are usually not translated and comprise a size range between 50 and 400 nucleotides in length. They can modulate transcription, translation, mRNA stability, and DNA maintenance or silencing. These diverse functions are achieved through a variety of mechanisms, including changes in RNA conformation, protein binding, base pairing with other RNAs and interactions with DNA (Waters and Storz 2009;Gottesman and Storz 2011). Due to the recent development and improvement of genome-wide RNAseq methods, there has been an explosion in the amount of sRNAs identified, but the characterization of their function in the diverse regulatory networks is still in its infancy [START_REF] Sorek | Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity[END_REF]. The major mechanisms of how sRNAs can act are as follows.

-Trans-encoded sRNA molecules are present on the chromosome in a location distinct from their targets and thus share only limited complementarity with their targets. The general mechanism of trans-acting sRNAs is to sequester the ribosome-binding site (RBS) of a target mRNA by base-pairing to the Shine-Dalgarno (SD) sequence or the start codon and may also interact with the coding sequence of the mRNAs [START_REF] Storz | Regulation by Small RNAs in Bacteria: Expanding Frontiers[END_REF]. In order to exploit their regulatory functions, most of the already characterized trans sRNAs are tightly coupled with the activity of RNases, which are enzymes involved in RNA turnover through RNA cleavage [START_REF] Viegas | Characterization of the role of ribonucleases in Salmonella small RNA decay[END_REF][START_REF] Viegas | Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway[END_REF][START_REF] Saramago | The role of RNases in the regulation of small RNAs[END_REF]. Furthermore, taking into account the limited complementarity between sRNAs and their mRNA targets, many trans-encoded RNA molecules engage the RNA chaperone protein Hfq (Vogel and Luisi 2011) (Fig. 1A). -Cis-encoded antisense RNAs are complementary to their target RNA as they are transcribed from the DNA strand opposite to the genes they regulate, hence they can interact autonomously. They are often located in the untranslated regions (UTRs) of the corresponding gene where the RNA duplex formation can affect ribosome-binding/translation, termination events or overall stability of the mRNA by rearranging the secondary structures, such as hairpins in the target RNA (Caldelari et al. 2013) (Fig. 1B). -Alternatively to the RNA-RNA regulation, sRNA can also interact with regulatory proteins, influencing their activity directly. The best understood system in this context is the global carbon storage regulator of the CsrA/RsmA family.

A common feature in this network present in numerous pathogenic and non-pathogenic bacteria is the transcription of at least one or more sRNAs named CsrBC or RsmYZ, depending on a two-component system (TCS). These sRNAs can interact with CsrA/RsmA and sequester it from its specific position on the target mRNA often in the immediate vicinity of the RBS [START_REF] Babitzke | CsrB sRNA family: sequestration of RNAbinding regulatory proteins[END_REF]. This leads to the translation of the previously blocked transcripts. Alternatively, it is discussed that CsrA might also have positive effects on the turnover of mRNAs due to stabilization of the target transcript and protection against RNase attacks. -A more recently described class of sRNAs participates in the clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas) system, which provides the bacteria with an RNA-mediated adaptive immune system against nucleic acids deriving, for example, from bacteriophages, plasmids or mobile genetic elements [START_REF] Brouns | Small CRISPR RNAs guide antiviral defense in prokaryotes[END_REF][START_REF] Jinek | Structures of Cas9 endonucleases reveal RNA-mediated conformational activation[END_REF]. The variable crRNAs originating from the foreign DNA are located in an array, each of which is flanked by an identical repeat sequence (Fig. 2). Together with the conserved Cas proteins, the crRNA can recognize the complementary DNA target and mediate its degradation.

By highlighting some of the recent studies about these different RNA-based regulations in intracellular bacteria we show the remarkable contribution of sRNAs and their broad diversity and functionality in regulatory networks (Table 1).

THE RNA CHAPERONE HFQ AND ITS INTERACTION WITH TRANS-ENCODED SMALL RNAs

Oliva et al.

333 Fernandez, Eoyang and August 1968). Hfq is now known as a central mediator of sRNA-based gene regulation in bacteria (Aiba 2007;Waters and Storz 2009;Gottesman and Storz 2011;[START_REF] Storz | Regulation by Small RNAs in Bacteria: Expanding Frontiers[END_REF]Vogel and Luisi 2011;Wagner 2013). It is a member of the Sm protein family that is ubiquitous in prokaryotes and in eukaryotes where it is implicated in RNA splicing and decay, suggesting an ancient origin [START_REF] Moller | Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction[END_REF]Wilusz and Wilusz 2005). However, in some cases there is no known Hfq homologue, as in the bacterial clades: Chlamydia-Spirochaetes, Actinomycetes-Deinococcus-Cyanobacteria and Green sulfur bacteria-Cytophagales and in those that have experienced a massive genome reduction due to their parasitic lifestyle, e.g. Buchnera sp., Rickettsia prowazekii and Brucella melitensis [START_REF] Sun | Predicted structure and phyletic distribution of the RNAbinding protein Hfq[END_REF]Chao and Vogel 2010). Briefly, Hfq in association with an sRNA might block the binding of 30S and 50S ribosomal subunits to the RBS of a target mRNA, leading to repression of protein synthesis (Fig. 3A). Conversely, Hfq can bolster protein translation by accompanying the sRNA to the 5 ′ end of its mRNA target and disrupting a secondary structure which otherwise could mask the RBS and prevent mRNA translation (Fig. 3B). Moreover Hfq can act alone, protecting sRNAs from ribonuclease cleavage by RNase E by binding to many RNase E cleavage sites and prohibiting its degradation in a post-target recognition manner (Vogel and Luisi 2011). On the other hand, Hfq can also stimulate the cleavage of sRNAs and their target mRNAs by directly interacting with RNase E, forming a degradasome-like complex that digests the bound RNA [START_REF] Hajnsdorf | Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I[END_REF][START_REF] Morita | RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs[END_REF]) (Fig. 3C andD). Furthermore, Hfq may induce 3 ′ end polyadenylation of an mRNA by the enzyme poly(A)polymerase (PAP) and the subsequent 3 ′ to 5 ′ degradation by the exoribonucleases, governing the RNA turnover (Fig. 3E). However, it should be mentioned that not all trans-encoded RNAs require Hfq (e.g. RprA or RNAII). These sRNAs free of Hfq binding are preferably degraded by polynucleotide phosphorylase (PNPase) [START_REF] Andrade | The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq[END_REF].

Taken together, Hfq is establishing dynamic interactions with a plethora of diverse RNA molecules and affects physiological functions and virulence of bacterial pathogens. Indeed, deletion of hfq led to growth defects and dramatic virulence phenotypes in several intracellular bacteria, such as Francisella tularensis (Meibom et al. 2009), Legionella pneumophila (McNealy et al. 2005), Neisseria gonorrhoeae [START_REF] Dietrich | The effect of hfq on global gene expression and virulence in Neisseria gonorrhoeae[END_REF], S. enterica serovar Typhimurium [START_REF] Sittka | The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium[END_REF]) and Listeria monocytogenes (Christiansen et al. 2004). Intriguingly, in a growing number of bacteria such as Burkholderia cenocepacia (Ramos et al. 2013), multiple Hfq proteins indicating potential functional diversification have been identified. Studies of Hfq in these different organisms will help to uncover its full role in post-transcriptional regulation. 6-15% of their genes were affected by the deletion of hfq (Chao and Vogel 2010). For S. enterica serovar Typhimurium, the intracellular pathogen, where Hfq has been studied probably the most using several different techniques, it has been shown that Hfq might directly or indirectly regulate 20% of all Salmonella genes. These genes include those required for host cell invasion, motility, central metabolism, lipopolysaccharide biosynthesis, two-component regulatory systems and fatty acid metabolism (Sittka et al. 2008;[START_REF] Ansong | Global systems-level analysis of Hfq and SmpB deletion mutants in Salmonella: implications for virulence and global protein translation[END_REF]. Thus Hfq causes diminished fitness and pleiotropic phenotypes and has been shown to be required for virulence of an increasing number of bacterial pathogens. For many intracellular bacteria, it was reported that Hfq deficiency dramatically impacts on virulence and fitness. For example Hfq is required for the expression of the VirB Type IV secretion system that is indispensable for full virulence of Brucella abortus [START_REF] Caswell | The RNA chaperone Hfq independently coordinates expression of the VirB type IV secretion system and the LuxR-type regulator BabR in Brucella abortus 2308[END_REF]. Hfq is also necessary for the stabilization of two small regulatory RNAs, called AbcR1 and AbcR2 (Table 1). Although single mutants are not defective in intracellular growth in macrophages, double mutants are significantly attenuated. Thus AbcR1 and AbcR2 together are important for survival of B. abortus in macrophages and for virulence in a mouse model of infection. Although less is know about their mechanism of action, AbcR1 and AbcR2 directly regulate gene expression by degradation of the mRNA targets, which may involve the recruitment of a ribonuclease (Caswell et al. 2012). These two sRNAs seem to perform redundant and compensatory regulatory functions probably as a consequence of an evolutionary adaption. This is an example where functional redundancy of RNA molecules may ensure the proper expression of genes required for the pathogenic lifestyle and is reminiscent of the CsrA system of, for example, L. pneumophila where the two sRNAs RsmY and RsmZ have a very strong virulence phenotype only when both are missing (Sahr et al. 2009).

Genome-wide identification of

Furthermore, the Hfq RNA chaperone of L. monocytogenes was shown to contribute to stress tolerance and virulence.

Co-immunoprecipitation with L. monocytogenes Hfq followed by sequencing of the Hfq-binding RNA molecules identified initially three novel sRNAs (Christiansen et al. 2006). Interestingly, Hfq is dispensable for sRNA-mediated ribo-regulation in most Gram-positive bacteria. However, in the Gram-positive, intracellular pathogen L. monocytogenes there is evidence for Hfqdependent translational repression. Hfq regulates the expression of the gene lmo0850 in two ways. First, Hfq stabilizes the sRNA LhrA (Table 1), encoded antisense to its target gene, and consequently down-regulates the lm0850 mRNA in an LhrAdependent manner. Secondly, Hfq facilitates the association of LhrA with its mRNA target, defining a structure amendable for RNA duplex formation. However, the exact biological role of the LhrA sRNAs in L. monocytogenes remains to be characterized (Nielsen et al. 2010).

In the last years several approaches have been developed to identify those sRNAs that are interacting with Hfq genome-wide. NGS of RNA enriched by co-immunoprecipitation with chromosomally encoded, epitope-tagged Hfq proteins has been used successfully for several bacteria [START_REF] Sharma | Experimental approaches for the discovery and characterization of regulatory small RNA[END_REF]. The first time it was applied to study sRNAs and mRNAs bound to Hfq of S. enterica serovar Typhimurium it allowed differentiation between transcriptional and post-transcriptional effects of Hfq and definition of the exact sequence of these RNA molecules (Sittka et al. 2008). This approach confirmed the previously described Hfq-associated RNAs identified by other techniques but revealed also that a fifth of all Salmonella genes are governed by the action of Hfq, including: the mRNAs of hilD, the master regulator of Salmonella invasion genes; flhDC, the flagellar master regulator; and two sigma factor regulons (Sittka et al. 2008). Recently this study was expanded by deep sequencing of Hfq-bound transcripts from multiple stages of growth of S. enterica serovar Typhimurium (Chao et al. 2012). This revealed a plethora of new small RNA species from within mRNA loci, and showed that the synthesis of the sRNA DapZ is controlled by HilD. Additionally, this study discovered that an mRNA locus might have a double functional output by producing both a

Mycobacterium tubercolosis

Mcr7

Trans-acting RNA Regulation of the TAT secretion system [START_REF] Solans | The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis[END_REF] protein and an Hfq-dependent trans-acting RNA. Furthermore, the genome-wide map established for Hfq targets suggested that the 3 ′ regions of mRNA genes constitute a reservoir of regulatory small RNAs (Chao et al. 2012). Despite the fact that not all bacteria encode an Hfq protein, the high percentage of sRNAs regulated by this RNA-binding protein in many bacteria and several bacterial pathogens led to the explosion of studies aiming not only at identifying Hfq-dependent sRNAs and their targets but also at characterizing their roles to understand the diverse and often speciesspecific cellular functions and their implications in pathogenesis. However, not all trans-encoded sRNAs need Hfq. As examples of the versatility of trans-encoded sRNAs, Hfq-independent sRNAs and their role in the iron level and life cycle regulation in different intracellular pathogens will be discussed in greater detail in the following paragraphs.

TRANS-ENCODED BACTERIAL sRNAs IMPLICATED IN THE INTRACELLULAR LIFE

Trans-encoded sRNAs respond to iron levels in the host cell

The impact of sRNAs on many aspects of bacterial physiology is known. Here we will concentrate on one important aspect, the regulation of iron levels in the cell. Iron participates in many biological functions and it is essential for bacterial growth but it is toxic at higher concentrations. Thus, iron homeostasis is tightly regulated. A bioinformatics screen identified the small RNA NrrF [for neisserial regulatory RNA responsive to iron (Fe)] in Neisseria meningitidis, which was demonstrated to be both responsive to iron in the environment as encountered in host tissues of fluids and Fur regulated. NrrF has a well-conserved orthologue in Neisseria gonorrhoeae [START_REF] Mellin | A novel fur-and ironregulated small RNA, NrrF, is required for indirect fur-mediated regulation of the sdhA and sdhC genes in Neisseria meningitidis[END_REF]) (Table 1). In order to monitor the level of iron within the cells, bacteria engage a tight iron-responsive regulation, mediated by the ferric uptake repressor Fur, an iron-dependent transcriptional repressor. In N. meningitidis Fur can indirectly activate gene expression by repressing the regulatory sRNA NrrF. NrrF is able to bind to limited complementarity regions of the sdh transcripts and downregulate the expression of the sdhA and sdhC genes, encoding subunits of the succinate dehydrogenase complex. Thus, the sRNA NrrF regulates Fur-dependent, iron-activated gene transcription in N. meningitidis. Interestingly, the Fe-regulated sRNA RyhB, first characterized in E. coli [START_REF] Masse | A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[END_REF][START_REF] Masse | Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli[END_REF][START_REF] Masse | Effect of RyhB small RNA on global iron use in Escherichia coli[END_REF], needs the binding of Hfq to exploit its function. In contrast, the NrrF-mediated iron regulation of the sdhA and sdhC genes in Neisseria does not require the chaperone Hfq [START_REF] Mellin | Role of Hfq in iron-dependent and -independent gene regulation in Neisseria meningitidis[END_REF]. Two RybB homologues, named RfrA and RfrB, predicted in Salmonella enterica serovar Typhi are important for optimal intracellular replication in macrophages (Leclerc, Dozois and Daigle 2013) (Table 1). Furthermore, Fur, a repressor of both of these sRNAs, was shown to play a role in phagocytosis and intracellular survival of S. Typhi in human macrophages. Interestingly, loss of either RfrA or RfrB resulted in distinct phenotypes, with respect to siderophore production, salmochelin production, or fur expression in low iron conditions, suggesting a non-redundant role for these regulatory RNAs [START_REF] Leclerc | Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells[END_REF]. Similarly, deletion of either rfrA or rfrB did not affect bacterial uptake, survival or replication; however, deletion of both sRNAs resulted in a significantly reduced intracellular replication ability, suggesting a complementary role for these two sRNAs for intracellular replication [START_REF] Leclerc | Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells[END_REF]. Indeed, RfrA and RfrB were induced inside THP-1 macrophages (Leclerc, Dozois and Daigle 2013), inside murine macrophages [START_REF] Padalon-Brauch | Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence[END_REF]) and inside HFQ AND SMALL REGULATORY RNAs ! ! Oliva et al. 337 fibroblasts [START_REF] Ortega | Dynamics of Salmonella small RNA expression in non-growing bacteria located inside eukaryotic cells[END_REF]. In addition to their central role in iron homeostasis regulation, these two sRNA molecules and their homologues have additional functions such as protecting against oxidative stress, bactericidal antibiotics and acid resistance [START_REF] Kim | Identification of target transcripts regulated by small RNA RyhB homologs in Salmonella: RyhB-2 regulates motility phenotype[END_REF]. Indeed, it was shown that the homologous sRNAs in S. enterica, named RyhB-1 and RyhB-2, are controlled by OxyR and are important for the oxidative stress response of this pathogen [START_REF] Calderon | Role of Salmonella Typhimurium small RNAs RyhB-1 and RyhB-2 in the oxidative stress response[END_REF]) (Table 1). This suggests that multiple bacterial pathogens that share these conserved sRNAs may employ a similar mechanism to regulate iron and/or oxidative stress within the host cells.

Trans-encoded sRNAs regulate intracellular differentiation of Chlamydia trachomatis

Another example of regulation of important functions of intracellular life by trans-encoded sRNAs is found in the obligate intracellular bacterium Chlamydia trachomatis. This pathogen employs the sRNA IhtA (inhibitor of hctA translation) to regulate its differentiation processes (Grieshaber et al. 2006) (Table 1). IhtA is a trans-encoded RNA, which is implicated in controlling the differentiation from the replicative reticulate bodies (RBs) to the infectious elementary bodies (EBs). IhtA is highly expressed in the replicative phase where it represses the translation of the hctA gene encoding the histone-like protein Hc1. Hc1 binds to and compacts the bacterial chromosome, making the EBs transcriptionally and translationally inactive. In contrast, as soon as IhtA transcription decreases during the late infectious phase, the hctA transcript is highly expressed, allowing the pathogen to transit and differentiate to EBs, where it endures in a metabolically inert stage (Grieshaber et al. 2006;[START_REF] Tattersall | Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia[END_REF][START_REF] Ortega | Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells[END_REF]. Thus, the sRNA IhtA is part of a global regulatory circuit that controls differentiation of RBs to EBs during the chlamydial life cycle. Recently it was shown that regulation of HctA by IhtA is a conserved mechanism found across pathogenic chlamydial species [START_REF] Tattersall | Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia[END_REF].

Trans-encoded sRNAs are implicated in pathogen entry into and replication within host cells

The advances in RNA sequencing and the possibility to undertake these analyses in infection-relevant conditions led to the discovery of several sRNAs implicated in virulence in cellular and animal infection models. An excellent example is the Rli27 sRNA that was identified by whole-genome tiling array analyses as a sRNA induced in L. monocytogenes in vivo [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]) (Table 1). Further functional analysis showed that Rli27 mediates the regulation of the cell wall-associated protein, Lmo0514 during the intracellular infection cycle [START_REF] Quereda | The Listeria Small RNA Rli27 regulates a cell wall protein inside eukaryotic cells by targeting a long 5 ′ -UTR variant[END_REF]. Interestingly, in response to environmental cues sensed in the eukaryotic intracellular niche, distinct promoters located upstream of the lmo0514 gene generate two alternative lmo0514 transcript isoforms containing 5 ′ UTRs of different length. These two transcript isoforms, which display two distinct target sites within the 5 ′ UTR for sRNAs, are differentially expressed in intracellular and extracellular bacteria. During the intracellular infection cycle, the long 5 ′ UTR lmo0514 transcript isoform, bearing the Rli27-binding site, is targeted by the Rli27 sRNA, leading to the release of the occluded SD sequence and allows therewith Lmo0514 protein production. Thus, L. monocytogenes promotes Rli27-mediated post-transcriptional regulation of its specific target, a cell wall-associated protein Lmo0154, only in response to its entry into eukaryotic cells [START_REF] Quereda | The Listeria Small RNA Rli27 regulates a cell wall protein inside eukaryotic cells by targeting a long 5 ′ -UTR variant[END_REF].

Another sRNA-dependent temporal and tissue-specific regulation of virulence has been reported in the S. enterica serovar Typhimurium pathogenicity islands (SPIs) [START_REF] Gong | A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors[END_REF]. This sRNA, called IsrM, is expressed in vitro under conditions comparable with those during infection in the gastrointestinal tract (Table 1). Moreover, in vivo assays revealed a higher expression of IsrM in the ileum, suggesting an involvement of this sRNA in Salmonella pathogenicity. Indeed, IsrM targets the SPI-effector SopA and the global regulator of the SPI-1 genes, HilE. HilE is the major regulator of Salmonella virulence crucial for bacterial evasion. Specifically IsrM targets the 5 ′ UTRs and masks the nearby SD sequence of both hilE and sopA mRNAs, hindering the production of the two proteins. Thus, Salmonella probably uses the fine-tuned expression of IsrM as a mechanism to regulate specifically SPI-1 protein expression in vivo temporally and spatially.

REGULATORY 5 ′ UTR ELEMENTS

Two diverse classes of 5 ′ UTRs of mRNAs have gained recent attention: RNA thermometers and RNA riboswitches. To some extent, these two types of regulatory molecules share common features but, most importantly, both are complex RNA molecules that sense a particular chemical or physical signal and accordingly alter their conformation to control the expression of downstream genes. 5 ′ UTR elements are another striking example of a refined RNA-dependent mechanism, allowing bacteria to respond rapidly and cost-effectively to physical alterations. Riboswitches are regulating the use of different nutrient sources that seem to be important for growth of bacterial pathogens inside host cells. Thermosensors may allow a pathogen to recognize the host environment by sensing the temperature shift encountered when extracellular bacteria invade host cells to replicate intracellularly. An example is L. monocytogenes (discussed below) that regulates virulence gene expression indispensable for intracellular growth in response to temperature by a thermosensor.

RNA thermometers

RNA thermometers control gene expression by temperatureinduced conformational changes. All cellular processes are temperature dependent, but virulence genes, cold shock genes and heat shock genes are particularly disposed to thermoregulation. Specifically, the response to an increase in temperature is often used by pathogenic bacteria when they enter their mammalian host. The temperature of 37 • C causes induction of virulence genes whose products are only needed in the host environment. The advantage of regulation via RNA thermometers lies in the fact that they respond to temperature changes in a more immediate manner because they control the translation of already existing or nascent mRNAs [START_REF] Kortmann | Bacterial RNA thermometers: molecular zippers and switches[END_REF]. Despite the fact that cis-acting regulatory 5 ′ UTR elements modulate gene expression either at the transcriptional or translational level [START_REF] Mandal | Gene regulation by riboswitches[END_REF], all RNA thermometers described so far act at the level of translational initiation. In most of the cases, the SD region and/or the initiation codon are involved in base-pairing interactions forming a secondary structure at low temperature. An increase in temperature facilitates the mRNA-ribosome interaction after the full liberation of the SD sequence and the AUG start codon, leading to the formation of the translation initiation complex (Narberhaus, [START_REF] Narberhaus | RNA thermometers[END_REF]. The first RNA thermometer described controls the expression of the cIII protein and also governs the development of phage λ. Strikingly, it by guest on August 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from CHAPTER TWO ! 338 FEMS Microbiology Reviews, 2015, Vol. 39, No. 3 represents the only thermometer which turns on the translation of the cIII protein when the temperature is decreased and, furthermore, it does not operate by gradual melting but switches between two exclusive conformations [START_REF] Altuvia | Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation[END_REF]). However, RNA thermometers have little or no sequence conservation and are thus difficult to predict from genome sequences. Therefore, the bioinformatics prediction has remained a major challenge [START_REF] Waldminghaus | Genome-wide bioinformatic prediction and experimental evaluation of potential RNA thermometers[END_REF].

The mechanisms of function of the different RNA thermometers known to date have recently been reviewed in great detail [START_REF] Kortmann | Bacterial RNA thermometers: molecular zippers and switches[END_REF], and thus here we will give a brief overview of those that are implicated in regulatory processes in intracellular bacteria.

A specific family of RNA thermometers is characterized by the presence of a short sequence motif composed of four uridines, also known as fourU. The fourU element is located at the 5 ′ UTRs of several virulence and heat shock genes and pairs particularly with AGGA in the SD sequence. FourU elements have been identified and characterized in several pathogenic bacteria such as the lcrF thermometer in Yersinia pestis, the agsA fourU thermometer sensor of Salmonella Typhimurium and the prfA sensor of the intracellular pathogen L. monocytogenes (Waldminghaus et al. 2007;[START_REF] Johansson | An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes[END_REF][START_REF] Bohme | Concerted actions of a thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence[END_REF]. PrfA of L. monocytogenes, the master regulator of virulence gene expression, is expressed at 37 • C, the temperature of mammalian host cells, but not at lower temperatures that L. monocytogenes encounters mainly when growing extracellularly in the environment. Indeed the 5 ′ UTR of the prfA mRNA adopts a stable stem-loop structure at low temperature, thereby blocking the SD sequence and preventing binding of the ribosome (Table 1). At 37 • C, the temperature encountered when infecting humans, the stem-loop melts into an alternative secondary structure, allowing the ribosome to access the SD sequence, leading to the translation of the prfA mRNA and to the subsequent induction of a number of virulence genes [START_REF] Johansson | An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes[END_REF][START_REF] Johansson | RNA-mediated control of virulence gene expression in bacterial pathogens[END_REF]. Another fourU RNA thermometer was identified in the 5 ′ UTR of the small heat shock agsA gene in S. enterica serovar Typhimurium. The 5 ′ UTR of the agsA gene, which codes for a small heat shock protein, consists of only two stem-loop structures, one of which contains the fourU motifs with imperfect complementarity to the SD sequence. As all RNA thermometers, the stability of this structure was calculated to decrease with increasing temperatures, thereby leading to a high level of agsA expression and subsequent availability of the small heat shock protein [START_REF] Waldminghaus | Genome-wide bioinformatic prediction and experimental evaluation of potential RNA thermometers[END_REF].

RNA riboswitches regulating metabolic function during intracellular growth

Riboswitches are widespread cis-acting RNA elements in mR-NAs that sense metabolites and modulate transcription or translation of mRNAs in response to these metabolites by forming alternative metabolite-free and metabolite-bound conformations [START_REF] Serganov | A decade of riboswitches[END_REF][START_REF] Peselis | Themes and variations in riboswitch structure and function[END_REF]. In prokaryotes, most of the riboswitches are at the 5 ′ UTR of the main coding region of a particular mRNA and typically consist of two distinct functional domains. The evolutionarily conserved aptamer element serves as the ligand-binding domain (sensor) whereas a variable sequence, termed the expression platform, is responsible for exerting an influence on the downstream coding sequences, regulating their expression [START_REF] Serganov | A decade of riboswitches[END_REF]. Riboswitches can control a broad range of genes including those involved in the biosynthesis and transport of prokaryotic metabolites such as amino acids, nucleotides, metal ions and cofactors [START_REF] Henkin | Riboswitch RNAs: using RNA to sense cellular metabolism[END_REF]. Therefore, to some extent the ligand represents not only the stimulus but also the final product of the biosynthetic pathway regulated by the downstream genes of the riboswitch, leading to a feedback control.

Riboswitches have been extensively studied in the last decade and many classical ones have been identified in prokaryotes. However, recently, the first ribsowitch controlling the transcription of non-coding RNAs was described in the intracellular pathogen L. monocytogenes [START_REF] Mellin | A riboswitch-regulated antisense RNA in Listeria monocytogenes[END_REF][START_REF] Mellin | Sequestration of a two-component response regulator by a riboswitchregulated noncoding RNA[END_REF]. In these studies two vitamin B12-binding (B12) riboswitch-dependent mechanisms were identified that control the catabolism of propanediol and ethanolamine molecules [START_REF] Mellin | A riboswitch-regulated antisense RNA in Listeria monocytogenes[END_REF][START_REF] Mellin | Sequestration of a two-component response regulator by a riboswitchregulated noncoding RNA[END_REF]. They showed that the B12 riboswitch was transcribed as part of a non-coding antisense RNA, named AspoC, and surprisingly was located downstream and in the antisense orientation of the adjacent gene, pocR (Table 1). PocR is a transcriptional factor that is activated by propanediol, a molecule produced in the intestine by commensal bacteria as a byproduct of the fermentation of rhamnose and fucose. Propanediol catabolism requires a B12-dependent diol dehydratase encoded by the pduCDE genes whose expression is induced by the transcriptional regulator PocR [START_REF] Bobik | Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase[END_REF]). In the absence of B12, the riboswitch induces the formation of an aspocR long transcript, which in turn inhibits in trans pocR expression. Furthermore it was suggested that this regulation relies on binding of AspocR to pocR mRNA. As such, the pdu genes are not expressed when vitamin B12 is not available. In contrast, low B12 conditions partially repress pocR expression and appear sufficient to induce the expression of the B12 biosynthesis genes; however, to induce the pdu gene expression maximally, accumulation of vitamin B12 is required. In response to sufficient accumulation of B12 and upon the ligandaptamer interaction, the riboswitch leads to the production of a short aspocR truncated transcript that does not prevent the expression of the pocR mRNA, whose product enables propanediol catabolism. Thus, the B12 riboswitch does not operate in a blackor-white switch but acts more as a fine-tuning regulatory device. Markedly, regulation by the B12 riboswitch-dependent AspocR allows the propanediol catabolism pathway to be highly activated only when both propanediol and B12 are available [START_REF] Mellin | A riboswitch-regulated antisense RNA in Listeria monocytogenes[END_REF].

Furthermore, in L. monocytogenes, vitamin B12 is also required for the utilization of ethanolamine [START_REF] Mellin | Sequestration of a two-component response regulator by a riboswitchregulated noncoding RNA[END_REF]. Ethanolamine, like the closely related propanediol molecule, is an important nutrient source for pathogens during infection [START_REF] Buchrieser | Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity[END_REF]. Recent studies suggested that propanediol and ethanolamine both play a role in L. monocytogenes pathogenesis as L. monocytogenes up-regulates the pdu and ethanolamine utilization (eut) genes during intracellular growth in intestinal epithelial cells [START_REF] Joseph | Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening[END_REF], and in the intestine of germ-free mice pre-treated with lactobacilli [START_REF] Archambaud | Impact of lactobacilli on orally acquired listeriosis[END_REF]. Related to these findings, an in-depth whole-genome transcriptome analysis revealed the presence of a B12 riboswitch upstream of the first gene of the eut locus, coding for the ethanolamine utilization pathway [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]. Indeed, according to the availability of the B12 cofactor, the riboswitch modulates eut gene expression through the production of a long or a short truncated sRNA transcript, called Rli55 (Table 1). Furthermore, four ANTAR (AmiR and NasR transcriptional antiterminator regulator) elements were found upstream of the eutA and eutV genes, and two in the Rli55 locus, which are transcribed as part of the 3 ′ end of the Rli55 transcript. Very elegant experiments showed that in the presence of ethanolamine by guest on August 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from HFQ AND SMALL REGULATORY RNAs ! ! Oliva et al. 339 but absence of the B12 cofactor, a long Rli55 together with the first ANTAR element is transcribed to sequester the two-component response regulator EutV, which consecutively leads to the down-regulation of the eut genes. Conversely, the accumulation of B12 and the successive interaction with the riboswitch triggers the production of a truncated Rli55 transcript lacking the first ANTAR element, which prevents binding to the EutV regulator. As such, the EutV protein co-ordinates the activation of the eut genes, leading to the utilization of ethanolamine [START_REF] Mellin | Sequestration of a two-component response regulator by a riboswitchregulated noncoding RNA[END_REF]. Thus, these studies highlighted a new, non-classical role for regulatory sRNAs, which is sequestering of a two-component system element dependent on a B12 riboswitch.

Recently trans-regulatory riboswitch elements that can function as sRNAs and link virulence and nutrient availability have been discovered in L. monocytogenes [START_REF] Loh | A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes[END_REF]. Listeria monocytogenes harbours seven putative S-adenosylmethionine (SAM) riboswitches that may, when SAM binds, form a termination structure that terminates transcription and inhibits synthesis of the downstream mRNA [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]). In L. monocytogenes, two riboswitches, named SreA and SreB, have been shown to function in trans and to act as sRNAs controlling expression of PrfA, the major regulator of virulence gene expression (Table 1). Elegant in vitro and in vivo experiments suggest a noncoding RNA function for SreA and SreB. They bind to the 5 ′ UTR of prfA, and thereby reduce prfA transcript stability and/or prfA mRNA translation. Thus, both SreA and SreB act together to control expression of prfA and thus the virulence of this intracellular pathogen. The identification of many diverse riboswitches, facilitated by the rapid expansion of computational data analysis together with experimental analyses will reveal new mechanisms employed by bacteria to regulate their transcriptional response when growing inside host cells.

CIS-ENCODED SMALL RNAS

In contrast to the trans-encoded sRNAs, the cis-encoded sRNAs are located in the antisense strand of their target RNA regions and their mechanism of action leads to the post-transcriptional down-regulation of the target gene(s) through a high degree of sequence complementarity. In general, this class of sRNAs does not require the help of the RNA-binding protein Hfq (Caldelari et al. 2013). However, some cis-encoded sRNAs may also stabilize their mRNA target, such as, for example, the AsdA sRNA discussed below. Although the first described cis-antisense RNAs were mobile elements such as phages, transposons and plasmids [START_REF] Wagner | Antisense RNA control in bacteria, phages, and plasmids[END_REF], few chromosomally encoded cis-antisense transcripts had been reported by 2007 [START_REF] Brantl | Regulatory mechanisms employed by cis-encoded antisense RNAs[END_REF]. With the use of tiling arrays and RNAseq to analyse the transcriptional landscape of a bacterial genome widely and at a high level of depth, antisense transcription that was thought to be simply an irregularity turned into a rule. Antisense sRNAs (asRNAs) are present in Gram-positive and Gram-negative bacterial species, but a high variability in their prevalence and genomic density was reported [START_REF] Georg | cis-antisense RNA, another level of gene regulation in bacteria[END_REF][START_REF] Raghavan | Antisense transcription is pervasive but rarely conserved in enteric bacteria[END_REF][START_REF] Lybecker | The double-stranded transcriptome of Escherichia coli[END_REF]. Thus new attention to asRNAs within the bacterial kingdom emerged. As-RNAs can be classified according to their position as internally located, 5 ′ -overlapping or 3 ′ -overlapping asRNAs, or according to their size as short asRNAs (ranging from 100 to 300 nucleotides) or as long asRNAs reaching several kilobases in size [START_REF] Georg | cis-antisense RNA, another level of gene regulation in bacteria[END_REF]. However, the lack of functional characterization of many of the identified asRNAs hinders their annotation according to their role in cells. To date, asRNAs have been reported to act at the level of transcription, mRNA stability or translation. Their diverse regulatory mechanisms, asRNA regulation has been reviewed recently in several articles [START_REF] Thomason | Bacterial antisense RNAs: how many are there, and what are they doing?[END_REF][START_REF] Georg | cis-antisense RNA, another level of gene regulation in bacteria[END_REF]. We will thus concentrate here on a particular asRNA mechanism newly described in Listeria, the excludon, and asRNAs important for Salmonella replication.

A new concept in antisense RNA-mediated regulation: the excludon

Recently a sophisticated asRNA-dependent mechanism that achieves a regulatory connection between neighbouring genes that often have opposite functions was reported in L. monocytogenes. The name 'excludon' has been coined to describe this new regulatory mechanism. A single transcript serves in parallel as an antisense repressor of one gene or group of genes and as an mRNA promoting the expression of the adjacent genes. This results in the regulation of adjacent genes of opposite functions [START_REF] Sesto | The excludon: a new concept in bacterial antisense RNA-mediated gene regulation[END_REF].

The 'excludon' in L. monocytogenes takes part in the control of flagellum biosynthesis. The asRNA, Anti0677 has a proximal region that is antisense to the lmo0675-lmo0676-lmo0677 locus and a distal region that is part of the gene encoding the regulator MogR (Toledo-Arana et al. 2009) (Table 1). The lmo0676-lmo0677 locus encodes FliP and FliQ that together with FliR (encoded by the adjacent gene lmo0678) constitute the flagellum export apparatus. The MogR regulator binds the fli operon promoter and down-regulates the flagellum and thus motility in L. monocytogenes. When Anti0677 is expressed, it inhibits the synthesis of lmo0675 (its function is not known yet) and the entire flagellum export apparatus and leads simultaneously to the expression of MogR. Thus the flagellum apparatus is concurrently turned off by the increasing expression of MogR and by the repression of the lmo0676-lmo0677 locus driven by the asRNA Anti0677. Furthermore, the inhibition of the flagellum export apparatus is likely to involve the formation of a double-stranded RNA when Anti0677 is transcribed and the subsequent cleavage by RNase III (Wurtzel et al. 2012). The above-described asRNA Anti0677 was the first example for the excludon concept; however, at least 13 additional, putatively excludon-like regulated genomic loci were detected through comparative transcriptomic analysis of L. monocytogenes, suggesting that excludon-mediated regulation might represent a frequent mechanism. Furthermore, similar organizations have been found in other bacteria such as L. pneumophila (unpublished data), thus this mechanism of antisense regulation is probably not restricted to the genus Listeria but extends to other bacteria.

Cis-encoded sRNAs: regulation of replication

Although transcription of asRNAs occurs genome-wide in many bacteria, only a small fraction of asRNAs is shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. Thus researchers are questioning whether these asRNAs are all functional or not [START_REF] Raghavan | Antisense transcription is pervasive but rarely conserved in enteric bacteria[END_REF]. Further functional analyses and in-depth characterization of these asRNAs will be necessary to answer this open question. For example, one of the cis-encoded RNAs, termed AsdA, was characterized in S. enterica serovar Typhi where it was shown to be involved in regulating the replication process (Table 1 gene that encodes a site-specific DNA-binding protein [START_REF] Dadzie | Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi[END_REF]. DnaA serves as a bacterial replication initiation factor and is capable of recognizing the origin of replication (oriC), mediating the open complex formation, and therefore it induces the assembly of the DNA replication machinery. Moreover DnaA acts as a transcriptional factor, promoting the transcription of target genes [START_REF] Messer | DnaA as a transcription regulator[END_REF]. Non-coding RNA elements are often transcribed in response to specific conditions. Indeed, the AsdA sRNA is highly expressed during the stationary growth phase after growth in rich medium but it is already expressed in very early exponential growth when iron limitation or osmotic stresses are exerted. In these conditions, the expression of the AsdA antisense transcript enhances the stability of the dnaA mRNA, which in turn increases its translational rate [START_REF] Dadzie | Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi[END_REF]. These observations indicated that the AsdA sRNA, through stabilization of the dnaA mRNA, is a crucial regulator of DNA replication in S. enterica serovar Typhi.

Cis-encoded sRNAs: regulation of virulence gene expression

Interestingly, a long asRNA of 1.2 kb, named AmgR, was identified in the antisense strand of the mgtCBR operon and complementary to the 5 ′ terminus of this polycistronic mRNA in S. enterica serovar Typhimurium (Table 1). AmgR is transcribed from a promoter that is located in the intergenic region between the mgtC and mgtB genes. The aforementioned operon encodes the MgtC protein implicated in Mg 2+ homeostasis and virulence, the Mg 2+ transporter MgtB and the 30 amino acid peptide MgtR that mediates degradation of MgtC by the FtsH protease. Intriguingly, the transcription of the polycistronic mgtCBR mRNA and the AmgR sRNA is under the positive control of the two-component regulatory system PhoP/PhoQ. In detail, when PhoQ senses low extracytoplasmatic Mg 2+ , it phosphorylates PhoP, which then binds to the mgtC and amgR promoters, mediating the transcription of the mgtCBR mRNA and the long sRNA.

As such, the long RNA regulatory element limits the MgtC and MgtB protein levels, promoting the MgtR binding to the MgtC protein and the subsequent degradation by the FtsH protease. Markedly, AmgR serves as a timing device to attenuate virulence in mice mediated by the sense-encoded MgtC protein [START_REF] Lee | An antisense RNA that governs the expression kinetics of a multifunctional virulence gene[END_REF]. The finding that the AmgR asRNA is located inside a polycistronic region revealed that the repertoire of regulatory sRNA elements might be larger than thought and raises the possibility that additional asRNAs exist which have been overlooked.

Another example of an asRNA, named lesR-1, encoded within the pSLT virulence plasmid has been reported recently in the bacterial pathogen S. enterica serovar Typhimurium (Gonzalo-Asensio et al. 2013) (Table 1). RNA expression has been studied using oligonucleotide microarrays containing probes in each of the two strands of every intergenic region testing a variety of culture conditions. Remarkably, lesR-1 was preferentially expressed in non-growing dormant bacteria during colonization of fibroblasts. The deletion of this asRNA not only influenced the control of bacterial growth within the fibroblast cell lines but also impaired virulence in a mouse infection model. As an as-RNA that is overlapping the PSLT047 transcript, the lesR-1 regulatory mechanism involves the direct interaction of the 3 ′ end of the RNA molecule leading to the subsequent remodelling of the PSLT047 protein levels. Given the virulence phenotype, this suggests that PSLT047 is important for the intracellular lifestyle of S. enterica serovar Typhimurium [START_REF] Ortega | A novel antisense RNA from the Salmonella virulence plasmid pSLT expressed by non-growing bacteria inside eukaryotic cells[END_REF].

DYNAMIC EXPRESSION CHANGES OF sRNAs DURING INTRA-AND EXTRACELLULAR GROWTH

The use of whole-genome tiling arrays and RNA deep sequencing has revealed that sRNAs, similar to protein-coding genes, show dynamically changing expression profiles during growth in laboratory media but also during infection. An example is L. pneumophila as this intracellular pathogen is known to have a pronounced biphasic life cycle. Schematically, pathogenic L. pneumophila encode one set of genes dedicated to transmission that includes all major virulence traits and another set that promotes replication in phagocyte vacuoles, a characteristic that is reflected in a major shift of gene expression during the switch from replicative to transmissive bacteria (Molofsky and Swanson 2004;[START_REF] Bruggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF]. Recently, RNAseq analyses showed that ∼700 sRNAs are expressed in the L. pneumophila genome. Similar to the protein-coding genes, >60% of the sRNAs are growth phase dependently regulated (Sahr et al. 2012). Most interestingly, transcriptional start site mapping identified in addition a high number of sRNAs with tandem promoters, 30% of which are used growth phase dependently (Sahr et al. 2012). These strong changes in sRNA expression profiles indicate an important role for sRNAs in regulating the biphasic life cycle of L. pneumophila and thus virulence.

Similarly, transcriptome data using a 70mer oligonucleotide array of the S. enterica Typhimurium genome revealed that ∼2% (98 genes) of all genes are differentially expressed in nongrowing intracellular bacteria, when the fibroblast infection model was used [START_REF] Nunez-Hernandez | Genome expression analysis of nonproliferating intracellular Salmonella enterica serovar Typhimurium unravels an acid pH-dependent PhoP-PhoQ response essential for dormancy[END_REF]. Later work also identified sRNAs that are differentially expressed. As an example, SraL shows higher expression levels in non-growing bacteria than in actively growing ones [START_REF] Ortega | Dynamics of Salmonella small RNA expression in non-growing bacteria located inside eukaryotic cells[END_REF].

A comprehensive picture of growth phase-and condition-dependent expression of sRNAs was reported for L. monocytogenes. The analyses of L. monocytogenes expression profiles using tiling arrays and bacteria grown in exponential phase, stationary phase, hypoxia and low temperature or isolated from the intestine of axenic mice or bacteria grown in the blood of human donors provided important information about their expression conditions and thus hints of their putative functions [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]. Similarly, RNA sequencing of L. monocytogenes grown in macrophages showed that 29 of the 150 described sRNAs are specifically induced during intracellular growth, pointing to functional roles during infection. Indeed, knockout mutants of three of these RNAs, named rli31, rli33-1 and rli50, were defective in intracellular growth [START_REF] Mraheil | The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages[END_REF]. Recently the non-coding genome was characterized in multiple growth conditions that are relevant for the infection process using RNA deep sequencing. This led not only to the characterization of condition-dependent expression of sRNAs but also to the identification of new regulatory mechanisms such as the excludon described above (Wurtzel et al. 2012).

Using a combined experimental and in silico approach, 34 sRNAs have been identified in Mycobacterium bovis BCG, many of which are responsive to changing environmental growth conditions. Homologues of several of these sRNAs are also present in Mycobacterium tuberculosis and Mycobacterium smegmatis [START_REF] Dichiara | Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis[END_REF]. Recently one of these, the sRNA homologous to Mcr7, has been functionally characterized in M. tuberculosis. To analyse the regulon of PhoPR, a two-component system essential for virulence of M. tuberculosis, a ChIPseq analyses using PhoP antibodies was undertaken. This allowed identification by guest on August 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from HFQ AND SMALL REGULATORY RNAs ! ! Oliva et al. 341 of all directly PhoP-regulated genes, but also a PhoP-regulated sRNA named Mcr7. In elegant experiments, it was shown that Mcr7 modulates the TAT secretion system of M. tuberculosis and impacts its activity. This leads to a diminished secretion of the immunodominant AG85 complex, implicated in successful infection of the host [START_REF] Solans | The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis[END_REF]. Taken together, sRNAs show dynamic changes of expression according to the conditions in which they are functioning and many are crucial contributors to the virulence process of intracellular bacteria.

PROTEINS REGULATING SMALL RNAs

RNA-binding proteins are major players in the regulation of gene expression. Certain of these proteins involve an association with sRNAs. One of the best known is the 6S RNA that interacts with σ 70 -containing RNA polymerase and regulates transcription at specific promoters and that recently has also been shown to be involved in virulence regulation of intracellular bacteria. Another well-known system present in many bacteria is the CsrA/RsmA protein, whose activity is regulated by sRNAs.

The RNA polymerase-interacting 6S RNA

First identified in E. coli in the late 1960s, the 6S RNA is a double-stranded sRNA transcribed by the ssrS gene and ubiquitously expressed, with a remarkable accumulation during the late stationary phase of the bacterial growth cycle [START_REF] Hindley | Fractionation of 32P-labelled ribonucleic acids on polyacrylamide gels and their characterization by fingerprinting[END_REF][START_REF] Wassarman | 6S RNA regulates E. coli RNA polymerase activity[END_REF]. The conserved secondary structure consisting of two irregular stem structures and a single-stranded central bulge has led to the prediction of the presence of this sRNA in >100 bacterial species so far [START_REF] Barrick | 6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter[END_REF]. Although the characterization and the identification of physiological functions of 6S RNA remained elusive for three decades as no appreciable growth phenotype in cells depleted or overexpressing 6S RNA was observed, the conserved secondary structure suggested an important role for this sRNA. The first time, a detectable phenotype was observed when E. coli was grown under stringent stress and limiting nutrient conditions, suggesting that the bacteria, under these conditions, engage 6S RNA to allocate the nutrients [START_REF] Trotochaud | 6S RNA function enhances long-term cell survival[END_REF]). Strikingly, the 6S RNA is the first sRNA that was shown to alter gene expression by binding directly to the active site of the housekeeping holoenzyme form of the RNA polymerase, the σ 70 -RNA polymerase, to hinder the access to the promoter DNA. In E.coli it was shown that the 6S RNA contributes to the regulation of a large number of genes, by inhibiting their transcription during the late stationary phase [START_REF] Wassarman | 6S RNA regulates E. coli RNA polymerase activity[END_REF][START_REF] Willkomm | 6S RNA-an ancient regulator of bacterial RNA polymerase rediscovered[END_REF]. The specificity of the binding strictly resides in the conserved secondary structure and in the internal stem bulge region of the 6S RNA [START_REF] Trotochaud | 6S RNA function enhances long-term cell survival[END_REF]. Secondly, further investigations on the nature of the secondary structure of the 6S RNA revealed a remarkable resemblance to the conformation of DNA, suggesting a shared mechanism of binding. Additional structural requirements, related to the 6S-dependent inhibition, involve the presence of a promoter with an extended -10 element and/or a weak -35 element [START_REF] Cavanagh | Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis[END_REF]. Furthermore, Wassarman and Saecker showed that 6S RNA can be used as a template for RNA synthesis during the stationary phase, generating a 14-20 nucleotide long RNA product, named pRNA [START_REF] Wassarman | Synthesis-mediated release of a small RNA inhibitor of RNA polymerase[END_REF]. The synthesis of the pRNA seems to be required to induce the release of the 6S-RNA polymerase complex, which consequently would lead to the formation of the 6S RNA-pRNA duplex. Thus, during outgrowth, the inhibitory machin-ery is switched off when necessary and the 6S RNA is destabilized and alternatively degraded post-pRNA synthesis (Wassarman and Saecker 2006). Although a down-regulation of the pRNA depending on the NTP concentration is suggested, many questions concerning the potential cellular function of this pRNA still need to be answered. The function of the 6S RNA has been studied in the intracellular pathogens L. pneumophila, S. typhimurium and Coxiella burnetii [START_REF] Ortega | Dynamics of Salmonella small RNA expression in non-growing bacteria located inside eukaryotic cells[END_REF][START_REF] Ortega | Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells[END_REF][START_REF] Warrier | Identification of novel small RNAs and characterization of the 6S RNA of Coxiella burnetii[END_REF]. In L. pneumophila the exact role of the 6S RNA (ssrS) is not known but it has been shown that it is important for intracellular multiplication in human macrophages and in Acanthamoeba castellanii but not for growth in laboratory media (Table 1). In contrast to the above-discussed function of the 6S RNA as a repressor of the transcription of genes during stationary phase of growth through the binding to the σ 70containing RNA polymerase, the 6S RNA of L. pneumophila serves mainly as a positive regulator. Several genes including those involved in stress adaptation, amino acid metabolism and genes encoding Dot/Icm effectors, important for Legionella's ability to survive in the host, were identified [START_REF] Faucher | Legionella pneumophila 6S RNA optimizes intracellular multiplication[END_REF]. This different regulatory mechanism of the L. pneumophila 6S RNA could be due to an only weak binding to the RNA polymerase, adding another regulatory level to the current knowledge of the regulatory mechanism by which 6S RNAs function [START_REF] Faucher | Legionella pneumophila 6S RNA optimizes intracellular multiplication[END_REF]. Interestingly, a second 6S RNA copy, named 6S2 RNA, was reported to be present in L. pneumophila strain Philadelphia [START_REF] Weissenmayer | Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs[END_REF]. According to their results these two 6S RNAs are expressed at different stages during the biphasic life cycle of L. pneumophila [START_REF] Weissenmayer | Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs[END_REF]). However, this second copy does not seem to be present in L. pneumophila strain Paris (Cazalet et al. 2004), indicating strain-specific differences (Cazalet et al. 2004;[START_REF] Sahr | Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence[END_REF]. Similarly, the 6S RNA homologues of S. enterica serovar Typhimurium and Coxiella burnetii were described as important for intracellular growth especially during stress conditions (Ortega, Gonzalo-Asensio and Garcia-del Portillo 2012; [START_REF] Ortega | Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells[END_REF][START_REF] Warrier | Identification of novel small RNAs and characterization of the 6S RNA of Coxiella burnetii[END_REF]. In C. burnettii the 6S RNA also interacts with the σ 70 -containing RNA polymerase, but it can serve as both a positive and a negative regulator during stationary phase growth (Table 1).

In conclusion, the different homologues of 6S RNA have multiple regulatory functions in intracellular pathogens with respect to both the genes they regulate and the mode of action, as they may induce or repress gene expression. Further investigations are necessary to determine when it acts as a negative or positive regulator and how this different mechanism is achieved.

The Csr/Rsm network and its interacting sRNAs

One of the best described RNA-binding proteins is the CsrA/RsmA (Carbon storage regulator/Regulator of secondary metabolism) ribonucleoprotein complex which controls different metabolic pathways and the repression of a variety of stationary-phase genes, including virulence-linked traits in pathogenic bacteria. CsrA was identified first in E. coli and defined as a carbon storage regulator system as the mutant strain of the csrA gene exhibited increased levels of glycogen compared with the wild-type strain [START_REF] Romeo | Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties[END_REF]. CsrA is a global bacterial regulator, as many homologous highly conserved proteins have now been identified in numerous bacteria but not in eukaryotes or Achaea. CsrA is a homodimer, in which each subunit is composed of five β-strands, one α-helix and an unstructured C-terminal part [START_REF] Gutierrez | Solution structure of the carbon storage regulator protein CsrA from Escherichia coli[END_REF][START_REF] Rife | Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 A resolution reveals a new fold[END_REF]. The major RNA-binding site is predicted to be the GxxG motif between strands β3 and β4, but other structures such as the β4 strand, the region between β2 and β3 and several conserved surfaceexposed residues are also most probably involved in the interaction. This RNA-binding site allows CsrA to recognize specific A(N)GGA motifs in the RNA sequence, apparently with much higher affinity if these motifs are represented in the loop of a hairpin structure [START_REF] Duss | Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition[END_REF]. Typically, the SD sequence in the RNA leader region is rich in GA motifs; therefore, it is assumed that CsrA is predominantly regulating translation initiation by inhibiting the ribosome binding and furthermore favouring the degradation of the transcript (Seyll and Van Melderen 2013) (Fig. 4). However, there is also evidence that CsrA interactions influence the fate of the RNA not only by blocking translation but also in various other ways, such as stabilization of the RNA by protecting it from RNase degradation, affecting transcription elongation/termination or also as a positive regulator of translation by forming alternative RNA structures which even support ribosome binding [START_REF] Liu | The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability[END_REF][START_REF] Figueroa-Bossi | RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination[END_REF]. Interestingly, the regulation of the intracellular level of CsrA itself is also controlled predominantly at the posttranscriptional level, in particular by two or more sRNAs that efficiently bind and sequester the CsrA protein [START_REF] Romeo | Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB[END_REF][START_REF] Babitzke | CsrB sRNA family: sequestration of RNAbinding regulatory proteins[END_REF]. These specific sRNAs, named CsrB/C or RsmY/Z in different bacteria, are composed of several repetitive sequence elements containing numerous conserved A(N)GGA motifs, most of which are located in hairpin loops (Table 1). Hence, almost the entire sRNA becomes covered with protein, making them a highly efficient antagonist to the interaction between CsrA and its target RNAs. Thus, the transcript level of these specific, small, untranslated RNAs is the key determinant of CsrA activity in the cell and already their moderate increase in expression can dramatically influence the equilibrium between target RNA-or sRNA-bound CsrA (Fig. 4). In all Csr/Rsm-regulatory networks known to date, the transcription of the small RNAs sequestering CsrA is regulated by a two-component system (TCS) that is homologous to the BarA/UvrY system in E. coli, and at least partially also by the alternative stress sigma factor RpoS (Romeo, Vakulskas and Babitzke 2013). This cascade of TCS/sRNA/CsrA is involved in the regulation of numerous cellular functions including carbon metabolism, motility, biofilm formation and adaptation to environmental stress responses, and production of virulence traits in the pathogen [START_REF] Lucchetti-Miganeh | The posttranscriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts[END_REF][START_REF] Timmermans | Post-transcriptional global regulation by CsrA in bacteria[END_REF]Romeo, Vakulskas and Babitzke 2013) (Fig. 4). Even though the overall processes of CsrA regulation and its influence on regulation of the stationary phase and virulencerelated pathways are better and better understood, not many direct target transcripts of CsrA, except its two regulatory sRNAs, have been identified to date in pathogenic bacteria. Most of the studies concentrated so far on indirect effects due to the loss of CsrA expression or overexpression of CsrA but rarely on physical interaction between the protein and the RNA.

The Csr/Rsm system is implicated in regulation of growth and motility Among intracellular bacteria, the Csr-type regulatory system is well characterized in L. pneumophila and S. enterica serovar Typhimurium [START_REF] Altier | Regulation of Salmonella enterica serovar typhimurium invasion genes by csrA[END_REF]Fettes et al. 2001;Molofsky and Swanson 2003;[START_REF] Jonas | The RNA binding protein CsrA controls c-di-GMP metabolism by directly regulating the expression of GGDEF proteins[END_REF]Rasis and Segal 2009;Sahr et al. 2009;[START_REF] Jonas | Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium[END_REF]Edwards et al. 2011;[START_REF] Knudsen | The role of ClpP, RpoS and CsrA in growth and filament formation of Salmonella enterica serovar Typhimurium at low temperature[END_REF]Nevo et al. 2014). The Csr system is very versatile, as even though the regulatory impact observed for Legionella and Salmonella is similar to what is known for other pathogens such as Pseudomonas, Yersinia, Vibrio or E. coli, there are also striking differences. One commonly observed feature of the CsrA cascade is its strong effect on the cell morphology of the bacteria. Due to the loss of the csrA gene, the cells are converted during replication into smaller sized cells with coccoid shape instead of the typical rod-shaped structure of Salmonella and Legionella, while overexpression of CsrA resulted in highly elongated cells. Furthermore, mutation in csrA causes a drastic reduction in growth rate in particular at lower temperature [START_REF] Forsbach-Birk | Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii[END_REF][START_REF] Knudsen | The role of ClpP, RpoS and CsrA in growth and filament formation of Salmonella enterica serovar Typhimurium at low temperature[END_REF]. Also the transition from a sessile to a motile life form of S. enterica serovar Typhimurium is strongly affected by CsrA [START_REF] Jonas | Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium[END_REF]). In S. enterica serovar Typhimurium CsrA seems to act positively on swarming motility and it is required for accurate flagella expression by stabilizing the flhDC and the fliA mRNAs, which are the regulators of the flagella operon. This stabilizing effect leads to an increased production of flagellar proteins [START_REF] Jonas | Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium[END_REF]. In Legionella, CsrA also affects the flagella sigma factor FliA, but in contrast to S. enterica serovar Typhimurium, overexpression of CsrA in L. pneumophila resulted in lower fliA transcription and subsequently to reduced levels of FlaA, the major structural flagellar protein controlled by FliA [START_REF] Forsbach-Birk | Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii[END_REF][START_REF] Bruggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF]. Additionally, the knockout mutation of the TCS LetA/LetS, which is necessary for the transcription of the CsrA antagonists RsmY/RsmZ, completely abolishes motility in the post-exponential phase (Molofsky and Swanson 2003;Rasis and Segal 2009;Sahr et al. 2009). Taken together, although CsrA is apparently a common regulator for flagella expression in different bacteria, the regulatory function differs significantly between them, as it can be either positive or negative.

HFQ AND SMALL REGULATORY RNAs ! ! Oliva et al. 343 The Csr/Rsm system is implicated in regulation of virulence and stress response

The control of stress responses and expression of virulence traits by the Csr system is a common feature among pathogens [START_REF] Lucchetti-Miganeh | The posttranscriptional regulator CsrA plays a central role in the adaptation of bacterial pathogens to different stages of infection in animal hosts[END_REF]. The drastic changes in morphology and motility are closely related to the expression of genes which are important during stress response, such as heat/cold shock, acid resistance, osmotic stress and iron starvation. For example, in L. pneumophila, the inhibition of CsrA expression plays a major role during the transition from the replicative to transmissive phase, which is associated with adaptation to environmental stress and virulence formation (Molofsky and Swanson 2004;Sahr et al. 2009). This finding was also supported by the recent identification of 26 effectors under the control of the LetA-RsmYZ-CsrA cascade [START_REF] Nevo | Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking[END_REF]). These effectors were identified by comparing the expression levels of several effector-encoding genes in the wild type and a letA knockout mutant strain, and selecting those that showed a strong reduction of the expression level in the mutant strain.

Then the presence of a putative CsrA-binding site was searched for by using the consensus sequence A(N)GGA. To confirm the regulation by CsrA, mutation of these putative CsrA-binding sites was performed. Indeed, during the exponential phase elevated expression levels of 22 of the 26 tested effector genes were observed. In contrast, in the wild-type strain, these effectors were more highly expressed in the stationary phase, suggesting that they might participate in the early steps of a new infection cycle most probably during the establishment of the Legionella-containing vacuole (LCV). Indeed, at least 19 of these 26 L. pneumophila effectors under the control of CsrA are involved in the modulation of endoplasmic reticulum (ER)-Golgi vesicular trafficking in S. cerevisiae (Nevo et al. 2014). The effectors RalF, YlfA and VipA had already been shown to cause growth defects in S. cerevisiae and to affect vesicular trafficking in the host cell directly (Shohdy et al. 2005;Heidtman et al. 2009). Nevu and colleagues proposed that these effectors are probably the first to translocate into the host cell when L. pneumophila begins a new infection cycle and they then participate in the establishment of the LCV, by modulating different components of the ER-Golgi trafficking pathway. The finding that secreted effectors important for forming the LCV are under the control of CsrA further highlights the important role of this RNA-binding protein in survival and replication of intracellular bacteria.

In Salmonella, virulence gene expression is also under the control of CsrA as it binds near to the SD sequence of the hilD mRNA. HilD is the master regulator of the expression of virulence genes located on the SPIs. This negative regulation of HilD translation results in an overall down-regulation of the two pathogenicity islands SPI-1 and SPI-2, both required for intestinal infection in animal models [START_REF] Martinez | Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons through HilD[END_REF]. This effect is abolished by the activation of the SirA/BarA two-component system, resulting in a high level of transcription of the sRNAs CsrB/CsrC that sequester the CsrA binding to the hilD untranslated region (Table 1).

Given that CsrA is a very important post-transcriptional regulator, several attempts have been made to develop bioinformatics approaches to identify the RNAs that CsrA is targeting. A sequence-based approach to predict genes directly regulated by CsrA, CSRA TARGET, was published recently [START_REF] Kulkarni | A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa[END_REF]. This computational algorithm predicted >100 mRNA targets in E. coli, S. enterica serovar Typhimurium, P. aeruginosa and L. pneumophila. In addition to new targets, already known and confirmed CsrA targets of these pathogens were identified, giving high confidence in its predictions. Furthermore, some predicted targets were tested and confirmed experimentally [START_REF] Kulkarni | A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa[END_REF]. The predicted CsrA targets indicated the important roles of CsrA in diverse processes such as stress response, quorum sensing and virulence factor regulation or metabolism. Also CsrA targets specific for intracellular pathogens, such as, for example, hilD, encoding the master regulator for the induction of invasion genes located on SPI-1 or sipA encoding a type III effector protein of S. enterica serovar Typhimurium necessary to induce a proinflammatory response in epithelial cells, were identified [START_REF] Kulkarni | A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa[END_REF].

Taken together, the Csr-type regulatory system is important for the expression of the virulence phenotype of intracellular bacteria by controlling a large variety of physiological and stress responses that are implicated directly or indirectly in host cell infection and the overall biological fitness during changing environmental conditions. However, although very similar cellular functions are regulated by this post-transcriptional control mechanism in many pathogens, the impact of the regulation, whether inducing or inhibiting, differs considerably between different bacterial species.

THE CRISPR/CAS SYSTEM AND THE IMPLICATION OF crRNAs IN VIRULENCE

Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR), has been identified which provides acquired immunity against viruses and plasmids in bacteria and Archaea [START_REF] Barrangou | CRISPR provides acquired resistance against viruses in prokaryotes[END_REF]. Unlike the restriction-based defence mechanism, the CRISPR/Cas system is highly specific, adaptive and heritable. It consists of an array of conserved short DNA repeat sequences, which are interspaced by variable spacer regions, originating from foreign DNA. Additionally, several cas (CRISPR-associated) genes are located in close proximity to the repeat-spacer array. The composition of the CRISPR/Cas locus can vary greatly between different microbial species and accordingly can be divided into types I-III. However, the basic mechanism to mediate adaptive immunity is common to all types of CRISPR/Cas systems and is composed of three main steps: the acquisition of foreign DNA, the processing and crRNA generation and the interference or silencing (for a review, see Sorek, Lawrence and Wiedenheft 2013) (Fig. 2). Thus CRISPR/Cas is a DNA-encoded, RNA-mediated defence system that provides sequence-specific recognition, targeting and degradation of exogenous nucleic acids. Going into the details of functionality and mechanistic differences between species would exceed the scope of this review, thus we place our main focus here on a newly described role for the CRISPR/Cas systems during infection and virulence formation of pathogens.

In a model it was proposed that the pathogenic potential of bacteria is associated with or even controlled by bacteriophage infections as CRISPR activity might interfere with the uptake of foreign DNA potentially carrying virulence traits, such as genes coding for toxins or antibiotic resistance [START_REF] Mojica | Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[END_REF]. Furthermore, it was found that the spacer regions contain sequence homologous not only to foreign DNA but also, even if in minor quantity, to endogenous chromosomal regions of the bacteria itself. This self-targeting was proposed to lead to a form of autoimmunity, suggesting a role in regulation of endogenous gene expression [START_REF] Stern | Self-targeting by CRISPR: gene regulation or autoimmunity?[END_REF]. Furthermore, changes in cas gene expression seem to be correlated with stress response, e.g. observed in L. pneumophila where an induction of the cas operon is dependent on the growth phase and typically up-regulated in the transmissive phase (Sahr et al. 2009). This phenomenon is abolished in a letA and a rpoS knockout mutant, suggesting a connection with the stringent response. Assuming an increased transcript level of RNAs in conditions relevant for their biological role, it suggests that CRISPR/Cas might have relevance for Legionella virulence formation. Indeed, similar results were observed in vivo during intracellular growth in macrophages and amoeba. Furthermore, cas2 mutants were significantly impaired during intracellular growth in amoebae, indicating a pivotal role for the CRISPR/Cas system during amoeba infection (Gunderson and Cianciotto 2013) (Table 1). Also in Listeria, the rliB locus composed of five small copies of expressed CRISPR repeats was shown to be involved in virulence [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]. Interestingly, in L. monocytogenes, RliB-CRISPR is not related to an adjacent cas operon but is instead expressed and processed by the PNPase that is needed for RliB-CRISPR-mediated DNA interference [START_REF] Sesto | A PNPase dependent CRISPR system in Listeria[END_REF]) (Table 1).

In both examples, a strong correlation between CRISPR expression and pathogenicity is evident. However, the functionality and the role are not yet established. CRISPR/Cas can promote bacterial fitness through protection against bacteriophages and thus increase indirectly the survival rate and ability to infect host cells. Alternatively, it might have a direct influence by regulating endogenous virulence factors and effectors similar to a mechanism identified recently in Francisella novicida [START_REF] Sampson | A CRISPR/Cas system mediates bacterial innate immune evasion and virulence[END_REF]. Therein it was shown for the first time that the Cas9 complex of this intracellular pathogen directly represses the production of an immunogenic lipoprotein, BLP, by stimulating its mRNA degradation. The BLP protein is recognized by the Toll-like receptor 2, triggering the antibacterial proinflammatory immune response. Therefore, Cas9-dependent repression of Blp production is essential for innate immune evasion and intracellular survival of F. novicida during infection of eukaryotic host cells. This process involves additionally two other newly described small RNAs that mediate the sequencespecific recognition of the blp transcript [START_REF] Sampson | A CRISPR/Cas system mediates bacterial innate immune evasion and virulence[END_REF]). These two sRNAs, named trans-activating crRNA (tracrRNA) and CRISPR/Cas-associated RNA (scaRNA), are not part of the crRNA cluster but closely related to the CRISPR/Cas region (Table 1). The knockout mutants of either one of them were not capable of establishing infection in a mouse model.

This outstanding observation demonstrates that the CRISPR/Cas system can also have an endogenous function independent from classical bacterial defence via an antisense RNA silencing mechanism. As CRISPR/Cas is widespread in bacteria, a similar mechanism might exist in numerous other pathogens, suggesting that this system might have a more general involvement in gene regulation, particularly during stress response and virulence formation. Unravelling this phenomenon and a deeper knowledge of the functionality of the CRISPR/Cas system will lead to a better understanding of gene regulation and virulence formation in pathogens and possibly to a new chance to fight bacterial infection independently of preceding antibiotic multiresistance.

CONCLUDING REMARKS

Several decades of research revealed the high variability, wide distribution and diverse functionality of sRNAs and demonstrated their crucial role in many biological processes such as in environmental sensing and stress adaptation, virulence and infectivity of intracellular bacteria, as well as development and metabolism. Here we aimed to highlight some of the most striking aspects that make sRNAs such versatile regulators in bacteria in particular for intracellular bacteria during the interaction, infection and growth within their eukaryotic host cells. However, considering the outstanding plethora and variety of sRNA regulatory elements, one may speculate about their functional redundancy, mainly as a consequence of their nature, within pathogenic and non-pathogenic bacterial species. In this context we highlighted several and very diverse sRNAs that can act independently or simultaneously to ensure the bacteria a favourable adaptation to adverse conditions and a benefit during the infection of the host cells (Fig. 5). On the other hand, only little is known about the opposite scenario, that one single sRNA may combine multiple regulatory mechanisms. Excitingly, it was recently shown that the same sRNA might indeed have several functions. The sRNA Qrr is able to fulfil four distinct regulatory mechanisms to fine-tune the regulation of quorum sensing in Vibrio [START_REF] Feng | A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics[END_REF]. Qrr can act as a by guest on August 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from HFQ AND SMALL REGULATORY RNAs ! ! Oliva et al. 345 negative control element to repress its targets through sequestration, catalytic degradation or coupled degradation, and at the same time as a positive regulatory element by setting free the RBS, allowing the target expression to occur while promoting its own degradation. The four different mechanisms are the results of diverse base-pairing strategies, employed by the Qrr sRNA to target defined quorum-sensing mRNAs [START_REF] Feng | A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics[END_REF]. Thus, this new view of multifunctional sRNA may be employed by other bacterial pathogens, representing more than a mere peculiarity but a common role not yet thoroughly examined. Recently it has been shown that sRNAs of bacteria can have an interspecies role, as two sRNAs, OxyS and DsrA of E. coli, have been reported to have physiological effects on Caenorhabditis elegans via regulating its gene expression (Liu et al. 2012), and that plant mi-croRNAs in the food of animals could have interspecies roles in mammalian cells [START_REF] Zhang | Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of crosskingdom regulation by microRNA[END_REF]. It also has been shown that L. monocytogenes releases nucleic acids and that such secreted bacterial RNA/DNA is recognized by cytosolic sensors such as RIG-I, MDA5 and STING, leading to the triggering of interferon-β production [START_REF] Abdullah | RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids[END_REF]. However, until now the main virulence factors identified, which are secreted into the host cell by intracellular bacteria, are proteins. Thus another current challenge might be to discover whether specific sRNAs of intracellular bacteria might play a role in the host cells they infect.

Taking into account the huge flexibility and evolutionary variety of sRNA within pathogenic bacteria one can imagine that bacterial sRNAs may also be secreted during infection to manipulate host cell function. In particular these bacterial sRNAs might mimic eukaryotic miRNAs to subvert their functions in the cells they infect. The identification of sRNAs secreted or released into the host cell to subvert its functions may emerge as a new, exciting topic of research in host-pathogen interactions. Thus, even though the analyses of bacterial RNAs is an ever-growing field of study, many questions still remain unanswered and new regulatory mechanisms might be discovered allowing us to appreciate more and more the diversified world of sRNA and its regulatory potential. 1972). The following studies on the HF I protein focused mainly on its binding properties and only in the early 1990s the E. coli gene encoding HF I was identified and designated as hfq [START_REF] Carmichael | The host factor required for RNA phage Qbeta RNA replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography[END_REF]de Haseth & Uhlenbeck, 1980a;1980b;[START_REF] Senear | Site-specific interaction of Qbeta host factor and ribosomal protein S1 with Qbeta and R17 bacteriophage RNAs[END_REF]. Since then, the attention has changed to the role of Hfq in the control of gene expression within bacterial cells. Hfq has been characterized as a member of the conserved family of RNA binding proteins named LSm (like-Sm)/Sm, that is present in all three domains of life. In eukaryotes, the Sm and LSm proteins have been associated with mRNA splicing, RNA decapping and RNA stabilization (reviewed in (C. J. Wilusz & Wilusz, 2005). Hfq is missing from three bacterial clades, the Chlamydia-Spirochaetes, Actinomycetes-Deinococcus-Cyanobacteria and Green sulfur bacteria-Cytophagales but is present in all alpha, beta, gamma and delta proteobacteria except some that have undergone massive genome reduction due to their parasitic lifestyle, like Buchnera sp., Rickettsia prowazekii and Brucella melitensis (Sun, Zhulin, & Wartell, 2002).

CHAPTER

In terms of topology, (L)Sm proteins are characterized by forming a ring-like multimeric complex that binds RNA and by the presence of a conserved protein fold, named LSmdomain [START_REF] Achsel | The Sm domain is an ancient RNA-binding motif with oligo(U) specificity[END_REF]. In detail, the LSm-fold structure consists of an N-terminal α-helix (α1), followed by five β-strands (β1-5), which are separated by five loops (L1-5) of variable length. The antiparallel β-strands form a half-open barrel structure with the N-terminal α-helix on top (Figure 7A). At the primary sequence level, the LSm proteins contain two conserved sequence motifs, named Sm1 and Sm2. The Sm1 signature resides in the first three β-strands and is more similar to the one of the eukaryotic LSm-proteins, whereas the Sm2 motif is located in the strands β4 and β5 (Figure 7C). The Sm1 motif is composed of diverse highly phylogenetically conserved residues such as acidic aspartate and glycine, which are involved in the fold maintenance. In contrast the Sm2 signature is divergent in the bacterial Hfq proteins and is composed of several residues, which CHAPTER TWO ! are essential for the protein stability [START_REF] Moskaleva | The structures of mutant forms of Hfq from Pseudomonas aeruginosa reveal the importance of the conserved His57 for the protein hexamer organization[END_REF]Møller et al., 2002;Sobrero & Valverde, 2012). The eukaryotic Sm proteins classically form heteromultimeric rings, whereas in bacteria there is usually only one Hfq protein, which assembles into ring-shaped homohexamers, as revealed from the crystal structures of several bacterial Hfq proteins [START_REF] Baba | Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer[END_REF]Beich-Frandsen, Vecerek, Sjöblom, Bläsi, & Djinovic-Carugo, 2011b;[START_REF] Bøggild | Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA-binding properties[END_REF][START_REF] Nikulin | Structure of Pseudomonas aeruginosa Hfq protein[END_REF][START_REF] Sauer | Structural basis for RNA 3'-end recognition by Hfq[END_REF][START_REF] Sauter | Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli[END_REF][START_REF] Schumacher | Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein[END_REF]. The intersubunit interactions are made up from residues in the β4 strand of one subunit and the β5 strand of the neighboring subunits. Adjacent monomers interact in an oriented way so that the doughnut-like structure assembles with N-terminal α-helices on the same face of the oligomer (Figure 7B).

HFQ AND SMALL REGULATORY RNAs

Interestingly, Hfq of enterobacteria contains unusually long C-terminal extensions (Figure 7C). Moreover, the sequence of these disordered C-terminal regions, which extend outwards from the hexameric ring, is not conserved and its biological function in E. coli is associated with protein stability but this is controversial in other microorganisms [START_REF] Arluison | The Cterminal domain of Escherichia coli Hfq increases the stability of the hexamer[END_REF] Beich-Frandsen, Vecerek, Konarev, Sjöblom, Kloiber, Hämmerle, et al.

The RNA binding-features of Hfq

Hfq works as chaperone and RNA-binder. Its surface properties are crucial for the binding and the discrimination of different targets. Hfq proteins are circular hexamers that delimit a central pore (Figure 8) and display two asymmetric faces, usually with a net positive electrostatic potential, an expected feature for a nucleic acid-binding protein. Thus, the two exposed faces -denoted distal and proximal -show distinct properties and binding specificities (Vogel & Ben F Luisi, 2011a) (Figure 8). Co-crystal structures of S. aureus, E. coli and L. monocytogenes Hfq in complex with short oligonucleotides demonstrated that the proximal face binds U-rich sequences with uredines accommodated around the pore in a constricted conformation that is stabilized by water molecules [START_REF] Sauer | Structural basis for RNA 3'-end recognition by Hfq[END_REF][START_REF] Schumacher | Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein[END_REF][START_REF] Wang | Hfq-bridged ternary complex is important for translation activation of rpoS by DsrA[END_REF]. Analysis of E. coli strains expressing Hfq proteins with mutations in conserved proximal face residues, such as Q8, F42 and K56, have highlighted the relevance of these residues for selection of U-rich HFQ AND SMALL REGULATORY RNAs ! ! segments [START_REF] Mikulecky | Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs[END_REF][START_REF] Sauer | Structural basis for RNA 3'-end recognition by Hfq[END_REF]. On the target side, sRNAs contain typical Rho-independent terminators, whose stretches usually have a poly-U 3' terminus, thus in vitro and in vivo elegant data have demonstrated that the specificity and affinity of the so-called proximal face relies on the recognition of the 3' hydroxyl group in the RNA conformation. As such, it is likely that binding in the proximity of the sRNA terminators affects the sRNA stability and turnover [START_REF] Otaka | PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action[END_REF][START_REF] Wilson | Transcription termination at intrinsic terminators: the role of the RNA hairpin[END_REF]. The model proposed is that the interaction of Hfq with the sRNA 3'end anchors the sRNA on the proximal face, whereas the U-rich sequences stabilize the complex by binding the lateral surface of the hexamer. This so-called lateral surface comprises six patches of conserved polar residues (R16, R17 and R19), constituting the lateral RNA binding motif. The number of the arginine residues affects the ability of Hfq to facilitate RNA interactions and defines different chaperone functions depending on the host organism [START_REF] Zheng | Arginine patch predicts the RNA annealing activity of Hfq from gram-negative and gram-positive bacteria[END_REF]. Strong arginine patches such as the ones in E. coli, Salmonella or Pseudomonas correlate with strong RNA activity of Hfq, typical for Gram-Negative bacteria. In contrast, low arginine content in the lateral motif has little effect on the physiology of Gram-positive bacteria [START_REF] Zheng | Arginine patch predicts the RNA annealing activity of Hfq from gram-negative and gram-positive bacteria[END_REF]. Furthermore, in E.coli it has been shown that acidic residues adjacent to this arginine patch affect the annealing and thus the selective recognition of the complementary RNA molecules [START_REF] Panja | Acidic residues in the Hfq chaperone increase the selectivity of sRNA binding and annealing[END_REF]. In addition to the lateral motif, another In contrast, the so-called distal face has preference for A-rich sequences, which avidly bind in a circular conformation the Hfq surface [START_REF] Link | Structure of Escherichia coli Hfq bound to polyriboadenylate RNA[END_REF]. The distal face seems to be important for the interaction with internal adenosine-rich sequences of mRNAs and in addition with poly-(A) n sequences of the 3'end of RNA degradation molecules [START_REF] Hajnsdorf | Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I[END_REF][START_REF] Salim | Requirement of upstream Hfq-binding (ARN)x elements in glmS and the Hfq C-terminal region for GlmS upregulation by sRNAs GlmZ and GlmY[END_REF][START_REF] Salim | An upstream Hfq binding site in the fhlA mRNA leader region facilitates the OxyS-fhlA interaction[END_REF][START_REF] Soper | The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA[END_REF]. Particularly, crystal structures of the E. coli Hfq protein with poly -(A) 15 RNA showed CHAPTER TWO ! the each Hfq monomer contains a tripartite RNA binding motif (A-R-E motif) with the 5'adenosine binding to Site A (specific for adenosines), the second nucleotide binding to the R site (specific for purine) and the following to a nondiscriminatory RNA-entrance/exit (E) site [START_REF] Link | Structure of Escherichia coli Hfq bound to polyriboadenylate RNA[END_REF]. Interestingly, Hfq of S. aureus binds to the distal surface to an (AA) 3 -A oligonucleotide, whereas the B. subtilis Hfq showed a more strictly preference for AG repeated sequences, suggesting the existence of species-specific RNA binding motifs [START_REF] Horstmann | Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract[END_REF][START_REF] Someya | Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer: insight into RNA-binding properties of bacterial Hfq[END_REF].

Generally, Hfq proteins of Gram-negative bacteria comprise a tripartite binding motif for (ARN) n sequences, whereas the Hfq homologs in Gram-positive bacteria have propensity for (AN) n repeats [START_REF] Horstmann | Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract[END_REF][START_REF] Sauer | Structure and RNA-binding properties of the bacterial LSm protein Hfq[END_REF]. A typical Hfq binding scenario comprises sRNAs carrying U-rich sequences preferentially binding the Hfq proximal face, on the other hand mRNA targets with A-rich motifs often found in the 5'UTRs bind preferentially to the distal face. However, additional evidence indicated that a simple two-face model does not necessarily occur [START_REF] Sauer | Structure and RNA-binding properties of the bacterial LSm protein Hfq[END_REF].

Mechanisms of Hfq riboregulation

The systematic discovery of sRNAs and the growing understanding of their Hfq-dependent action in bacterial pathogens, paved the way to analyze in molecular details the specific interactions of Hfq with sRNAs. Indeed, first in-vitro data demonstrated that the Hfq protein is facilitating sRNA-mRNA target interactions (Møller et al., 2002;[START_REF] Sittka | Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes[END_REF][START_REF] Soper | The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA[END_REF]. Interestingly, the sRNA-mRNA complex could be assembled also in absence of the Hfq RNA-binder, however its presence strongly accelerated simultaneous binding of two RNAs to Hfq through the formation of a ternary sRNA-mRNA-Hfq complex, in which Hfq "passively" serves as platform for the RNA interactions [START_REF] Mikulecky | Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs[END_REF]. This mechanism has been shown for E. coli Hfq, which forms a stable complex with the DsrA sRNA and its rpoS mRNA target [START_REF] Soper | The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA[END_REF].

Although the simultaneous interaction with Hfq has been reported also for fhlA mRNA and OxyS sRNA, whether the ternary complexes, for both fhlA and rpoS, is a prerequisite for the mRNA-sRNA duplex formation remains still to be clarified [START_REF] Salim | An upstream Hfq binding site in the fhlA mRNA leader region facilitates the OxyS-fhlA interaction[END_REF] [START_REF] Chao | An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs[END_REF][START_REF] Moon | Competition among Hfq-binding small RNAs in Escherichia coli[END_REF]. Intriguingly, Hfq and RNase E display similar binding preferences for AU-rich sequences and the interaction of the proximal face of Hfq with the 3'-terminal U of a terminator eventually makes the sRNA inaccessible for RNase Edependent cleavage [START_REF] Folichon | The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation[END_REF][START_REF] Moll | Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs[END_REF][START_REF] Sauer | Structural basis for RNA 3'-end recognition by Hfq[END_REF].

Overall, Hfq-facilitated sRNA-mRNA target pairing leads to distinct fates and activities. On CHAPTER TWO ! one hand, Hfq facilitates sRNA binding to the mRNA ribosome-binding site (RBS) on its 5' untranslated region, thus arresting the binding of the ribosome subunits and consequently the translation initiation. On the other hand, Hfq favours the interaction of the sRNA with the 5' region of the mRNA target and its translation by setting free the RBS, which alternatively would be locked by the formation of a secondary structure. In some cases, the formation of the sRNA-mRNA target duplex bound by Hfq results in an accelerated degradation due to RNase E cleavage of the mRNA or both RNA molecules (Aiba, 2007;[START_REF] Caron | Small RNA-mediated regulation at the level of transcript stability[END_REF]Morita, Maki, & Aiba, 2005). In addition, mRNA cleavage can occur by promoting the adenylation of the 3'ends of the mRNA by the enzyme poly(A)polymerase (PAP) and consequently triggering the 3' to 5' exonucleolytic cleavage, governing the RNA turnover [START_REF] Hajnsdorf | Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I[END_REF]. As such, Hfq acts as a central mediator of sRNA-based gene regulation in bacteria (Aiba, 2007;Storz, Vogel, & Wassarman, 2011;Vogel & Ben F Luisi, 2011b;Waters & Storz, 2009). Thus the loss of Hfq has strong effects on the physiology and fitness of bacteria, however Hfq effects are not the same in all bacteria, but species-specific phenotypes have been observed. With the exception of F. tularensis, where Hfq acts only as repressor, Hfq may be an activator or repressor of gene expression (Meibom et al., 2009). Hfq as pleiotropic regulator is involved in the altered expression of genes of diverse functions such as in metabolism, transport, energy production and conversion or membrane proteins (Boudry et al., 2014;[START_REF] Chiang | Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae[END_REF][START_REF] Cui | Impact of Hfq on global gene expression and intracellular survival in Brucella melitensis[END_REF][START_REF] Geng | Involvement of the posttranscriptional regulator Hfq in Yersinia pestis virulence[END_REF][START_REF] Kendall | Hfq virulence regulation in enterohemorrhagic Escherichia coli O157:H7 strain 86-24[END_REF].

Regulation of hfq expression

Despite the fact that it has been shown that the activity of a large pletora of sRNAs and their mRNA targets is governed by Hfq, how hfq gene and protein expression itself is regulated remains still poorly understood. The E. coli hfq gene is part of the amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon, which contains four σ 70 -dependent promoters and three σ 32 -dependent heat-shock promoters [START_REF] Tsui | Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock[END_REF]. This superoperon organization is well conserved in most γ-proteobacteria (Sobrero & Valverde, 2012). Within the α-and βproteobacteria and in some bacillales, the operon is composed of only the hfq-hflX tandem, the same organization that is found in L. pneumophila (described below). Only in few bacterial species such as E. coli and Francisella tularensis evidence of hfq-hflX co-transcription has been shown (Meibom et al., 2009;Tsui, Leung, & Winkler, 1994). hfq expression has been best studied in E. coli, in which Hfq negatively regulates its own expression through binding HFQ AND SMALL REGULATORY RNAs ! ! of the protein to its own mRNA at two distinct sites of the 5' UTR, competing with the translation machinery for the ribosome-binding site (Vecerek, Moll, & Bläsi, 2005). In addition, RNase E is involved in the regulation of the hfq transcript level, which accumulates 3-fold in the rne mutant strain compared to the transcript level detected in the wild type strain.

In detail, the binding of Hfq to its own transcript results in the exposition of the RNase E binding sites on the hfq mRNA and consequently to the cleavage by the endonuclease RNase E (Tsui et al., 1994). In this context, the Carbon Storage Regulator CsrA was demonstrated to inhibit the formation of the translational initiation complex by blocking the RBS and overlapping the hfq mRNA Shine-Dalgarno sequence [START_REF] Baker | CsrA inhibits translation initiation of Escherichia coli hfq by binding to a single site overlapping the Shine-Dalgarno sequence[END_REF]. Despite the evolutionary divergence in the tree of life of the bacterial species E. coli and Sinorhizobium meliloti, in the latter the Hfq protein has also been reported to govern its own expression by analyzing a hfq'-'lacZ translational fusion [START_REF] Sobrero | Evidences of autoregulation of hfq expression in Sinorhizobium meliloti strain 2011[END_REF]. However, whether this process involves also sRNAs targeting of the hfq mRNA is not known. This regulatory circuit might control the expression of hfq precisely to keep the concentration of this global regulator within a defined range. Furthermore, the analysis of RNAs co-immunoprecipated with Hfq in the γ-proteobacterium Rhodobacter sphaeroides has revealed the presence of the hfq mRNA, which might suggest a evolutionary conservation of the autoregulation of Hfq in other bacteria [START_REF] Berghoff | Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides[END_REF]. In addition to the abovementioned Hfq translational autocontrol, evidence has emerged that Hfq is post-translationally regulated by titration mechanisms. Examples for such a mechanism have been described in diverse bacteria such as the titration of the Pseudomonas aeruginosa Hfq by the sRNA CrcZ [START_REF] Sonnleitner | Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa carbon catabolite repression[END_REF] or the altered number of Hfq-bound mRNAs upon overexpression of the sRNA ArcZ in Salmonella or the competitive binding of the E.coli sRNAs on Hfq, leading to the modification of protein availability [START_REF] Moon | Competition among Hfq-binding small RNAs in Escherichia coli[END_REF].

In some bacterial species, hfq expression is growth phase dependently regulated by pleiotropic regulators or sigma factors. An example is Hfq of Shigella flexneri, which is positively controlled by the pleiotropic regulator DksA during the exponential phase of growth and also under stringent conditions (A. K. [START_REF] Sharma | Induction of expression of hfq by DksA is essential for Shigella flexneri virulence[END_REF]. In L. pneumophila hfq expression was reported to be upregulated by the sigma factor S during exponential growth and aureus pathogenicity (Y. [START_REF] Liu | Hfq is a global regulator that controls the pathogenicity of Staphylococcus aureus[END_REF]. Moreover, the transcriptome data from the wt strain and the hfq deletion mutant strain revealed that the expression of many genes, most of which were related to virulence, was altered. Nevertheless, an in depth investigation on the regulation of hfq is still missing what would probably explain why Hfq could not be detected in some strains. A particular case are bacteria from the Burkholderia cepacia complex, which encode two distinct and functional Hfq-like proteins that are differentially regulated in a growth-phase dependent manner, however the mechanism is not known [START_REF] Sousa | The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans[END_REF]. The hfq1 transcript is maximally expressed at the early exponential growth phase, whereas hfq2 expression is highly active during the stationary phase [START_REF] Ramos | Regulation of Hfq mRNA and Protein Levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA[END_REF][START_REF] Sousa | The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans[END_REF]. Similarly, Panda and colleagues reported recently the presence of three hfq-like genes in Bacillus anthracis, of which hfq1 and hfq2 are encoded on the chromosome, while hfq3 is encoded on a plasmid [START_REF] Panda | Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis[END_REF]. The expression of hfq1 and hfq2 is also growth-phase dependent but again, the regulators responsible are not described [START_REF] Panda | Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis[END_REF].

Role of Hfq in bacterial pathogens

Hfq is implicated in the regulation of virulence in several bacteria as described in chapter two.

Another not described example for the implication in virulence gene regulation of Hfq is Yersinia enterocolitica. Indeed, it was shown that Hfq governs the production of the heatstable enterotoxin, a urease, the siderophore yersiniabactin and the LpxR/SfpA lipopolysaccharide deacylase, all of which are important virulence factors [START_REF] Kakoschke | The RNA chaperone Hfq impacts growth, metabolism and production of virulence factors in Yersinia enterocolitica[END_REF]. Another example is Bordetella pertussis, where Hfq is required for the expression of the adenylate cyclase toxin, the pertussis toxin and the hemagglutinin virulence factors [START_REF] Bibova | The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis[END_REF]. Hfq activity is often associated with alternative sigma factors, which redirect gene expression to accomplish stress and virulence responses. In some pathogens like Salmonella Typhimurium and E. coli, Hfq was reported to be required for the efficient translation derepression of the alternative sigma factor RpoS, leading to increased availability HFQ AND SMALL REGULATORY RNAs ! ! of the protein and activating its targets (L. [START_REF] Brown | Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene[END_REF][START_REF] Muffler | The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli[END_REF]. Also in P. aeruginosa, about two-thirds of the RpoS-dependent genes are regulated by Hfq [START_REF] Sonnleitner | Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1[END_REF]. In K. pneumoniae, the Hfq-dependent regulation of rpoS appears more elaborate than the one in E. coli and S. Typhimurium [START_REF] Chiang | Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae[END_REF]. Particularly the absence of Hfq not only affects the transcript level of rpoS but the loss of Hfq causes also a reduced translation efficiency of rpoS probably due to the absence of a positive regulation by sRNAs. Besides RpoS, the expression of the K.

pneumoniae envelope stress sigma factor RpoE was also affected by the absence of Hfq.

Overall, 19,5 and 17,3% of Hfq-dependent target genes are part of the RpoE and RpoS regulons, respectively.

In S. Typhimurium, the Hfq-dependent sRNA SdsR, which is an enterobacterial "core" sRNA, is part of the large operon controlled by the stationary phase sigma factor σ S . σ S engages the sRNA SdsR to inhibit the synthesis of the OmpD, StcD, EnvE and TolC membrane proteins in a Hfq-mediated manner [START_REF] Fröhlich | The target spectrum of SdsR small RNA in Salmonella[END_REF]. In addition, Salmonella utilizes further sRNAs to control the expression of outer membrane proteins (OMPs). Some of them are under the control of the sigma factor RpoE, which in order to avoid a compromised envelope integrity, induces the expression of MicA, RybB or MicL for the repression of the OMP synthesis (Gottesman & Storz, 2011;[START_REF] Papenfort | SigmaEdependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay[END_REF]Vogel & Papenfort, 2006).

The RNA binding proteins Hfq and CsrA may work together

Besides the connection of Hfq with sigma factors, there is now evidence of an association of Hfq-regulated sRNAs also with other RNA binders. For example, a small RNA in E. coli named McaS has been demonstrated to serve both the Hfq and CsrA regulons [START_REF] Jørgensen | Dual function of the McaS small RNA in controlling biofilm formation[END_REF]. Like Hfq, the post-transcriptional regulator CsrA was reported to impact more than 20% of all mRNAs in E. coli, including those involved in motility and biofilm formation (A. N. Edwards et al., 2011;[START_REF] Lawhon | Global regulation by CsrA in Salmonella typhimurium[END_REF][START_REF] Wei | Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli[END_REF][START_REF] Yakhnin | CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage[END_REF]. Particularly, the McaS sRNA was shown to display a conserved region as potential hotspot of sRNA-based regulation, which base-pairs the csgD mRNA, leading to its inhibition of the translational initiation in an Hfq-dependent manner [START_REF] Holmqvist | Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis[END_REF]Jørgensen et al., 2012;[START_REF] Mika | Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli[END_REF][START_REF] Thomason | A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli[END_REF]. CsgD is a transcriptional activator of genes required for the formation of the biofilm matrix [START_REF] Povolotsky | Life-style" control networks in Escherichia coli: signaling by the second messenger c-di-GMP[END_REF]. In contrast, McaS activates the CHAPTER TWO ! translation of the FlhD 2 C2 master regulator of flagellar synthesis, also in an Hfq-dependent mechanism [START_REF] Thomason | A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli[END_REF]. Thus, this sRNA displays a dual function governing the bacterial biofilm and its motility. In addition, CsrA was previously shown to repress translation of the pgaA mRNA, whose protein is required for the production of the biofilmpromoting adhesion poly-β-1,6-N-acetyl-d-glucosamine (PGA)(X. [START_REF] Wang | The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation[END_REF]) and more recent evidence indicated that McaS is implicated in the activation of the PGA production by decoying the CsrA protein [START_REF] Jørgensen | Dual function of the McaS small RNA in controlling biofilm formation[END_REF]. Thus, McaA through Hfq and CsrA, represses one type of biofilm while promoting the other one [START_REF] Holmqvist | A small RNA serving both the Hfq and CsrA regulons[END_REF][START_REF] Jørgensen | Dual function of the McaS small RNA in controlling biofilm formation[END_REF]. Taken together, among the displayed pleiotropic functions, this chaperone regulates a plethora of sRNAs, which are implicated in diverse posttranscriptional regulatory circuits, including metabolism, virulence or biofilm formation.

The L. pneumophila life cycle

In the natural environment, L. pneumophila resides in biofilm communities together with grazing amoebae. When engulfed by these protozoa, the bacterium is not digested as food, but is able to protect itself from the lysosomal digestion. It builds a protected vacuole, called

Legionella containing vacuole (LCV) where it replicates to high numbers before escaping the spent host to search for a new one. As a facultative intracellular microbe, L. pneumophila transits between an intracellular and an extracellular habitat, displaying a multiphasic life cycle composed of at least two distinct stages. Besides an extracellular form when persisting in aquatic environments, L. pneumophila alternates between two morphologically distinct and reversible forms within the host cell: a non-infectious replicative form where virulence genes are down regulated, and a virulent, trasmissive form where virulence genes are upregulated and flagella are expressed leading to high motility (Molofsky & Swanson, 2004) (Figure 9). growth in rich medium; now replicative traits are expressed, whereas traits that promote transmission are repressed. In contrast, when nutrients become limiting (end of the infection cycle inside the host cell or in broth culture when the bacteria reach the post exponential growth phase) this leads to the induction of the transmissive phase. This phase is characterized by bacteria that become highly motile, express many virulence traits allowing them to survive osmotic stress, to lyse the spent host cell and to leave the host cell and to be infection competent for a new host. This transition from the replicative phase to the transmissive phase is reversible as the infection of a new host stimulates the repression of the virulent traits and the return to the replicative phenotype (Figure 10). The reciprocal expression of replicative or transmissive traits is a rational strategy for this intracellular pathogen to limit energy costs. In agreement, in a nutrient-rich environment, the infectious traits are not required or built. Conversely, during the transmissive phase, the biochemical pathways dedicated to the expression of the replicative aspects are neither demanded nor activated (Molofsky & Swanson, 2004). The transition between the mutually exclusive phases is reflected by a major change in the gene expression [START_REF] Brüggemann | Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins[END_REF]. In particular, the bacteria coordinately fine-tune in a growth phase dependent manner the expression of motility, cytotoxicity, sodium sensitivity, resistance to stresses such as UV light, heat or osmotic pressure, metabolic pathways and evasion from the CHAPTER THREE ! lysosomal digestion (Brüggemann et al., 2006;[START_REF] Byrne | Expression of Legionella pneumophila virulence traits in response to growth conditions[END_REF][START_REF] Hammer | Co-ordination of legionella pneumophila virulence with entry into stationary phase by ppGpp[END_REF]Hammer, Tateda, & Swanson, 2002;Molofsky & Swanson, 2003;2004).

3.2

Regulatory network governing Legionella differentiation

Metabolic triggers

The transition from the replicative to the transmissive phase is triggered by a stringent response like mechanism. In E. coli the stringent response is induced by a limited amino acid supply to allow a long-term survival in adverse conditions. This includes the rapid arrest of growth and the inhibition of ribosome, protein and stable RNA synthesis by guanosine tetraphosphate (p)ppGpp. The alarmone (p)ppGpp is converted from GTP by the (p)ppGpp synthetase RelA, which in turn is activated by the binding of uncharged transfer RNAs (tRNAs) to ribosomes. As result, the accumulation of the second messenger (p)ppGpp leads to the activation of the stationary-phase σ factor RpoS and the expression of the stationary-phase genes. Thus the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium [START_REF] Nazir | p)ppGpp and the bacterial cell cycle[END_REF]. By analogy to E. coli, in L. pneumophila two observations supported the activation of a similar stringent response pathway for the entry into the virulent phase. Firstly, in response to amino acid starvation and upon entry into the post-exponential phase of growth, the bacteria answer with accumulation of the second messenger (p)ppGpp. Secondly, upon expression of the E. coli relA gene and in nutrientindependent manner, L. pneumophila accumulates (p)ppGpp and activates different virulence traits [START_REF] Hammer | Co-ordination of legionella pneumophila virulence with entry into stationary phase by ppGpp[END_REF]. In support to this hypothesis, genetic data demonstrated that a L. pneumophila relA mutant replicates efficiently within amoeba and macrophages and upon exit from the exponential phase, the mutant strain does not accumulate (p)ppGpp and the infectious traits are poorly expressed [START_REF] Zusman | Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression[END_REF]. However, the fact that the relA mutant displays milder effects on the transmissive traits than those of other regulatory mutants such as letA and rpoS (described in the following paragraph) suggests that beside the stringent response, additional signals and redundant strategies are employed by L. pneumophila to govern its differentiation [START_REF] Zusman | Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression[END_REF]. Accordingly, other factors besides RelA, might trigger the microbial switch to the virulent phase. Indeed, in response to fatty acid biosynthesis perturbations, the bifunctional and essential enzyme SpoT mediates (p)ppGpp turnover via its hydrolase activity and weak synthase activity (Dalebroux,

REGULATION OF L. PNEUMOPHILA VIRULENCE ! ! 54
Svensson, Gaynor, & Swanson, 2010a;[START_REF] Potrykus | p)ppGpp: still magical?[END_REF]. Moreover, In E. coli, a relA and spoT double mutant does not lead to the production and accumulation of (p)ppGpp, resulting consequently in a state of rRNA transcription activation and in the synthesis of stable RNA [START_REF] Paul | rRNA transcription in Escherichia coli[END_REF][START_REF] Stent | A genetic locus for the regulation of ribonucleic acid synthesis[END_REF]. By analogy to E. coli, the L. pneumophila strain depleted of relA and spoT lacks (p)ppGpp synthetase activity [START_REF] Dalebroux | SpoT governs Legionella pneumophila differentiation in host macrophages[END_REF][START_REF] Trigui | A regulatory feedback loop between RpoS and SpoT supports the survival of Legionella pneumophila in water[END_REF]. Taken together, in vitro analyses demonstrated that the two (p)ppGpp synthetases confer plasticity to L. pneumophila, enabling transition to the transmissive phase when either amino acid or fatty acid synthesis is compromised. In addition, after the conditions are again favorable for establishing a protective vacuole, L. pneumophila employs SpoT to hydrolyse (p)ppGpp to tightly regulate the alarmone abundance and reverses the state from the transmissive to the replicative form [START_REF] Dalebroux | SpoT governs Legionella pneumophila differentiation in host macrophages[END_REF].

Transcriptional control by sigma factors

The signaling alarmone (p)ppGpp is a key player at the top of the regulatory network governing the transition of L. pneumophila from the replicative state to the transmissive state.

Generally, bacteria dealing with changing conditions reorganize their transcriptome in order to activate genes necessary for the new condition and repress the ones no longer required [START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF]. This is often achieved by the activity of sigma factors that are essential for modulating gene expression upon entry into a particular state or condition (T. M. [START_REF] Gruber | Multiple sigma subunits and the partitioning of bacterial transcription space[END_REF][START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF][START_REF] Navarro Llorens | Stationary phase in gram-negative bacteria[END_REF]. For example, the alternative sigma factor RpoS (σ S /σ 38 ), the master regulator of the stress response in E. coli, is implicated in resistance to heat, high osmolarity, acid and oxidative stress and in the expression of virulence traits [START_REF] Dong | Role of RpoS in virulence of pathogens[END_REF]. Similarly, L. pneumophila RpoS controls multiple pathways associated with motility and pathogenic functions as well as the activity of transcriptional regulators and of Dot/Icm effectors leading to a global influence on intracellular multiplication [START_REF] Bachman | RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase[END_REF][START_REF] Hales | The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii[END_REF][START_REF] Hovel-Miner | SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila[END_REF][START_REF] Trigui | A regulatory feedback loop between RpoS and SpoT supports the survival of Legionella pneumophila in water[END_REF]. The accumulation of (p)ppGpp increases the amount of L. pneumophila RpoS, however the molecular mechanism remains to be elucidated.

Biochemical studies on E. coli (p)ppGpp demonstrated the this alarmone, by biasing the competition among sigma factors for the binding to the RNA core polymerase, acts as global regulator of transcription [START_REF] Farewell | Negative regulation by RpoS: a case of sigma factor competition[END_REF][START_REF] Jishage | Regulation of sigma factor competition by the alarmone ppGpp[END_REF][START_REF] Laurie | The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase[END_REF]. In detail, (p)ppGpp is suggested to destabilize the binding

CHAPTER THREE

! of the vegetative sigma factor σ D/70 to the core and endorses the recruitment of alternative sigma factors and the expression of their targets [START_REF] Hales | The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii[END_REF][START_REF] Jishage | Regulation of sigma factor competition by the alarmone ppGpp[END_REF]Molofsky & Swanson, 2004).

Many of the physiological effects of the alarmone (p)ppGpp are mediated in cooperation with the RNA polymerase (RNAP) secondary channel interacting protein DksA [START_REF] Haugen | Advances in bacterial promoter recognition and its control by factors that do not bind DNA[END_REF][START_REF] Potrykus | p)ppGpp: still magical?[END_REF]. This protein was shown in E. [START_REF] Albert-Weissenberger | Control of flagellar gene regulation in Legionella pneumophila and its relation to growth phase[END_REF]Brüggemann et al., 2006;Dalebroux, Yagi, Sahr, Buchrieser, & Swanson, 2010b;[START_REF] Heuner | The flagellum of Legionella pneumophila and its link to the expression of the virulent phenotype[END_REF][START_REF] Jacobi | Characterization of the alternative sigma factor sigma54 and the transcriptional regulator FleQ of Legionella pneumophila, which are both involved in the regulation cascade of flagellar gene expression[END_REF]. Moreover, L. pneumophila engages the flagellar sigma factor FliA not only for the expression and synthesis of the flagellum, but also for the expression of pathways related to cytotoxicity, lysosome evasion and replication [START_REF] Heuner | Influence of the alternative sigma(28) factor on virulence and flagellum expression of Legionella pneumophila[END_REF]. Thus FliA can act both as regulator of virulence factors and together with FleQ and RpoN to coordinately initiate the expression of the flagellum machinery (Hammer et al., 2002;[START_REF] Heuner | Influence of the alternative sigma(28) factor on virulence and flagellum expression of Legionella pneumophila[END_REF][START_REF] Heuner | The flagellum of Legionella pneumophila and its link to the expression of the virulent phenotype[END_REF]Molofsky & Swanson, 2004).

Post-transcriptional regulation of the transmissive traits

An additional layer of regulation of the L. pneumophila biphasic life cycle occurs at posttranscriptional level. Post-transcriptional regulation is often controlled by two-component systems (TCS), which use protein phosphorylation cascades for signal transduction (J. [START_REF] Stock | Signal transduction: response regulators on and off[END_REF]. TCS control diverse cellular pathways such as chemotaxis, stress and metabolic responses, virulence and differentiation [START_REF] Mascher | Stimulus perception in bacterial signal-transducing histidine kinases[END_REF].

Typical TCS are composed of two elements: a membrane-bound sensor histidine kinase, which detects the external signal and a cytoplasmatic protein, which upon activation by the cognate kinase induces the cellular response. The mechanism of activation of the TCS is triggered by the binding of the physiological signal, which leads to a conformational change and the autophosphorylation on a conserved histine residue of the sensor kinase. The cognate response regulator is then stabilized and phosphorylated by the kinase leading to its activation and the subsequent adaptive response (A. H. [START_REF] West | Histidine kinases and response regulator proteins in twocomponent signaling systems[END_REF]. Indeed, the TCS LetA/LetS (Legionella transmission activator and sensor, respectively) of L. pneumophila is an important regulator in its life cycle switch. Originally, the LetA/LetS TCS of L.

pneumophila was identified in a genetic screen for mutants lacking the flagellum and only later the adaptive response induced by the LetA/LetS pathway was reported to include also the activation of a large set of virulence phenotypes and the control of the progression into the transmissive state (Gal-Mor & Segal, 2003b;Hammer et al., 2002;Lynch, Fieser, Glöggler, Forsbach-Birk, & Marre, 2003). Whether, the alarmone (p)ppGpp directly actives the sensor

CHAPTER THREE

! 57 histidine kinase LetS is not yet known, however it has been reported that during the stationary phase LetS activates LetA by a multi-step phosphorelay (R. L. Edwards, Jules, Sahr, & Buchrieser, 2010). By analogy to other bacteria harboring this TCS, it was postulated that the L. pneumophila LetA/S TCS modulates the carbon metabolism when the conditions are not favorable, which are genes that are regulated in parallel by the small RNA binding protein CsrA [START_REF] Heeb | Regulatory roles of the GacS/GacA two-component system in plantassociated and other gram-negative bacteria[END_REF]. Indeed, genetic data later demonstrated that the major function of

LetA is to relieve the repression exerted by the global regulatory RNA-binder CsrA, ensuring thereby the expression of the transmissive traits [START_REF] Hovel-Miner | SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila[END_REF]Rasis & Segal, 2009;Sahr et al., 2009). Thus the TCS LetA/LetS has a central role in the regulatory network governing the L. pneumophila biphasic life cycle and virulence.

CsrA belongs to a family of global regulators that in L. pneumophila repress diverse traits related to virulence, including motility, pigmentation, sodium resistance or cytotoxicity (Fettes, Forsbach-Birk, Lynch, & Marre, 2001;Molofsky & Swanson, 2003). L.

pneumophila CsrA is essential thus only conditional mutants or partial mutants have been obtained, which are all strongly attenuated for intracellular multiplication. First, four effectorencoding genes have been shown to be regulated by the LetAS-CsrA regulatory cascade (Rasis & Segal, 2009;[START_REF] Shi | The Legionella pneumophila global regulatory protein LetA affects DotA and Mip[END_REF]. These four effectors, named RalF, VipA, YlfA and YlfB, are involved in vesicular trafficking during the establishment of the LCV [START_REF] De Felipe | Legionella eukaryotic-like type IV substrates interfere with organelle trafficking[END_REF][START_REF] Franco | The Legionella pneumophila effector VipA is an actin nucleator that alters host cell organelle trafficking[END_REF][START_REF] Nagai | A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes[END_REF].

Recently, by applying a bioinformatics approach and validation by expression analyses, 26

effector-encoding genes have been identified to be regulated by the LetAS-CsrA regulatory cascade, all of which were expressed at higher levels during the stationary phase [START_REF] Nevo | Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking[END_REF], further supporting the important role of CsrA in the expression of the virulent phenotype.

In addition, another TCS, named PmrA/B, has been described to be important for virulence of L. pneumophila [START_REF] Zusman | The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii[END_REF]. L. pneumophila PmrA/B activates the expression of 43 effector-encoding genes, representing the largest effector regulon known (Al-Khodor, Kalachikov, [START_REF] Al-Khodor | The PmrA/PmrB two-component system of Legionella pneumophila is a global regulator required for intracellular replication within macrophages and protozoa[END_REF][START_REF] Zusman | The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii[END_REF]. In addition an interplay between PmrA/B TCS and CrsA in a growth-dependent manner has been reported (Rasis & Segal, 2009) Another player in this complex regulatory network, is the Lqs (Legionella quorum sensing)

system that also plays a role in the regulation of gene expression during the transmissive phase [START_REF] Spirig | The Legionella autoinducer synthase LqsA produces an alpha-hydroxyketone signaling molecule[END_REF]. The Lqs system consists of the LqsR response regulator and the LqsS sensor histidine kinase [START_REF] Tiaden | The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA[END_REF]. The regulation is initiated by the production of the diffusible signaling molecule 3-hydroxypentadecan-4-one (Legionella auto inducer-1-LAI-1) by the autoinducer synthase LqsA, which is detected by the sensor kinase LqsS, which in turn activates the response regulator LqsR [START_REF] Spirig | The Legionella autoinducer synthase LqsA produces an alpha-hydroxyketone signaling molecule[END_REF][START_REF] Tiaden | The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila[END_REF]. Interestingly, this system contains two histidine kinases, as a homologue of LqsS, named LqsT, was identified and shown to also respond to LAI-1 [START_REF] Kessler | The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit[END_REF].

Although the mechanism of how LqsR affects gene expression is currently not known, it has been reported that LqsR influences the expression of genes involved in virulence including 12

effector-encoding genes [START_REF] Kessler | The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit[END_REF][START_REF] Tiaden | The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA[END_REF]. Furthermore, LqsR expression depends on the alternative sigma factor RpoS and to a smaller extent on the response regulator LetA [START_REF] Tiaden | The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA[END_REF]. Considering that in L. pneumophila the transmissive traits are repressed by CsrA, it is not surprising that the production of LqsR is regulated at the post-transcriptional level by the global repressor CsrA (Sahr et al., 2009). In addition to the abovementioned systems, a forth TCS, named CpxR/A has been analyzed in L.

pneumophila (Gal-Mor & Segal, 2003a). CpxR/A was shown to repress few Dot/Icm effector genes and to activate few others [START_REF] Altman | The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates[END_REF]Gal-Mor & Segal, 2003a). Taken together, the study of the complex regulatory system governing the L. pneumophila life cycle uncovered four TCS (LetA/S, PmrA/B, LsqR/Q and CpxR/A), sigma factors (RpoS) and RNA-binding proteins (CsrA) as important regulatory elements which act at the posttranscriptional level to fine tune gene expression according to the environmental conditions.

Implication of regulatory sRNAs on L. pneumophila virulence

This fine-tuned and hierarchical regulation of the L. pneumophila life cycle includes also small RNAs, which ensure a fast and more cost-effective regulation compared to the one controlled by polypeptides [START_REF] Altuvia | A small, stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator[END_REF].

Previous evidences in E. coli showed that the BarA/UvrY TCS (the L. pneumophila LetA/S

CHAPTER THREE

! homolog) controls the expression of two sRNAs, named CsrB and CsrC. Within these sRNAs GGA motifs were identified, which are the characteristic binding sequences for CsrA. Thus it was thought that such sRNAs might also exist in L. pneumophila. Indeed, two homologs of CsrB, named RsmY and RsmZ were identified by a bioinformatics search (P. R. [START_REF] Kulkarni | Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri[END_REF]. Functional analyses confirmed that these sRNAs were the missing regulatory elements between the LetA/S TCS and the RNA binding protein CsrA in L. pneumophila and that LetA induces the expression of these small RNAs (Rasis & Segal, 2009;Sahr et al., 2009). Deep RNA sequencing from exponentially (replicative) and post! exponentially (virulent) in vitro grown L. pneumophila have identified more than 700 sRNAs, 60% of which are growth-phase dependently regulated, suggesting that a set of these yet uncharacterized sRNAs, coordinately with RsmY/Z/X might regulate gene expression and influence the expression of virulence determinants (Sahr et al., 2012). Probably Hfq, acting as RNA chaperone regulates a number of these sRNAs implicated in bacterial virulence.

Indeed, Hfq seems to play a role in this regulatory network. However, to date little is known of the functional role and the regulation of L. pneumophila Hfq. First results published in 2005 suggested that Hfq plays a role the iron uptake, and that it interacts not only with the exponential gene fur, but also with the global regulator gene csrA, influencing thus genes under the control of this RNA-binding protein. The authors reported that the hfq transcript is highly expressed in the mid-exponential phase of growth and that it is under the control of the sigma factor RpoS. By contrast, hfq transcription was shown to be down regulated in the stationary phase of L. pneumophila growth probably due to a regulatory role of the twocomponent regulator LetA (McNealy et al., 2005). Recently, Tiaden and colleagues have shown that the quorum sensing regulator LqsR, influences the expression of hfq upon entry into the stationary growth phase, as hfq expression was upregulated in an lqsR mutant strain [START_REF] Tiaden | The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA[END_REF]. Thus L. pneumophila Hfq seems also to play a role in the regulatory network governing L. pneumophila differentiation from the replicative/non infectious to the transmissive/infectious phase.

Regulatory network governing L. pneumophila bi-phasic life cycle

The present view on the regulatory cascade governing L. pneumophila virulence is as follows:

Upon entry into the post-exponential phase, the detection of uncharged tRNAs due to amino acid starvation leads to the activation of RelA, whereas SpoT recognizes fatty acid starvation.

REGULATION OF L. PNEUMOPHILA VIRULENCE ! ! 60

Together RelA and SpoT activation leads to the synthesis of (p)ppGpp which by an unknown mechanism results in the activation of the LetA/S system. LetA binds directly to a conserved consensus sequence upstream the rsmX/Y/Z genes, leading to their expression (Rasis & Segal, 2009;Sahr et al., 2009). These sRNAs contain multiple repeated CsrA binding motifs and act as sponge to bind and sequester CsrA from their target mRNAs, leading to the expression of virulence traits. The expression of RsmX, RsmY and RsmZ is strongly reduced in letS or rpoS deletion mutants, suggesting that these two regulators might coordinately regulate expression of these sRNAs positively [START_REF] Hovel-Miner | SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila[END_REF]Rasis & Segal, 2009). Moreover, it is important to mention that although Hfq was reported to influence L. pneumophila differentiation by interacting with the major regulatory elements of the cascade, it is expected that Hfq, acting as RNA chaperone and RNA binder might regulate a number of sRNAs implicated in bacterial virulence (Figure 11). However, the role of Hfq in this regulatory network is not known yet. Taken together, the expression of L. penumophila virulent determinants is fine-tuned both at the transcriptional and post-transcriptional level, by a complex regulatory network ensuring a successful infection cycle. CsrA that is bound near the ribosomal binding site of its mRNA targets inhibits their translation. RsmX, RsmY and RsmZ titrate CsrA away from its targets, enabling translation of the mRNAs, leading to expression of transmissive phenotypes. Hfq also influences the different phenotypes, however its exact role is not known.

AIM OF THE PH.D. THESIS

!

Aim of the Ph.D. thesis

The elucidation of the complex regulatory network and its components that govern the L. pneumophila differentiation from the replicative phase to the transmissive phase is a major focus of Legionella research. Deep RNA sequencing analysis comparing the transcriptional profiles of L. pneumophila grown at the exponential (replicative) and post-exponential (virulent) phases defined the complete operon map of this pathogen and the presence of more than 700 sRNAs, many of which are growth phase dependently regulated (Sahr et al., 2012).

As reported for many pathogenic bacteria, a key factor in the regulation of small regulatory RNAs is the global posttranscriptional regulator Hfq. This RNA binder and chaperone is also growth phase-dependently regulated in L. pneumophila, however how it is regulated is not known. Our transcriptional start site mapping of the L. pneumophila genome lead to the identification of a putative sRNA transcribed at the antisense strand that overlaps the 5' untranslated region of the hfq transcript including its ribosome binding site (Sahr et al., 2012).

This sRNA, named Anti-hfq seemed to be more abundant in the replicative phase and its expression decreased at the beginning of the transmissive phase.

Based on these findings the aim of this work was I) to elucidate how the growth phase dependent expression of Hfq is regulated in L. pneumophila;

II)

to analyze the role of Hfq and its newly identified putative antisense RNA in L. pneumophila virulence; III) to decipher the regulatory mechanism by which this sRNA functions.

In the first part of the thesis a L. pneumophila deletion mutant of the hfq gene and a knockdown mutant of the sRNA were constructed and investigated by applying cell biological assays to characterize their regulation and intracellular growth phenotypes.

In the second part of the thesis RNA binding and stability assays were performed to provide insight into the mechanism of regulation by which the sRNA regulates Hfq expression.

!

! 62

CHAPTER FOUR

RESULTS-REGULATION OF HFQ IN L. PNEUMOPHILA

CHAPTER FOUR !

L. pneumophila hfq-chromosomal organization

In L. pneumophila lpp0009 encodes Hfq, an 85 amino acid protein. It is part of an operon together with the gene lpp0010, putatively coding a GTP-binding protein HflX (Figure 12A).

Hfq protein has homologs in all the L. pneumophila genomes sequenced to date, with at least 80% of conservation. Our transcriptional starting site analysis revealed the presence of a putative small RNA, which is divergently transcribed on the antisense strand of the hfq gene and overlaps its 5' UTR region as shown in the Figure 12. Further analysis of the deep sequencing data performed from bacteria grown at the replicative phase and transmissive phase indicated that the hfq transcript is highly expressed in transmissive phase and less in replicative phase, whereas the sRNA, later named Anti-hfq, seems to be highly abundant in replicative phase and less in transmissive phase of growth.

Thus, we hypothesized that the putative sRNA might act as a cis-encoded sRNA, regulating the expression of the hfq transcript in a growth phase-dependent manner.

RESULTS

!

! 64 ABSTRACT Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycledependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5= untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells.

Article published in the journal mBIO

Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria.

IMPORTANCE

The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra-and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated.

I n recent years, the discovery of a class of regulatory elements, called small noncoding RNAs (sRNAs) revealed a high complexity of posttranscriptional gene regulation in prokaryotes and eukaryotes [START_REF] Waters | Regulatory RNAs in bacteria[END_REF]. sRNAs were reported to exert a wide range of cellular functions in bacterial physiology, in which rapid and fine-tuned adaptations in response to environmental changes are required [START_REF] Caldelari | RNA-mediated regulation in pathogenic bacteria[END_REF]3)

RESULTS

!

! encoded sRNAs that modulate gene expression through complementarity to their adjacent or distant mRNA targets, respectively. In bacteria, trans-encoded sRNAs commonly require the assistance of the RNA chaperone Hfq to promote their interaction with the cognate mRNA targets. Although cis-encoded sRNAs share extended base pairing complementarity to their counterpart mRNAs, in a few cases, Hfq is required for their function [START_REF] Møller | Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction[END_REF]. First identified in Escherichia coli as a host factor essential for the replication of the Q␤ RNA phage, Hfq is now recognized as a global regulator of gene expression present in a wide variety of bacteria that impacts many molecular processes in bacterial physiology, stress response, and virulence [START_REF] Franze De Fernandez | Factor fraction required for the synthesis of bacteriophage Qbeta-RNA[END_REF][START_REF] Updegrove | Hfq: the flexible RNA matchmaker[END_REF]. The importance of the RNA-binding protein Hfq was uncovered by the characterization of hfq null mutants in diverse bacterial pathogens (7,[START_REF] Tsui | Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12[END_REF]. Further detailed research in its function in different bacteria showed that Hfq is a key posttranscriptional regulator, stabilizing sRNAs or facilitating sRNA/mRNA interactions that inhibit or enhance translation initiation. Furthermore, Hfq can act independently to modulate gene expression by affecting mRNA translation (for reviews, see references 6 and 9). Although deep sequencing approaches have revealed a high number and broad spectrum of sRNAs in diverse pathogens, such as Salmonella enterica serotype Typhimurium [START_REF] Sittka | Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq[END_REF], Pseudomonas aeruginosa [START_REF] Wurtzel | The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature[END_REF], Yersinia pseudotuberculosis [START_REF] Nuss | Transcriptomic profiling of Yersinia pseudotuberculosis reveals reprogramming of the Crp regulon by temperature and uncovers Crp as a master regulator of small RNAs[END_REF], or Legionella pneumophila [START_REF] Sahr | Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence[END_REF], the extent of Hfq-mediated riboregulation is highly complex and variable for each RNA type and in each organism. Furthermore, Hfq-associated sRNAs have been reported to control gene expression of multiple targets, thus regulating diverse cellular pathways, such as biofilm formation ( 14), catabolite repression [START_REF] Beisel | Discriminating tastes: physiological contributions of the Hfq-binding small RNA Spot 42 to catabolite repression[END_REF], quorum sensing ( 16), or the control of transcriptional factors [START_REF] Coornaert | MicA sRNA links the PhoP regulon to cell envelope stress[END_REF]. Hfq is closely related to the Sm family of RNA-binding proteins in archaea and eukaryotes and phylogenetically widespread among bacteria, as about half of the sequenced bacterial genomes harbor at least one copy of the hfq gene [START_REF] Møller | Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction[END_REF][START_REF] Sobrero | The bacterial protein Hfq: much more than a mere RNA-binding factor[END_REF]. Legionella pneumophila is an intracellular bacterium that inhabits environmental aquatic systems, like lakes and rivers where it replicates in aquatic protozoa, but it can also infect humans to cause a severe pneumonia, and it also carries a gene that encodes Hfq [START_REF] Cazalet | Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity[END_REF][START_REF] Mcnealy | The Hfq homolog in Legionella pneumophila demonstrates regulation by LetA and RpoS and interacts with the global regulator CsrA[END_REF]. However, little is known about the role of Hfq in the L. pneumophila life cycle or its regulatory function. The change between extra-and intracellular life and between replication in a host (replicative phase) and transmission to a new host (transmissive/virulent phase) demands a highly fine-tuned regulatory network [START_REF] Molofsky | Differentiate to thrive: lessons from the Legionella pneumophila life cycle[END_REF]. Indeed, the life cycle switch from replicating to transmissive/virulent L. pneumophila is governed through the function of several key regulators. Probably the most important ones are the two-component system (TCS) LetA/LetS (Legionella transmission activator and sensor, respectively) that induces traits necessary for efficient host transmission [START_REF] Edwards | The Legionella pneumophila LetA/LetS two-component system exhibits rheostate-like behavior[END_REF][START_REF] Hammer | A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila[END_REF][START_REF] Lynch | The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii[END_REF] and CsrA (carbon storage regulator) that is a posttranscriptional regulator, repressing transmissive/virulence traits during replication of L. pneumophila and releasing them in later stages of infection [START_REF] Fettes | Overexpression of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation[END_REF][START_REF] Molofsky | Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication[END_REF]T. Sahr,C. Rusniok,F. Impenes,G. Oliva,O. Sismeiro,J. Y. Coppee,and C. Buchrieser,unpublished data). Moreover, the three sRNAs RsmX, RsmY, and RsmZ that are sequestering CsrA in transmissive phase to allow virulence traits to be translated are indispensable in this regulatory cascade [START_REF] Rasis | The LetA-RsmYZ-CsrA regulatory cascade, together with RpoS and PmrA, post-transcriptionally regulates stationary phase activation of Legionella pneumophila Icm/Dot effectors[END_REF][START_REF] Sahr | Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila[END_REF].

Here we report that L. pneumophila Hfq is regulated in a life cycle-dependent manner by a unique sRNA, named Anti-hfq that is transcribed in the early phase of the L. pneumophila life cycle. Our data support a complex model of regulation of the hfq transcript by the Anti-hfq sRNA, in which the Hfq chaperone together with RNase III are engaged to ensure the growth phase-dependent expression of this RNA-binding protein. Moreover, our results show that Hfq affects intracellular multiplication in amoebae, and consequently L. pneumophila virulence.

RESULTS

Hfq is highly conserved within the genus Legionella and other bacterial species. In L. pneumophila, Hfq is an 85-amino-acid protein encoded by the gene lpp0009. The hfq gene is organized in an operon with the putative GTP-binding protein HflX encoded by gene lpp0010 (Fig. 1A). Although the L. pneumophila hfq gene shares the conserved chromosomal gene arrangement typical of other organisms like E. coli or

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Vibrio cholerae only partly, it shows high nucleotide and amino acid identity with Hfq of many Gram-negative bacteria (up to 70%) and Gram-positive bacteria (up to 50%). Furthermore, all residues that contribute to RNA binding are conserved in L. pneumophila (Fig. 1B). Comparison of the Hfq amino acid sequence among more than 300 L. pneumophila strains sequenced in the last years [START_REF] Cazalet | Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity[END_REF][START_REF] D'auria | Legionella pneumophila pangenome reveals strain-specific virulence factors[END_REF][START_REF] Gomez-Valero | Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes[END_REF][START_REF] Schroeder | Legionella pneumophila strain 130b possesses a unique combination of type IV secretion systems and novel Dot/Icm secretion system effector proteins[END_REF][START_REF] David | Multiple major diseaseassociated clones of Legionella pneumophila have emerged recently and independently[END_REF] revealed that Hfq is 100% conserved across the different L. pneumophila strains. Analyses of four nonpneumophila Legionella species [START_REF] Gomez-Valero | Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires' disease[END_REF] showed that Hfq is 80% conserved (Fig. 1C).

Hfq is highly expressed during postexponential/transmissive growth phase. In several pathogens, the level of expression of Hfq is growth phase dependent. In order to assess the transcriptional and posttranscriptional level of Hfq at different growth phases, we performed Northern and Western blot analyses of total RNA and whole protein lysates obtained from cultures of L. pneumophila (wild type [wt]) grown in liquid medium at 37°C. Northern blots using an hfq-specific probe showed very low hfq transcripts during exponential growth (optical density at 600 nm [OD 600 ] of 2), but high transcript levels upon entry into postexponential growth (OD 600 of 4). Protein expression followed the same pattern as shown by immunoblotting using anti-Hfq antibodies (Fig. 2A).

Hfq is necessary for efficient intracellular replication at environmental temperatures. In order to analyze the role and regulation of Hfq of L. pneumophila, we constructed an hfq deletion mutant (⌬hfq) by the insertion of an in-frame apramycin resistance cassette (Fig. 2B). The resistance cassette used does not contain a transcriptional terminator; thus, transcription of the downstream gene, hflX, was not negatively affected as verified by transcriptome analyses (described below). Furthermore, the ⌬hfq mutant strain was completely sequenced using the Illumina technique, which ascertained that no secondary mutations had been introduced during the mutant construction. Analyses of the ⌬hfq mutant confirmed that the expression of Hfq was indeed abolished (Fig. 2C). To complement the ⌬hfq mutant, a plasmid harboring the entire hfq gene and its own promoter was transformed into the ⌬hfq mutant, generating the

RESULTS

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! complemented strain ⌬hfq pBChfq. Western blot analyses using anti-Hfq antibodies confirmed the expression of Hfq in ⌬hfq pBChfq (Fig. 2D). In contrast to a previous report where the ⌬hfq mutant in another L. pneumophila strain showed a prolonged lag phase [START_REF] Mcnealy | The Hfq homolog in Legionella pneumophila demonstrates regulation by LetA and RpoS and interacts with the global regulator CsrA[END_REF], the growth pattern of the ⌬hfq mutant analyzed here was very similar to that of the wt strain at 37°C (see Fig. S1A in the supplemental material) and at 20°C (Fig. S1B), indicating that the growth defect of a strain lacking Hfq is due to the intracellular environment in amoeba, and not to a general growth defect at lower temperatures.

To learn whether Hfq is implicated in virulence of L. pneumophila as reported for other bacterial pathogens, we compared the ability of the wt L. pneumophila and ⌬hfq mutant to infect and multiply in Acanthamoeba castellanii and in the human monocytederived cell line THP-1. Similarly to what was reported previously, the ⌬hfq mutant strain showed only a minimal growth defect in A. castellanii and THP-1 cells at 37°C (Fig. 3A andB). In contrast, when replication in A. castellanii was monitored at 20°C, the ⌬hfq mutant showed a clear replication defect compared to the wt strain (Fig. 3C). Furthermore, complementation of the ⌬hfq mutant restored the intracellular replication pattern (Fig. 3D). Taken together, our data imply that Hfq plays a role in intracellular replication in amoeba at environmental temperatures and thus on the virulence of L. pneumophila.

Hfq expression is affected by RpoS and LetA. The activation of virulence traits of L. pneumophila is highly regulated at the transcriptional and posttranscriptional levels. Major regulators implicated are the sigma factor RpoS and the two-component system LetA/LetS (21) (Fig. 4D). Hfq is another candidate, as the mutant showed a replication defect (Fig. 3C). To determine the role and place of Hfq in this regulatory network, we analyzed the hfq transcript and protein levels in rpoS and letA mutants. Northern blot analysis showed that hfq transcripts were abolished in ⌬rpoS and ⌬letA mutants, confirming that RpoS and LetA are implicated in the regulation of hfq expression (Fig. 4A). This was also reflected in the protein level, as observed by immunoblot analysis, where Hfq expression in the ⌬rpoS and ⌬letA mutants was strongly decreased compared to the Hfq levels in the wt strain (Fig. 4B). Thus, RpoS and LetA are strongly

CHAPTER FOUR

! implicated in the regulation of Hfq expression at the transcript and protein levels.

Flagella and consequently motility are hallmarks of the transmissive/virulent phase of L. pneumophila. We thus analyzed FlaA expression in the ⌬hfq mutant strain and the ⌬rpoS and ⌬letA mutants in which FlaA expression is known to be reduced. As expected, FlaA was highly expressed in the late postexponential phase in the wt but strongly reduced in the ⌬hfq mutant strain, suggesting its involvement in the regulatory cascade governing L. pneumophila differentiation, motility, and virulence (Fig. 4C).

Taken together, the expression of Hfq in L. pneumophila is regulated in a growth phase-dependent manner and is influenced by RpoS and LetA. Furthermore, Hfq itself seems to be implicated in the activation of traits typical of the transmissive/virulent phase of L. pneumophila.

Transcriptome analyses of the ⌬hfq mutant strain reveal only few changes in gene expression. To analyze which genes Hfq is affecting that may lead to the decreased intracellular replication, transcriptome analysis at postexponential growth (OD 600 of 4 grown in vitro in BYE medium and in vivo after 96 h of infection of A. castellanii) when Hfq is expressed the highest was performed. The comparison of the wt and ⌬hfq mutant transcriptomes in vitro identified only 18 differentially expressed genes (Table S1). This is in accordance with an in vitro transcriptome analysis of an hfq mutant in strain L. pneumophila JR32, where only a few genes and a mobile genetic element that excised upon the deletion of hfq were differentially expressed (34). In vivo, 74 genes were differentially transcribed due to the loss of Hfq, the majority of which (69 genes) was upregulated in the absence of Hfq, whereas only five genes were downregulated (Table S2). Interestingly, CsrA (0.43ϫ), a major regulator of metabolic and regulatory functions during replication (Sahr et al., unpublished) was downregulated in In contrast, no effect of Hfq on other important regulators like RpoS or the two-component system LetA/LetS was seen on the transcript level, indicating no direct feedback cascade for this regulatory pathway. In total, eight genes were upregulated both in vitro and in vivo. Two of these genes are involved in flagellar assembly and motility (flgG and flgH), and two are coding for the enhanced entry protein EnhA (lpp2693) and EnhB (lpp2694), which are implicated in host cell infection [START_REF] Cirillo | Identification of novel loci involved in entry by Legionella pneumophila[END_REF]. Additionally, the macrophage infectivity potentiator Mip, at least four Dot/Icm effector proteins, transcriptional regulators Fis1 and Fis2, and the DNA-binding protein HU-beta are differentially transcribed in the ∆hfq mutant during in vivo growth. These data might suggest a direct influence of Hfq on virulence formation as seen in infection of A. castellanii. An antisense RNA is present in the 5= untranslated region of hfq. We had previously established a complete transcriptional map of the L. pneumophila genome that revealed the presence of a dynamic pool of sRNAs regulated in a growth phasedependent manner [START_REF] Sahr | Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence[END_REF]. Among these sRNAs, we identified a transcriptional start site (TSS) of a noncoding gene located in the reverse strand of the 5= untranslated region (5= UTR) of the hfq gene (Fig. 5A). In order to confirm experimentally the presence of a sRNA, we performed 3= rapid amplification of cDNA ends (RACE), which yielded only a single band around 100 bp from RNA samples isolated from a culture grown at the early exponential phase (OD 600 of 2). Cloning and sequencing of this cDNA amplimer that we named Anti-hfq showed that the noncoding RNA is 101 bp long (Fig. 5B and Fig. S2A). Using the program Mfold [START_REF] Zuker | Mfold web server for nucleic acid folding and hybridization prediction[END_REF], the anti-hfq secondary structure was predicted to be composed of a duplex, with a 5= overhang of 1 nucleotide (5= C) and a 3= overhang of 3 nucleotides (3= UUA) containing a putative Rho-independent terminator identified by FindTerm (Softberry) (Fig. 5C). Although other programs did not confirm this terminator structure, the RACE PCR results showed that the transcript terminated at 101 bp where FindTerm predicted the terminator; hence, under the given conditions, Anti-hfq is indeed an sRNA. Bioinformatic analysis revealed the presence of an identical

CHAPTER FOUR

! anti-hfq sequence among all L. pneumophila strains investigated. Anti-hfq homologues were also found among other Legionella species with a sequence identity of at least 80%, but no homologous sequences were found in other bacterial genomes. Thus, Anti-hfq represents a unique sRNA element within the genus Legionella. Anti-hfq is expressed at the early exponential phase of the Legionella growth cycle. To determine the pattern of the Anti-hfq transcripts during the L. pneumophila life cycle, total RNA was extracted at exponential growth (OD 600 of 1) and postexponential growth (OD 600 of 4) of wt L. pneumophila grown in liquid BYE medium. The total RNA was reverse transcribed, and quantitative PCR (qPCR) analysis on the obtained cDNA was performed. We used different primer pairs: primer pair 1 (hfq-qPCR-F [F stands for forward] and hfq-qPCR-R [R stands for reverse]) exclusively recognizing the hfq mRNA and primer pair 2 (anti-hfq-qPCR-F and anti-hfq-qPCR-R) recognizing both the hfq and Anti-hfq RNAs, as these two transcripts entirely overlap (Fig. 5A). To confirm the growth phase-dependent expression of Anti-hfq, we calculated the ratio between the hfq and Anti-hfq transcript levels in the two growth phases. This showed that in the exponential phase, the Anti-hfq transcript was expressed about 1.5-fold higher than the hfq transcript, whereas its expression levels decreased to 0.05-fold compared to hfq in the postexponential phase (Fig. 5D). This alternative expression of either hfq or Anti-hfq suggests a regulation in which the expression of the Anti-hfq transcript might inhibit the expression of the sense transcript due to the cis regulatory function of the Anti-hfq sRNA.

Anti-hfq affects intracellular replication. To analyze whether the Anti-hfq sRNA indeed impacts Hfq expression levels, we first constructed a strain overexpressing

RESULTS

! !

Anti-hfq sRNA, in which the anti-hfq gene was cloned under the control of an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible promoter. Upon induction with IPTG, the overexpression of the Anti-hfq sRNA decreased Hfq expression levels compared to the wt (Fig. 5E, top blot), supporting the idea that Anti-hfq sRNA is able to directly regulate Hfq expression. As the deletion of hfq resulted in a strongly decreased flagellin expression (Fig. 4C), we postulated that the overexpression of the Anti-hfq sRNA should also impact flagellin expression via the repression of Hfq. Indeed, when the Anti-hfq sRNA was overexpressed, the expression of FlaA was strongly reduced compared to the wt strain (Fig. 5E, bottom blot), further suggesting a cis regulatory function of the Anti-hfq sRNA on Hfq expression. This result is consistent with a model in which an antisense sRNA regulates the transcription of its sense protein-coding gene, here hfq. The L. pneumophila ∆hfq mutant is attenuated in intracellular growth of A. castellanii (Fig. 3C), and flagellin is less well expressed in comparison to the wt strain (Fig. 4C). Thus, to test whether Anti-hfq has a role in intracellular replication of L. pneumophila, we infected A. castellanii with the strain overexpressing anti-hfq. At 72 h postinfection, 10-fold fewer intracellular bacteria were recovered from amoeba infected with the Anti-hfq sRNA-overexpressing strain (pMMBantihfqOE) compared to the wt, similar to the replication rate seen for the ∆hfq mutant strain (Fig. 5F and3C). Thus, Anti-hfq sRNA plays a role in intracellular replication of L. pneumophila. Hfq expression is regulated by the Anti-hfq sRNA. In the ⌬hfq mutant used until now, the anti-hfq gene was still intact (Fig. 2B). Thus, to further study the function of Anti-hfq sRNA, we constructed a second mutant containing a larger deletion as the entire region spanning hfq and anti-hfq (⌬hfq ⌬anti-hfq) was replaced with an apramycin cassette (Fig. 6A). By complementing this mutant with the plasmid pBCanti-hfq (-10) in which two single mutations in the anti-hfq Ϫ10 box had been introduced, we were able to study the role of the Anti-hfq sRNA without disturbing Hfq expression. This complemented strain was named the ⌬anti-hfq(-10) strain (Fig. 6A). When analyzing the Hfq expression levels in the ⌬anti-hfq(-10) mutant, the Hfq expression pattern differed

CHAPTER FOUR

! compared to the wt strain, as the expression of the hfq transcripts started already during exponential growth of L. pneumophila (Fig. 2A, bottom blot, and Fig. 6B), indicating that Anti-hfq sRNA indeed represses hfq transcripts in exponential growth. In contrast, in the complemented mutant strain (∆hfq ∆anti-hfq pBChfq), Hfq expression was restored to wt levels (Fig. 6C), whereas in the control strain (∆hfq ∆anti-hfq mutant carrying the empty plasmid; ∆hfq ∆anti-hfq pBC), no expression of Hfq was seen, as expected (Fig. 6C). Thus, the antisense RNA Anti-hfq regulates Hfq expression levels in a growth phase-dependent manner by functioning as a cis-complementary sRNA.

The hfq and Anti-hfq RNA transcripts interact in vitro. Our previous results suggest a regulation of the hfq transcript through binding of its Anti-hfq antisense sRNA. To investigate a direct interaction of Anti-hfq and hfq mRNA in vitro, we performed electrophoretic mobility shift assays (EMSAs). Incubation with a radioactively labeled Anti-hfq RNA probe and increasing concentrations of cold hfq mRNA resulted in a slower-migrating complex, suggesting a direct interaction of the two RNA molecules (Fig. 7A). In contrast, when the EMSA was performed with Anti-hfq sRNA and a truncated hfq mRNA probe spanning the nucleotides 78 to 255 missing the 5= UTR region and the first 26 codons (hfq OUT), no changes in terms of migration were observed, consistent with the absence of formation of a complex (Fig. 7A). Similar results were obtained when using the mRNA of an unrelated gene (lpp0644 RNA probe) as a second negative control (Fig. S2B). Thus, Anti-hfq forms an RNA duplex with the hfq mRNA and most likely regulates hfq mRNA expression by direct binding due to complementarity.

Purified Hfq binds hfq and Anti-hfq sRNA with different affinity. Although the Hfq protein is known to facilitate the interaction between trans-encoded sRNAs and their mRNA targets, the Hfq chaperone may also function to stabilize/destabilize cis-encoded sRNAs and their complementary mRNA targets. Thus, we sought to determine whether the Hfq protein might be able to form complexes either with the hfq mRNA or with the Anti-hfq sRNA. The analysis of the hfq and anti-hfq sequences revealed the presence of (AAN) n triplets and AU-rich regions, which could be Hfq binding regions, further suggesting the hypothesis of an Hfq autoregulatory loop. To assess the ability of Hfq to bind hfq and Anti-hfq transcripts separately, we evaluated binding in vitro by EMSAs using recombinant Hfq protein. As shown in Fig. 7B andC, Hfq interacts with both RNA molecules but with different affinities.

To study the inhibitor complex formed by the hfq mRNA, Anti-hfq, and the Hfq protein in more details, we employed a gel-shift kinetic assay (Fig. 7D). A radioactively labeled Anti-hfq RNA probe was incubated with 25 nM of cold hfq mRNA in the absence (Fig. 7D, lanes 1, 3, and 5) or presence (Fig. 7D, lanes 2, 4, and 6) of Hfq protein for 0.5 (lanes 1 and 2), 1.5 (lanes 3 and 4), and 2.5 (lanes 5 and 6) minutes. As shown above, the two RNA molecules were able to interact. Additionally, we detected a strong band corresponding to the formation of a ternary complex already after 0.5 min of incubation. Moreover, the intensities of the shifted bands indicated that the affinity of Hfq for the RNA-RNA complex might be much stronger than for the single RNAs alone. The super shift and thus the formation of the ternary complex was increasing with longer incubation time (after 1.5 and 2.5 minutes). To test the specificity of this complex, radioactively labeled hfq or Anti-hfq probes were incubated alone in parallel with increasing amounts of Hfq confirming that Hfq is indeed able to bind each of the RNA molecules separately (Fig. 7D, lanes 7 to 9 and 10 to 12). Therefore, although Anti-hfq is complementary to its own target and thus it should not require Hfq for binding, Hfq is able to bind the two RNA molecules, forming a ternary complex.

RNase III might participate in the double-strand RNA (dsRNA) regulation. One of the regulatory functions of the Hfq RNA chaperone is the recruitment of RNases for the degradation of sRNA and/or mRNA targets. Thus, we wondered whether RNases might be in involved in the degradation of the ternary complex in L. pneumophila. To answer this question, we performed an RNA stability assay in the wt and an RNase III gene (lpp1834) deletion mutant that we constructed. Analysis of the hfq mRNA levels,

RESULTS

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! after the addition of rifampin, showed a half-life of 4.1 min in the wt strain and of 8.2 min in the RNase III deletion mutant (Fig. 7E). In contrast, when the half-life of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was determined in the same conditions, no significant differences were observed in the relative mRNA levels between the wt and the RNase III deletion mutant (Fig. 7F). This strongly suggests that RNase III is involved in the cleavage of the hfq-Anti-hfq RNA duplex and hence, affects the stability of the hfq mRNA, closing the Hfq regulation loop.

DISCUSSION

Legionella pneumophila needs to adapt to many different environmental conditions, including low-temperature and nutrient-poor aquatic and hostile intracellular environ-

CHAPTER FOUR

! ments of protozoa or human macrophages. To regulate the transition from one environment to another environment, L. pneumophila has evolved a complex regulatory cascade allowing it to switch from a replicative stage to a transmissive/virulent stage [START_REF] Molofsky | Differentiate to thrive: lessons from the Legionella pneumophila life cycle[END_REF]. This regulatory network is comprised of many global regulators like the RNA-binding protein CsrA and its small noncoding RNAs RsmX, RsmY, and RsmZ, the TCS LetA/LetS, and the stress sigma factor RpoS [START_REF] Edwards | The Legionella pneumophila LetA/LetS two-component system exhibits rheostate-like behavior[END_REF][START_REF] Hammer | A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila[END_REF][START_REF] Lynch | The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii[END_REF][START_REF] Fettes | Overexpression of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation[END_REF][START_REF] Molofsky | Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication[END_REF][START_REF] Rasis | The LetA-RsmYZ-CsrA regulatory cascade, together with RpoS and PmrA, post-transcriptionally regulates stationary phase activation of Legionella pneumophila Icm/Dot effectors[END_REF][START_REF] Sahr | Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila[END_REF]Sahr et al,unpublished). Here we demonstrate that the RNA chaperone Hfq is another major player in the regulation of the switch to transmissive/virulent L. pneumophila and that life cycle-dependent Hfq expression is regulated by an antisense RNA named Anti-hfq.

Comparative sequence analyses showed that Hfq is highly conserved and present in all L. pneumophila strains sequenced thus far (Fig. 1B). Our observation that the hfq transcript and the Hfq protein are barely expressed at early stages of growth but highly expressed at the postexponential phase of growth (Fig. 2A) establishes Hfq as a growth phase-dependent regulated protein and suggests its implication in the regulation of the expression of virulence traits, a feature of postexponential bacteria. Interestingly, in 2005, McNealy and colleagues (20) had reported that Hfq of L. pneumophila JR32 is expressed in the exponential phase of growth and is positively regulated by the stationary-phase sigma factor RpoS. Furthermore, they proposed that upon entry into stationary phase, Hfq expression is abolished through the regulatory function of the two-component regulator LetA, thereby ensuring that hfq transcripts are off when the infectious traits need to be activated [START_REF] Mcnealy | The Hfq homolog in Legionella pneumophila demonstrates regulation by LetA and RpoS and interacts with the global regulator CsrA[END_REF]. The differences from our results might be due to the different strains used or perhaps to the excision of the 100-kb plasmid pL100 when hfq is deleted, as reported by Trigui and colleagues (34). However, our results are in agreement with the hfq expression pattern observed in several other bacterial pathogens such as P. aeruginosa and Listeria monocytogenes [START_REF] Christiansen | Identification of small Hfq-binding RNAs in Listeria monocytogenes[END_REF][START_REF] Sonnleitner | Hfq-dependent alterations of the transcriptome profile and effects on quorum sensing in Pseudomonas aeruginosa[END_REF] but also with the life cycle of L. pneumophila [START_REF] Molofsky | Differentiate to thrive: lessons from the Legionella pneumophila life cycle[END_REF][START_REF] Brüggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF]. The regulation of virulence traits by Hfq, which demands its expression in the postexponential growth phase, is supported by the observation that the hfq mutant is defective in intracellular growth, a characteristic also reported by McNealy and colleagues, and the transcriptome results identifying virulence genes and virulence gene regulators to be differentially expressed upon deletion of hfq (see Table S1 and S2 in the supplemental material). By analyzing the protein and transcript levels of Hfq in different regulatory mutants, we show that Hfq expression is influenced by the stationary sigma factor RpoS and the response regulator LetA during the postexponential phase, as both directly or indirectly turn on hfq transcription (Fig. 4A andB). Thus, Hfq plays an important role in the regulatory cascade governing the switch to the transmissive phase of L. pneumophila (Fig. 4D).

In agreement with the position of Hfq in this regulatory network, the loss of Hfq impaired intracellular replication at 20°C, the optimal growth temperature of A. castellanii and a temperature that is close to environmental conditions (Fig. 3C). The transcriptome analysis of the ∆hfq mutant during infection of A. castellani supported this finding, as several secreted effector proteins, the enhanced entry proteins EnhABC, the global DNA-binding transcriptional regulators Fis1 and Fis2, and the DNA-binding protein HU-beta were differentially regulated in the hfq mutant. Moreover, the abovementioned regulators are all related to environmental adaptation, virulence, and stress response regulation and fitness in different pathogenic bacteria [START_REF] Duprey | Bacterial virulence and Fis: adapting regulatory networks to the host environment[END_REF]. Furthermore, a hallmark of transmissive/virulent L. pneumophila, the expression of flagellar protein FlaA that is intimately linked to virulence, was strongly reduced in the ⌬hfq mutant at an OD 600 of 4, similar to what is seen in LetA and RpoS mutants (Fig. 4C). Collectively, these results indicate that L. pneumophila requires Hfq to promote motility and to efficiently multiply within A. castellanii at environmental temperatures.

Most studies of Hfq analyzed its role in the regulation of sRNAs and their mRNA targets, but not how Hfq expression itself is regulated. L. pneumophila Hfq is clearly growth phase dependently regulated, as transcript and protein levels are low during replicative/exponential growth but are strongly expressed in transmissive/postexponential growth (Fig. 2A). This growth phase-dependent regulation is achieved by an sRNA that we named Anti-hfq as it is transcribed on the antisense strand of the hfq

RESULTS

! !

gene overlapping its 5= UTR (Fig. 5A). Anti-hfq is a 101-bp long sRNA that is highly expressed during exponential growth, but its expression is strongly decreased upon entry into the transmissive/postexponential growth phase. These opposite expression patterns of the hfq and Anti-hfq transcripts together with the fact that the sRNA is encoded antisense to hfq suggested that it has a role in regulating hfq expression. Furthermore, the identification of a partly conserved LetA binding site (two mismatches) suggested that the growth phase-dependent expression of Anti-hfq sRNA might be regulated by LetA. However, we could not firmly establish a specific interaction; thus, this regulatory pathway remains to be analyzed in the future. A detailed analysis of the anti-hfq sequence revealed the presence of a putative Rho-independent transcriptional terminator as described in a large part of functional Hfq binding modules of sRNAs [START_REF] Ishikawa | The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3= poly(U) tail[END_REF]. Furthermore, the ARN or ARNN (R is purine, and N is any nucleotide) motifs that are preferentially bound in the distal site of the Hfq homohexamer [START_REF] Sauer | Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition[END_REF] were also present in the proximity of the hfq ribosome binding site (RBS), and we showed that Anti-hfq sRNA binds the complementary region of the hfq mRNA. Furthermore, Hfq is able to interact separately with both RNA molecules, hfq and Anti-hfq (Fig. 7B andC), but it also forms a ternary complex, suggesting an autoregulatory circuit (Fig. 7D). Finally, the riboendonuclease RNase III takes part in the regulation of Hfq probably cleaving the double-strand RNA as suggested by RNA stability measurements in an RNase III mutant strain (Fig. 7E). Several studies of E. coli had suggested that Hfq binds two distinct sites of the 5= UTR of its own mRNA, hindering the formation of the translation initiation complex and thus negatively regulating its own expression. In E. coli, RNase E is recruited to exert its RNase function to degrade hfq mRNA [START_REF] Vecerek | Translational autocontrol of the Escherichia coli hfq RNA chaperone gene[END_REF]. Thus, collectively, the data suggest that binding of the cis-encoded Anti-hfq sRNA obstructs Hfq translation in exponential growth (Fig. 7A).

The regulation of Hfq by a cis-encoded sRNA is an unusual feature. We propose that binding of the cis-encoded Anti-hfq sRNA to hfq mRNA in exponential growth leads to low translation of Hfq, whereas when the expression of Anti-hfq sRNA decreases in the transmissive phase, high expression of Hfq is possible. This leads to the expression of several Dot/Icm secreted substrates, global regulators like Fis1 and Fis2 that are implicated in the regulation of virulence traits [START_REF] Zusman | Two Fis regulators directly repress the expression of numerous effector-encoding genes in Legionella pneumophila[END_REF] and probably of several of the many growth phase dependently regulated sRNAs that we identified earlier (13) (Fig. 8). Thus, L. pneumophila is equipped with a highly sophisticated regulatory mechanism further fine-tuning the regulation of the reciprocal expression of distinct sets of genes under different environmental conditions.

MATERIALS AND METHODS

Bacterial strains, growth media, and culture conditions used. The bacterial strains used in this study are listed in Table 1. L. pneumophila strain Paris and its derivatives were cultured in N-(2acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract broth (BYE) or on ACES-buffered charcoal-yeast (BCYE) extract agar [START_REF] Feeley | Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila[END_REF], and E. coli was grown in Luria-Bertani (LB) broth and agar. All strains were grown at 37°C. For the construction of knockout mutants and complementation plasmids, antibiotics were used at the following concentrations: ampicillin at 100 mg/ml, kanamycin at 50 mg/ml, and chloramphenicol at 20 mg/ml for E. coli; and kanamycin at 10 mg/ml, chloramphenicol at 20 mg/ml, and apramycin at 15 mg/ml for L. pneumophila. A. castellanii ATCC 50739 was cultured in PYG 712 medium [2% proteose peptone, 0.1% yeast extract, 0.1 M glucose, 4 mM MgSO 4 , 0.4 M CaCl 2 , 0.1% sodium citrate dihydrate, 0.05 mM Fe(NH 4 ) 2 (SO 4 ) 2 • 6H 2 O, 2.5 mM NaH 2 PO 3 , 2.5 mM K 2 HPO 3 ] at 20°C. THP-1 human monocytes were grown in RPMI 1640 GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 .

Mutant and plasmid constructions. The plasmids and oligonucleotide primers used in this study are listed in Table 1 and2, respectively. Mutant strains of L. pneumophila were constructed as previously described [START_REF] Brüggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF][START_REF] Hindré | Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation[END_REF]. In brief, the gene of interest was inactivated by introduction of an apramycin resistance (Apr r ) cassette. The mutant alleles were constructed using a three-step PCR. For the construction of the ⌬hfq deletion mutant strain, three overlapping fragments (lpp0009 upstream region primers hfq-Mut_F and hfq-apra_R, antibiotic cassette-primers apra_F and apra_R, lpp0009 downstream region primers hfq-apra_F and hfq-Mut_R; Table 2) were amplified independently and purified on agarose gels. The three resulting PCR products were mixed at the same concentration (15 nM), and a second PCR with flanking primers (primers hfq-Mut_F and hfq-Mut_R) was performed. This PCR product, the resistance marker cassette flanked by 300-bp regions homologous to lpp0009 was introduced into the L. pneumophila Paris strain by natural competence [START_REF] Buchrieser | Induction of competence for natural transformation in Legionella pneumophila and exploitation for mutant construction[END_REF]. Strains that had undergone allelic exchange were selected by plating on BCYE containing apramycin, and the mutant was verified by PCR and sequencing. For the

CHAPTER FOUR

! construction of the ⌬hfq ⌬anti-hfq double mutant strains and the RNase III mutant, the same cloning strategy was used, and the primers are listed in Table 2.

For complementation experiments, the region, including lpp0009 and lnc0003 was PCR amplified with primers containing HindIII and SalI restriction sites at their ends (Hfq_compl_F and Hfq_compl_R) and ligated to the pBC-KS plasmid, previously digested with the two restriction enzymes. The resulting plasmid, named pBChfq, was introduced into the ⌬hfq and ⌬hfq ⌬anti-hfq deletion mutant strains by electroporation. The wild-type (wt) L. pneumophila Paris, the ⌬hfq and the ⌬hfq ∆anti-hfq deletion FIG [START_REF] Tsui | Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12[END_REF] Model of the regulation of Hfq in replicative and transmissive L. pneumophila. During the replicative phase, the Anti-hfq sRNA is highly expressed and represses Hfq expression through binding to the hfq mRNA. This process also involves Hfq itself, which autoregulates its own expression and the riboendonuclease RNase III that likely cleaves the hfq mRNA product. In contrast, upon entry into the transmissive phase, Anti-hfq is not expressed, leading to high Hfq expression that now influences the expression of motility and virulence traits of L. pneumophila.

RESULTS

!

! mutant strains containing the empty plasmid pBC-KS were used as control. For constructing the Anti-hfq mutant strain, site-directed mutagenesis of anti-hfq was performed on the pBChfq plasmid as the template using the QuikChange site-directed mutagenesis kit (Qiagen) following the manufacturer's instructions. Two mutations were introduced in the Ϫ10 promoter region of the anti-hfq gene using the primers M-10anti-hfq_F and M-10anti-hfq_R. The resulting plasmid, pBCanti-hfq(-10), was introduced into the ⌬hfq ⌬anti-hfq deletion mutant, creating the ⌬anti-hfq(-10) mutant strain.

For overexpression of Anti-hfq sRNA in L. pneumophila, we used the pMMB207C (derived from pTS-10, kindly provided by H. Hilbi [START_REF] Weber | The inositol polyphosphate 5-phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE[END_REF]). The anti-hfq gene was amplified using primers containing XbaI and PstI restriction sites (anti-hfq_OE_F and anti-hfq_OE_R primers) and ligated into pMMB207C, linearized using the same restriction enzymes. The resulting plasmid (pMMBanti-hfqOE) and the control plasmid pMMB207C (here named pMMB) were introduced via electroporation into wt L. pneumophila Paris strain. For overexpression, IPTG (0.5 mM) was added at an OD 600 of 0.8.

Sequencing of the ⌬hfq mutant strain. For whole-genome sequencing, paired-end sequences and a read length of 100 bases were obtained from an Illumina HiSeq platform (Biomics pole Institut Pasteur).

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!

Sequence reads were mapped to a reference genome using SMALT v0.7.4, and single nucleotide polymorphisms (SNPs) were searched for using a standard approach.

A. castellanii and THP-1 infection assay. Infection of A. castellanii with L. pneumophila Paris and its derivatives was done as described previously [START_REF] Rolando | Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy[END_REF]. In brief, A. castellanii were washed once with infection buffer (PYG 712 medium without proteose peptone, glucose, and yeast extract) and seeded at a density of 4 ϫ 10 6 cells per 25-cm 2 flask. Wild-type and mutant strains of L. pneumophila were grown on BCYE agar to stationary phase, diluted in infection buffer, and mixed with A. castellanii at a multiplicity of infection (MOI) of 0.1 or 1 (as indicated in the figure legends). Intracellular multiplication was monitored by plating a 100-l sample that was centrifuged at 14,500 rpm and vortexed to break up amoeba, at different time points on BCYE plates. The number of bacteria recovered was counted as CFU. In THP-1 cell infection assays, cells were seeded in 12-well tissue culture trays (TTP) at a density of 2 ϫ 10 5 cells/well. THP-1 cells were pretreated with 10 to 8 M phorbol 12-myristate 13-acetate (PMA) (Sigma) for 72 h to induce differentiation into macrophage-like adherent cells. Stationary-phase L. pneumophila bacteria were resuspended in serum-free medium and added to cells at an MOI of 10. After 2 h of incubation, cells were washed with phosphate-buffered saline (PBS) before incubation with serum-free medium. At 2, 24, 48, and 72 h, the supernatant was collected and the cells were lysed with PBS-0.1% Triton X-100. The infection efficiency was monitored by determining the CFU of the different L. pneumophila strains recovered on BCYE agar plates. Each infection was carried out in triplicate.

RNA isolation and Northern blot analysis. Total RNA was extracted as previously described ( 50). Wild-type and mutant L. pneumophila Paris strains were grown in BYE medium and harvested for RNA isolation at exponential phase (OD 600 of 1.0 and 2.0) and postexponential phase (OD 600 of 3 and 4). Total RNA was treated with DNase I and purified using columns (Qiagen). Ten micrograms of total RNA isolated from different conditions (see above) were size separated on 10% denaturing polyacrylamide gels containing 8 M urea (Bio-Rad) and transferred onto positively charged nylon membranes (BrightStar-Plus; Ambion). The membranes were photographed under UV light to capture ethidium bromide staining of rRNA bands for loading controls. RNA was cross-linked to membranes by exposure to UV light for 2 min, and membranes were prehybridized in Ultrahyb buffer (catalog no. AM8670; Ambion) for 1 h. RNA probes radioactively labeled with [␣-33 P]UTP (catalog no. BLU007X500UC; PerkinElmer) were generated using the T7 Maxiscript kit (catalog no. AM1314; Ambion), and PCR templates were amplified from genomic DNA using primers listed in Table 2. The membrane was then hybridized at 65°C by adding the radiolabeled probes overnight. Blots were washed twice at the hybridization temperature in 2ϫ SSC-0.1% SDS (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and then washed twice in 0.1ϫ SSC-0.1% SDS. Membranes were wrapped in Saran Wrap and subsequently used to expose films (catalog no. 28906844; GE Healthcare).

RNA isolation, labeling, and microarray hybridization. For total RNA extraction, wild-type Paris and the ⌬hfq mutant strains were grown in BYE medium in vitro and harvested for RNA isolation at postexponential growth phase (OD 600 of 4). For in vivo experiments, A. castellanii amoebae were infected with wt or ⌬hfq mutant at an MOI of 100 as described above. Cells were cultivated at 20°C and harvested for RNA isolation after 96 h. RNA was prepared in biological triplicates for in vitro and biological duplicates for in vivo experiments as described above, and all samples were hybridized twice to the microarrays (dye swap). RNA was reverse transcribed with Superscript indirect cDNA kit (Invitrogen) and labeled with Cy5 or Cy3 (Amersham Biosciences, Inc.) according to the supplier's instructions. The design of microarrays containing gene-specific 70-mer oligonucleotides based on all predicted genes of the genome of L. pneumophila strain Paris (CR628336) and its plasmid (CR628338) was previously described [START_REF] Brüggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF]. Hybridization was performed following the manufacturers' recommendations (Corning) using 250 pmol of Cy3-and Cy5-labeled cDNA. Slides were scanned on a GenePix 4000A scanner (Axon Instruments). Laser power and/or the photomultiplier tube (PMT) were adjusted to balance the two channels, and the resulting files were analyzed using GenePix Pro 4.0 software. Spots were excluded from analysis in case of high local background fluorescence, slide abnormalities, or weak intensity.

Data normalization and differential analysis were conducted using the R software (http://www.Rproject.org). No background subtraction was performed, but a careful graphical examination of all the slides was conducted to ensure a homogeneous, low-level background in both channels. A loess normalization [START_REF] Yang | Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation[END_REF] was performed on a slide-by-slide basis (BioConductor package marray; https:// www.bioconductor.org/packages/release/bioc/html/marray.html). Differential analysis was carried out separately for each comparison between two time points, using the VM method (VarMixt package [START_REF] Delmar | VarMixt: efficient variance modelling for the differential analysis of replicated gene expression data[END_REF]), together with the Benjamini and Yekutieli P value adjustment method [START_REF] Reiner | Identifying differentially expressed genes using false discovery rate controlling procedures[END_REF]. Empty and flagged spots were excluded from the data set, and only genes with no missing values for the comparison of interest were analyzed.

Determination of RNA half-life and quantitative RT-PCR. Wild-type and RNase III gene deletion mutant strains of L. pneumophila were grown to an OD 600 of 2.5 in BYE medium. Cells were subsequently treated with rifampin (final concentration of 500 g/ml). Aliquots were removed at time zero (just before treatment) or after 5, 10, or 20 min of treatment. Cells were harvested by centrifugation in a tabletop centrifuge at 13,000 rpm for 1 min. Pellets were flash frozen in liquid nitrogen, and subsequently RNA was isolated as described above. Quantitative reverse transcription-PCR (qPCR) was then performed as described previously [START_REF] Brüggemann | Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila[END_REF] at cDNA concentrations ranging from 5 ng to 5 ϫ 10 Ϫ3 ng. Primers used are listed in Table 2. Primer efficiencies were evaluated by generating a standard curve with serial dilutions, which indicated an efficiency of 90% to 110% for all primers used. The specificity of the amplified product and primer dimer formation was verified for each primer set by the presence of a single peak in a disassociation step carried out after each run. The absence of contaminating DNA was verified using control samples for each RNA sample for which no prior reverse transcription reaction had been carried Western blot analysis. Samples were denatured at 90°C for 10 min and separated on a 4 to 20% gradient SDS-PAGE gel (Bio-Rad) and transferred using a Trans-Blot Turbo transfer system (Bio-Rad). The membrane was stained with black amide or red ponceau solutions for loading controls and blocked in 1.2% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBS-Tween) for 1 h at room temperature. Membranes were incubated overnight at 4°C with anti-Hfq or anti-FlaA primary antibodies that we generated. Briefly, Hfq and FlaA 6ϫHis protein production was induced at an OD 600 of 0.5 by 0.4 mM IPTG at 37°C for 4 h. Hfq and FlaA-6x-His proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose beads and a Poly-Prep chromatography column. The resulting proteins were injected into rabbits, and crude sera were recovered 90 days later (Thermo Fisher Custom Antibody Services). Specific immunoglobulins were purified from serum samples by using a 1.0-ml HiTrap affinity NHS column (GE Healthcare) according to the manufacturer's instructions. The antibody specificity and purity were assessed by Western blotting against the purified proteins. Membranes were incubated overnight at 4°C with Hfq or FlaA primary antibodies (diluted 1:2,000). The membranes were washed three times for 5 min each time in TBS-0.5% Tween at room temperature. The membrane was incubated for 1 h at room temperature with the secondary antibody, horseradish peroxidase (HRP)-labeled anti-rabbit (Dako) in TBS-0.5% Tween before the membrane was washed as described above. Signals were visualized using the ECL2 prime Western blot detection kit (Pierce) and the G-Box imaging system (Syngene).

Rapid amplification of the 3= end of cDNA (3= RACE). Amplification of the 3=-end region of anti-hfq was performed using total RNA purified from wt L. pneumophila Paris strain in the early exponential growth phase (OD 600 of 2) as described above. Total RNA was treated with DNase I (Roche), incubated at 37°C with 10 U tobacco acid pyrophosphatase (TAP) (Epicentre) as previously described ( 28), subjected to phenol-chloroform-isoamyl alcohol (IAA) extraction (25:24:1), and precipitated overnight at Ϫ20°C with 10% 3 M sodium acetate (pH 5.2), 2% glycogen (20 mg/ml), and 2.5 volume of ethanol. For the 3= adapter ligation, a mix of 3= RNA adapters P-UCGUAUGCCGUCUUCUGCUUG-UidT (100 M) was ligated to the processed RNA using the T4 RNA ligase (Epicentre) according to the manufacturer's instructions. cDNA was then synthesized as described above, and amplification (primers anti-hfq_3'RACE _F and anti-hfq_3'RACE_R) products were fractionated in a 2% agarose gel. After staining with ethidium bromide, the sole band obtained of about 100 nucleotides (nt) was cut from the gel and purified using the NucleoSpin Extract kit (Macherey-Nagel). The purified size-selected cDNA fragment was cloned into the pGEM-T Easy (Promega) plasmid, and the cloned fragment was sequenced.

RNA in vitro transcription and labeling. Anti-hfq (103-nt), hfq mRNA (335-nt), lpp0644 (137-nt), and hfqOUT (180-nt) genes used for electrophoretic mobility shift assay (EMSA) gel mobility assays and hfq mRNA (128 nt) used for Northern blot analyses were amplified from bacterial DNA with primers containing the T7 promoter at the 5= end (Table 2). The resulting fragments were used as the templates to produce in vitro RNA (MEGAscript T7 kit; Ambion) and radioactively labeled with [␣-33 P]UTP (PerkinElmer). The reaction mixture was incubated at 37°C for 30 min, and RNA was digested with Turbo DNase digestion (1 U, 15 min at 37°C) and purified using the Illustra Micro-Spin G-25 columns (GE Healthcare) according to the supplier's protocol.

EMSA gel mobility assay. RNA-RNA binding assays were performed to assess the binding affinity of the Anti-hfq transcript and hfq mRNA, hfqOUT (spanning the nucleotide sequence 78 to 255 and missing the 5= UTR and the first 26 codons of the hfq transcript), and lpp0644 as a control. Briefly, 25 nM anti-hfq, together with 0, 10, 15, 30, or 50 nM hfq full transcript or 0, 15, 30, or 50 nM hfqOUT probes was incubated with buffer containing 10 mM Tris-Cl (pH 8.3) and 0.1 mM EDTA, denatured at 70°C for 5 min, and cooled down for 15 min at room temperature. In vitro formation of complexes between Hfq and hfq mRNA or Anti-hfq sRNA (25 nM) in vitro was analyzed by EMSA using 0.05, 0.08, 0.1, 0.16, 0.22, 0.27, 0.5, and 1 M His-tagged Hfq (Hfq6XHis) and supplemented with 5ϫ structure buffer .0], 250 mM NaCl, 250 mM KCl, 200 ng/ml tRNA) and incubated at 37°C for 15 min.

For the formation of ternary complexes, 25 nM radioactively labeled Anti-hfq and 25 nM cold hfq mRNA probes were incubated alone or with 1 M His-tagged Hfq (Hfq6XHis) protein for 0.5, 1.5, or 2.5 min and supplemented with 5ϫ structure buffer .0], 250 mM NaCl, 250 mM KCl, 200 ng/ml tRNA). For a control, 25 nM radioactively labeled Anti-hfq or hfq mRNA probes were incubated with 0.5 and 1 M (Hfq6XHis) for 10 min at room temperature. Prior to loading, reactions were mixed with native loading buffer, and samples were loaded onto 6 or 8% polyacrylamide 1ϫ Tris-acetate-EDTA gel in 1ϫ Tris-acetate EDTA running buffer. Following electrophoresis at 4°C, the gels were wrapped in Saran Wrap and subsequently exposed to films (GE Healthcare).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/

CHAPTER FOUR

! 85

Supplementary results

LetA might regulate antihfq expression

As LetA influences positively the expression of the hfq transcript, we sought to determine whether LetA directly regulates the expression of these two oppositely transcribed genes.

Interestingly, a search for LetA binding sites in the hfq and anti-hfq sequences revealed the presence of a putative binding site within the anti-hfq sequence, very similar to the consensusbinding site deduced from the known LetA binding sites in the sRNAs RsmY, RsmZ and RsmX (Figure 12). Differently to the reported mechanism of action of this protein, we hypothesized that LetA binding to this sequence might lead to the repression of the expression of anti-hfq upon entry into the stationary phase, enabling thus the expression of the complementary target hfq. In order to investigate a direct interaction between LetA and the anti-hfq gene in vitro, I performed an electrophoretic mobility shift assays (EMSA). Incubation of a radioactive antihfq probe with increasing concentrations of the purified recombinant LetA protein resulted in a shift of a higher molecular weight, suggesting a direct interaction with the anti-hfq target.

However, under our conditions, the LetA protein displayed the same pattern of slower migrating complexes also for a negative control, suggesting an unspecific binding (Figure 13).

. In vitro binding of a LetA and anti-hfq DNA probe. EMSA assays using 50nM radioactively labeled anti-hfq with the indicated increasing molar amounts of LetA protein showed that LetA indeed binds anti-hfq, however this may be an unspecific binding as the control was also binding. The duplex complexes between LetA and the competitors lpp0533, the cold anti-hfq a derivate anti-hfq probe in which highly conserved nucleotides in the putative LetA binding sites were mutated, were pre-incubated 20 minutes at room temperature. A binding reaction was carried out for 15 min at room temperature, and samples were then loaded onto 6% polyacrylamide 0.5X Tris-acetate-EDTA gel in 0.5X Tris-acetate-EDTA running buffer. Symbols: (L) LetA6X His, (A) radioactively labeled anti-hfq DNA probe. Asterisks, radioactively labeled DNA probes, + cold RNA probes; -no RNA probes.

To further investigate the relationship between LetA and Anti-hfq, a controlled system in which the antihfq gene, including the putative LetA binding site, was cloned in frame with the blaM gene into the pXDC61 plasmid, has been constructed (Figure 14A). We aimed to evaluate the levels of Anti-hfq expression at the exponential and post-exponential growth phases. Betalactamase activity analyses revealed a down regulation of Anti-hfq after the entry of the bacteria in the post-exponential phase, confirming our hypothesis (Figure 14B).

Moreover the expression of the betalactamase, related to the Anti-hfq transcript level, was not affected in the ΔletA deletion mutant during bacterial growth, indicating that the lack of the DNA-binding protein LetA led to the accumulation of Anti-hfq even in the transmissive phase (Figure 14B). Although, this approach demonstrated that LetA has a negative influence on the anti-hfq gene in the post-exponential phase, I had problems with reproducibility and thus we could unfortunately not confirm our hypothesis. We then set up an in vivo approach using LetA Chromatin immunoprecipitation (ChIP) coupled to antihfq detection by quantitative real-time PCR. To generate a strain in which the LetA protein was overexpressed, the L. pneumophila wt strain was transformed with a plasmid in which the letA gene was under the control of a strong promoter and cloned in frame with 2 FLAG tags. Moreover, a strain in which the 2 FLAG tags were missing was used as negative control. Quantitative RT-PCR of the LetA-immunoprecipitated fractions revealed anti-hfq as direct target of the DNA-binding protein LetA. However, like for the other approaches used, several negative controls were also found among the LetA targets, and thus we could not confirm our hypothesis (Figure 15, data showing only one negative and one positive control). Taken together, the three different approaches used did suggest that LetA

RESULTS

CHAPTER FOUR

! 93 indeed regulates anti-hfq expression, but did not firmly establish that a direct interaction between LetA and anti-hfq occurs during the post-exponential phase. Thus it is also possible that the influence of LetA is indirect.

CONCLUSIONS AND PERSPECTIVES

! Legionella pneumophila persists in the environment in diverse and multiple forms, such as the viable-but-non-cultivable state, the biofilm-associated sessile state or the free-living planktonic state. However, the primary site for L. pneumophila replication is within environmental protozoa [START_REF] Fields | Legionella and Legionnaires' disease: 25 years of investigation[END_REF]. Exposure to contaminated aerosols generated by man-made water systems such as cooling towers, air-conditioning systems, humidifiers or even showers may lead to human infection, culminating eventually in Legionnaires' disease, mainly in susceptible individuals [START_REF] Fields | Legionella and Legionnaires' disease: 25 years of investigation[END_REF][START_REF] Yiallouros | First outbreak of nosocomial Legionella infection in term neonates caused by a cold mist ultrasonic humidifier[END_REF]. In order to survive and replicate in the environment, this intracellular bacterium must adapt to various stresses, including hostile intracellular as well as nutrient-limited extracellular habitats.

Adaptation to these changing conditions is achieved by a tight regulation of its life cycle in which L. pneumophila alternates between a replicative phase in the host and a transmissive phase, when nutrient depletion triggers the escape of the bacterium from the spent host (Molofsky & Swanson, 2004). Thus, L. pneumophila represents a paradigm of highly adapted intracellular pathogens that have evolved sophisticated mechanism to evade the host cell defense but also to persist in the environment [START_REF] Berjeaud | Legionella pneumophila: The Paradox of a Highly Sensitive Opportunistic Waterborne Pathogen Able to Persist in the Environment[END_REF]. The bacterium interferes with the host signaling pathways by employing a large set of effector proteins, many of which contain eukaryotic-like domains that are translocated to the host cell cytoplasm by a dedicated type-IVB (T4BSS) secretion system [START_REF] So | Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors[END_REF]. These effectors influence trafficking in the host cell, the signal transduction or the transcriptional regulation and ensure the establishment of the protective and replicative niche for the bacteria within the host cell (Heidtman et al., 2009;Hubber & Roy, 2010;[START_REF] Pearson | Post-modern pathogens: surprising activities of translocated effectors from E. coli and Legionella[END_REF][START_REF] Simon | Subversion of Cell-Autonomous Immunity and Cell Migration by Legionella pneumophila Effectors[END_REF]. Each infection stage requires the function of defined and specific sets of effectors and virulence traits to be produced, thus their expression needs also to be spatially and temporally controlled.

Indeed, transcriptome analysis of different L. pneumophila life cycle stages showed that in each phase L. pneumophila expresses a particular set of genes dependent on the environmental cues perceived (Brüggemann et al., 2006). To allow such a sophisticated regulation of its genes, L. pneumophila has evolved a complex and elaborated regulatory network, which for both sRNAs and their mRNA targets and the functional mechanisms of sRNA-mediated gene regulation [START_REF] Feliciano | Hfq: a multifaceted RNA chaperone involved in virulence[END_REF][START_REF] Sauer | Structure and RNA-binding properties of the bacterial LSm protein Hfq[END_REF]Sobrero & Valverde, 2012;Updegrove et al., 2016;Vogel & Ben F Luisi, 2011a). Global transcriptome and proteomic data of several pathogenic bacteria suggest a global impact of Hfq on the bacterial physiology as they revealed that Hfq directly or indirectly regulates for example 20% of all Salmonella genes, 18% of all K. pneumoniae genes or 6% of all Francisella genes [START_REF] Chiang | Impact of Hfq on global gene expression and virulence in Klebsiella pneumoniae[END_REF]Meibom et al., 2009;Sittka et al., 2008). Thus, it is not surprising that hfq mutations are associated with broadly pleiotropic phenotypes, with a remarkable impact on bacterial fitness, stress response and virulence (Sobrero & Valverde, 2012;Vogel & Ben F Luisi, 2011a). Moreover, the Hfq protein has been used as bait in many high-throughput RNA sequencing studies to pull down and identify new classes of regulatory Hfq-dependent sRNAs, so far the biggest class of sRNAs identified in prokaryotes. Although Hfq is a key player in the riboregulation network of several bacteria, a generalized model of which are Hfq targets cannot be established as most of our current knowledge on the role of Hfq is based on studies performed in E. coli and Salmonella (Sittka et al., 2008;Tsui et al., 1994). Thus only the in depth analysis of Hfq in a greater number of bacteria will allow to learn whether there are common regulatory pathways that are under the control of Hfq or whether these differ in each species.

In this study we showed that the hfq deletion mutant does not affect L. pneumophila growth in liquid medium as judged by the growth curve profiles comparing the wild type and the deletion mutant strains. When characterized in cell culture assays using THP-1 human monocyte-derived macrophages and A. castellanii cells Hfq was required for efficient replication within A. castellanii at 20° C, however no effect was observed when both THP-1

CHAPTER FIVE

! and amoebae were infected at 37° C. This observation suggests that Hfq is an important virulence factor in the environment, as temperatures in environmental waters rarely are much higher than 20°C. Thus, we suggest that Hfq regulates a defined set of transmissive traits, which this bacterium engages for an efficient replication in its natural host. Furthermore, we have shown that Hfq directly or indirectly influences bacterial motility, as judged by the loss of FlaA in the hfq deletion mutant compared to the wild-type in the post-exponential phase.

This observation is in agreement with what is known from other pathogens that engage Hfq to regulate motility during host cell colonization [START_REF] Kendall | Hfq virulence regulation in enterohemorrhagic Escherichia coli O157:H7 strain 86-24[END_REF][START_REF] Sonnleitner | Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1[END_REF][START_REF] Wilf | The RNA chaperone, Hfq, controls two luxR-type regulators and plays a key role in pathogenesis and production of antibiotics in Serratia sp. ATCC 39006[END_REF]. Thus, our data show, that the RNA-binding protein Hfq takes part in the regulatory network governing L. pneumophila virulence.

In agreement with a role of Hfq in virulence regulation, we showed that Hfq is regulated in a growth-phase dependent manner, with high expression of Hfq in post exponential/transmissve growth phase but low expression in exponential/replicative growth phase. Also our transcriptional analyses of the L. pneumophila genome showed that Hfq expression is high upon entry into the infectious form of L. pneumophila. This finding is different to what has been reported previously for Hfq of another L. pneumophila strain (McNealy et al., 2005).

Their model proposed a growth-phase dependent regulation of Hfq, in which during the exponential phase RpoS controls hfq expression to allow the bacteria to adapt and efficiently use the replicative nutrients. Upon entry into the transmissive phase, induced by nutrient depletion, LetA switches off the hfq transcription through an RpoS-independent pathway, enabling transmissive traits to be activated (McNealy et al., 2005).

The differences with this study may be due to the different strain used by the two groups, or may also lie in an erroneous interpretation of the results by McNealy and colleagues. Hales and Shumann [START_REF] Hales | The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii[END_REF] showed that RpoS is very poorly expressed in exponential growth but highly expressed in stationary phase cells, suggesting that it exerts its major regulatory role during stationary phase what is in agreement with our results but in contrast to the model proposed by McNealy where RpoS exerts its regulatory role in exponential phase. Furthermore, the clear impact on intracellular replication in A. castellanii also suggests that Hfq has a role in post-exponential phase and not in replicative/exponential growth. Thus the predominant production of Hfq upon onset of the late post-exponential phase as revealed in our study goes in hand with a role of this RNA-binding protein in the regulatory circuit that controls the switch from the replicative into the transmissive phase. We

CONCLUSIONS AND PERSPECTIVES! !

! also show that the expression of Hfq is dependent on RpoS and LetA, but in contrast to

McNealy and colleagues we found that RpoS regulates Hfq expression in post-exponential phase when RpoS is expressed the highest and in agreement with the role of Hfq in the regulation of the transmissive phase.

Our analysis of the L. pneumophila operon map lead also to the identification of a putative sRNA, later named Anti-hfq that is transcribed in the antisense strand of the hfq gene and overlaps its 5'UTR region. These observations supported a model in which the sRNA Antihfq may negatively regulate the hfq transcript, thus acting as a cis-encoded sRNA. Indeed, we show here that this sRNA, named Anti-hfq, is expressed and is 101 bp long as judged by amplification by 3' RACE PCR and sequencing. The Anti-hfq transcript is highly abundant in the early exponential phase and its expression level strongly decreases upon entry into the post-exponential phase, showing a reverse expression profile compared to the one of the hfq transcript. When Anti-hfq was overexpressed in L. pneumophila the expression of Hfq was downregulated whereas its knock-down resulted in the constitutive expression of Hfq throughout the L. pneumophila life cycle, suggesting a direct mechanism of regulation.

Indeed, the in-vitro transcribed Anti-hfq is able to bind the hfq transcript through complementary base pairing, suggesting that Anti-hfq acts as a cis-encoded sRNA and directly regulates hfq transcription. This binding imposes a negative regulation on the mRNA translation due to the fact that the base-pairing region is in the proximity of the ribosomebinding site. The regulation of Hfq by an antisense RNA is an unusual feature, however a similar mechanism seems to exist in Burkholderia cenocepacia. This bacterium encodes two distinct hfq genes. The hfq gene encodes a 79-amino acid protein and the hfq2 gene encodes an unusual Hfq-like protein of 188 amino acids [START_REF] Sousa | The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans[END_REF]. The analysis of the two hfq transcripts showed that these two mRNA molecules are differently transcribed dependent on the growth phase. The mRNA levels corresponding to hfq are maximal at the early exponential phase of growth and decrease upon entry into stationary phase, whereas hfq2 transcripts are strongly expressed in cells in the stationary phase [START_REF] Ramos | The second RNA chaperone, Hfq2, is also required for survival under stress and full virulence of Burkholderia cenocepacia J2315[END_REF]. This strict pattern of expression requires a tightly controlled regulation in the bacterial cell. Indeed, further studies proposed that the Hfq protein was negatively regulated by a sRNA named MtvR. This regulation is mediated by the binding of MtvR to the RBS of the hfq 5'UTR by an antisense mechanism, leading to the inhibition of the Hfq translation. Moreover, the binding of the MtvR on the 5'UTR of hfq is likely to be mediated through the help of the RNA chaperone Hfq2 (Ramos, Grilo, da Costa, Feliciano, & [START_REF] Ramos | MtvR Is a Global Small Noncoding Regulatory RNA in Burkholderia cenocepacia[END_REF]. Furthermore, a sRNA, named h2cR present in the antisense strand of the hfq2 gene and overlapping its 5' UTR region negatively regulates hfq2 mRNA and a crossregulatory loop has been suggested as the Hfq protein seems to stabilize the hfq2 mRNA [START_REF] Ramos | The Novel Cis-Encoded Small RNA h2cR Is a Negative Regulator of hfq2 in Burkholderia cenocepacia[END_REF]. However, as recently, three of the abovedescribed articles have been retracted, we do not know what of these results is correct.

CHAPTER FIVE

!

However, the mechanism described resembles the hfq regulatory mechanism we found in L.

pneumophila.

In our proposed model, during the exponential phase the abundantly transcribed sRNA ensures that the Hfq protein is not unnecessarily expressed. Indeed, we show that the RNase III is involved in the regulation of the hfq transcripts in exponential growth phase probably by a double-strand cleavage mechanism. Thus, as described in other bacterial species, also in L. pneumophila the sRNA-mRNA binding and the subsequent translational blockage results in the degradation of the RNA molecules by Hfq-dependent recruitment of RNase III (Aiba, 2007;[START_REF] Caron | Small RNA-mediated regulation at the level of transcript stability[END_REF]. Thus, the L. pneumophila Hfq protein might facilitate the binding of its own mRNA and anti-hfq, inhibiting its own translation. Such an Hfq auto regulatory loop has been described in E. coli, in which the binding of either Hfq or ribosomes to the hfq mRNA determines the fate of the hfq transcript [START_REF] Vecerek | The C-terminal domain of Escherichia coli Hfq is required for regulation[END_REF]. Indeed, in E. coli two binding sites in the 5'UTR, one of which overlaps the RBS of the hfq mRNA, act synergistically to bind Hfq and thereby repress its translation. This regulation renders the untranslated hfq mRNA a target for RNase E cleavage [START_REF] Vecerek | The C-terminal domain of Escherichia coli Hfq is required for regulation[END_REF]. In L.

pneumophila, the presence of a (ARN) n motif in the hfq 5'UTR and of an uridine-rich sequence in the 3' end of Anti-hfq that may represent putative Hfq-binding sites, suggested that also in L. pneumophila a Hfq auto regulatory mechanism may exist. Indeed, Hfq interacts in vitro with Anti-hfq and/or hfq mRNA transcripts as confirmed by EMSA assays. Thus Hfq does not only bind Anti-hfq RNA or its own mRNA but also forms a ternary complex with both RNA molecules. Since the sRNA-mRNA duplex appears to be formed in vitro also in absence of the protein, Hfq is not necessary but might contribute to the accelerated interaction with these RNAs.

Although the Hfq chaperone is known to mainly facilitate trans-encoded sRNA-mediated riboregulation, few examples of cis-encoded sRNA acting in trans have already been described. For example, the cis-encoded sRNA GadY of E. coli was reported to base pair with the 3' end of its target mRNA gadX and also to efficiently bind Hfq. Whether Hfq is required

CONCLUSIONS AND PERSPECTIVES! !

! 100

for the GadY and gadX mRNA interaction is not known, however the GadY RNA levels are strongly reduced in the hfq mutant, suggesting that this sRNA can act in trans [START_REF] Opdyke | GadY, a small-RNA regulator of acid response genes in Escherichia coli[END_REF]. Furthermore, the cis-encoded sRNA named ArrS targets and inhibits the gadE T3 mRNA, which is involved in acid resistance in E. coli [START_REF] Aiso | Overexpression of an antisense RNA, ArrS, increases the acid resistance of Escherichia coli[END_REF]. ArrS and GadX and their partners were later found to be enriched in an Hfqimmunoprecipitated pool of RNAs, confirming that these cis-encoded RNAs act as trans-sRNAs [START_REF] Melamed | Global Mapping of Small RNA-Target Interactions in Bacteria[END_REF]. In addition, a model in which Hfq acts as "passive" platform for the sRNA and mRNA duplex formation has been described. In E. coli, Hfq forms a ternary complex with the sRNA DsrA and its mRNA target rpoS. This ternary complex is likely to be an intermediate state during inter-molecular annealing and it needs to be disrupted in order to allow the base pairing. This mechanism would explain the nature of an unstable ternary and intermediate complex, which still is necessary for the RNA duplex binding (W. [START_REF] Wang | Hfq-bridged ternary complex is important for translation activation of rpoS by DsrA[END_REF]. Thus we can speculate that L. pneumophila engages Hfq for the efficient binding between hfq and Anti-hfq, allowing a temporal and spatial hfq regulation. Taken together, we have elucidating the tightly controlled regulation of the Hfq gene and protein expression during the bacterial life cycle and its role in the bacterial physiology. Thus the present work provides a novel and global comprehension of the regulatory mechanisms associated to virulence and intracellular adaptation of L. pneumophila.

To substantiate the proposed in vitro model a global analysis of the protein and RNA molecules bound by L. pneumophila Hfq will be performed in the future using RNA deep sequencing approaches. This analysis would not only confirm that the hfq mRNA and the Anti-hfq sRNA are direct targets of Hfq, supporting our Hfq auto regulatory model tested in vitro but it would also lead to the identification of all Hfq-dependent targets in L. pneumophila. Furthermore, as over 700 sRNAs are predicted to be present in L. pneumophila according to RNAseq analyses and transcriptional start site mapping (Sahr et al., 2012) we expect to identify many of these as bound by Hfq. Indeed, in analogy to the iPAR-CLIP or CLASH methodologies that were applied to detect protein-dependent miRNAtargets in eukaryotes, several RNA sequencing approaches have been established also in prokaryotes and provided a detailed and global knowledge about Hfq-binding sRNAs. For example the recently described RIL-seq approach (RNA interaction by ligand and sequencing) applied to E. coli led to the identification of Hfq-bound pairs of small RNAs showing sequence complementarity, revealing new insights into the sRNA-target interactions [START_REF] Melamed | Global Mapping of Small RNA-Target Interactions in Bacteria[END_REF]. This approach resulted also in the expansion and re-wiring of the

CHAPTER FIVE

! sRNA-target networks, as additional targets of established sRNAs as well as of new Hfqdependent sRNAs and their targets were identified. Moreover, RIL-seq contributed to a better understanding of the structure and dynamics of Hfq-bound sRNA interactions [START_REF] Melamed | Global Mapping of Small RNA-Target Interactions in Bacteria[END_REF]. Thus applying this methodology to L. pneumophila at different stages of its life cycle should allow characterizing Hfq-mediated sRNA-mRNA interactions. Further analyses would uncover the roles of these RNA molecules in the physiology of L. pneumophila, including virulence.

Most interestingly, based on the bulk of experimental data collected on the functional characterization and properties of the RNA chaperone Hfq of several bacterial species, this protein has also been used as an innovative tool for practical applications. Indeed, comparable to the silencing strategy by small-interfering RNAs in eukaryotes, artificial sRNAs (atsRNAs)

guided by the Hfq protein will lead to a stringent regulation of base pairing mRNA targets. As certain sRNAs directly mediate gene expression of multiple mRNA targets, the abovementioned silencing strategy might represent a double-edged sword, which on one hand may globally switch off crucial processes but on the other hand might results in undesired altered effects (Nielsen et al., 2010;Papenfort & Vogel, 2010;Sobrero & Valverde, 2012). A successful example of using atsRNAs has been addressed in E. coli, in which a series of artificial sRNAs were used for targeting and silencing essential genes in an Hfq and RNase Edependent manner, demonstrating that atsRNA was an powerful approach for specific gene silencing and thereby deciphering gene functions in prokaryotes [START_REF] Man | Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria[END_REF].

! !

Réseaux de régulation impliqués dans la virulence de Legionella pneumophila : le rôle de Hfq et du petit ARN non -codant Anti-hfq RÉSUMÉ Legionella pneumophila, responsable de la maladie du légionnaire, est une bactérie aquatique parasitant les amibes, mais aussi les macrophages alvéolaires humains. Legionella alterne entre une forme infectieuse non réplicative et une forme réplicative intracellulaire, qui n'exprime pas les facteurs de virulence. Ce cycle de vie biphasique est gouverné par un système de régulation complexe permettant son adaptation dans différents hôtes. Le but de mon projet de thèse était d'étudier un des facteurs clés dans la régulation des ARNm, le régulateur post-transcriptionnel global Hfq. L'expression de Hfq est régulée au cours du cycle infectieux chez L. pneumophila: Hfq est peu exprimée en phase réplicative, mais fortement exprimée lors de la phase de transmission, ce qui suggère un rôle dans la transition entre ces deux phases. J'ai identifié un petit ARN (sRNA) que j'ai nommé Anti-hfq puisqu'il est transcrit dans l'orientation antisens à Hfq et chevauche sa région 5' non traduite (UTR). Mes recherches ont mis en évidence un mécanisme sophistiqué par lequel Anti-hfq régule l'expression de Hfq: Anti-hfq interagit avec l'ARNm du gène hfq par sa région complémentaire et ainsi contrôle la stabilité de la protéine. De plus, j'ai montré que la protéine Hfq auto-réprime sa propre traduction en facilitant l'interaction entre Anti-hfq et son propre ARNm. Finalement, Hfq régule son propre renouvellement par le recrutement de la RNase III. De plus, des tests de réplication intracellulaire ont montré que Hfq et Anti-hfq sont nécessaires pour la multiplication intracellulaire de L. pneumophila, ce qui a mis en évidence un rôle important de Hfq et Anti-hfq dans la virulence de cette bactérie.

!

Mots clés: Legionella pneumophila; cycle biphasique; Hfq; virulence; Anti-hfq; régulation génique.

Regulatory circuits involved in Legionella pneumophila virulence : the role of Hfq and the cis-

encoded sRNA Anti-hfq SUMMARY Legionella pneumophila, the causative agent of the pneumonia-like Legionnaires' disease, is commonly found in aquatic habitats worldwide where it multiplies within protozoa. To adapt between intra-and extracellular environments, L. pneumophila evolved a biphasic lifecycle wherein it alternates between an infectious and non-replicative form and an intracellular form, which does not express the virulent phenotypes. This biphasic life cycle is governed by a complex regulatory network that comprises transcriptional and post-transcriptional regulatory elements, enabling the bacteria to adapt in diverse hosts. During my Ph.D., I focused my attention of the post-transcriptional regulator Hfq, a hexameric, RNA-binding protein and chaperon of small RNAs (sRNA). The expression of this fascinated protein is life cycle regulated: poorly expressed during the replicative phase of growth, whereas significantly upregulated upon entry into the transmissive phase of growth. Moreover, my research research lead to the identification of a sRNA transcribed antisense to the hfq gene overlapping its 5'UTR region. This antisense RNA, named Anti-hfq, was found to regulate hfq expression by basepairing complementarity, describing a sophisticated mechanism of regulation. In addition, the Hfq protein controls its own translation by facilitating the interaction between Anti-hfq and its own mRNA. Thus, Hfq regulates its turnover, recruiting the endoribonuclease RNaseIII. Furthermore, infection assays revealed that Hfq and Anti-hfq are necessary for efficient replication of L. pneumophila in amoeba revealing an important role of both in bacterial virulence. Key words: Legionella pneumophila; biphasic life cycle; Hfq; virulence; Anti-hfq; gene regulation.

FIGURE 1 .

 1 L. PNEUMOPHILA GROWTH ON AGAR AND MICROSCOPIC VIEW.. ...................................

FIGURE 2 .

 2 FIGURE 2. FROM THE ENVIRONMENT TO HUMANS.. ......................................................................

FIGURE 3 .

 3 FIGURE 3. EVOLUTION OF THE LEGIONNAIRES' DISEASE INCIDENCE RATE.. .................................

FIGURE 4 .

 4 FIGURE 4. SCHEMATIC REPRESENTATION OF THE DIFFERENT STEPS OF THE INTRACELLULAR LIFE CYCLE OF L. PNEUMOPHILA IN PHAGOCYTIC CELLS.. .......................................................... FIGURE 5. INTRACELLULAR MULTIPLICATION OF L. PNEUMOPHILA WITHIN MACROPHAGES. ..... FIGURE 6. THE DOT/ICM TRANSLOCATION SYSTEM OF L. PNEUMOPHILA. ..................................

FIGURE 7 .

 7 FIGURE 7. SCHEMATIC REPRESENTATION OF THE SM FOLD AND THE HEXAMERIC STRUCTURE OF THE HFQ PROTEIN. . ...........................................................................................................

FIGURE 8 .

 8 FIGURE 8. STRUCTURE OF THE HFQ HEXAMERS SHOWING ITS MAIN SURFACES.. ........................ FIGURE 9. THE L. PNEUMOPHILA LIFE CYCLE. ............................................................................ FIGURE 10. LIFE CYCLE OF L. PNEUMOPHILA IN BROTH CULTURE.. ............................................

FIGURE 11 .

 11 FIGURE 11. SCHEMATIC REPRESENTAION OF THE REGULATORY NETWORK CONTROLLING THE L. PNEUMOPHILA LIFE CYCLE.. ...............................................................................................

FIGURE 12 .

 12 FIGURE 12. SCHEMATIC REPRESENTATION OF THE L. PNEUMOPHILA HFQ AND ANTI-HFQ LOCUS.

FIGURE 13 .

 13 FIGURE 13. CONSENSUS LETA BINDING SITE AND PUTATIVE LETA BINDING SITE OF ANTI-HFQ..

FIGURE 14 .

 14 FIGURE 14. IN VITRO BINDING OF A LETA AND ANTI-HFQ DNA PROBE.. .................................... FIGURE 15. ACTIVITY ASSAY TO MEASURE THE LEVEL OF ANTI-HFQ EXPRESSION ..................... FIGURE 16. CHIP EXPERIMENT IN LETA2X FLAG CLONE USING ANTIBODIES AGAINST IGG AND FLAG. ...............................................................................................................................

Figure 1 .

 1 Figure 1. L. pneumophila growth on agar and microscopic view. Left panel L. pneumophila growing on BCYE agar, right panel, electron microscopy image of L. pneumophila in liquid.

Figure 3 .

 3 Figure 3. Evolution of the Legionnaires' disease incidence rate. Scheme representing the number of cases (left axis) and incidence rate (right axis) in France according to the mandatory reporting data from 1988 to 2015 (http://invs.santepubliquefrance.fr/Dossiers-thematiques/Maladiesinfectieuses/Infections-respiratoires/Legionellose/Donnees-de-surveillance).

Figure 4 .

 4 Figure 4. Schematic representation of the different steps of the intracellular life cycle of L. pneumophila in phagocytic cells.After uptake in protozoa or macrophages, the bacteria persist within the so-called Legionella-containing vacuole (LCV), which subsequently evades the lysosomal network. Within minutes of uptake, numerous vesicles derived from the mitochondria and the endoplasmic reticulum (ER) are docked onto the LCV membrane. Within this ER-like vacuole, the bacteria replicate and at the end of the replication cycle flagellated bacteria are released. Adapted from[START_REF] Isberg | The Legionella pneumophila replication vacuole: making a cosy niche inside host cells[END_REF].

Figure 5 .

 5 Figure 5. Intracellular multiplication of L. pneumophila within macrophages. Confocal images (60X objective) of human monocyte-derived macrophages (hMDMs) infected with L. pneumophila strain Philadelphia JR32 expressing constitutively GFP, at 2h (left) and 20h (right) post-infection. Cyan: nucleus (DAPI), Red: cytoplasm, Green: L. pneumophila, Bar: 10 μm (Pictures kindly provided by P. Escoll, Institut Pasteur).

Figure 6 .

 6 Figure 6. The Dot/Icm translocation system of L. pneumophila. Proposed location and interaction of the various Dot/Icm components in the L. pneumophila membrane, based on a protein stability study[START_REF] Buscher | The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02[END_REF]. Individual letters represent Dot protein names, whereas letters preceded by an "I" indicate Icm protein names. Adapted from[START_REF] Isberg | The Legionella pneumophila replication vacuole: making a cosy niche inside host cells[END_REF].

Figure 1 .

 1 Figure 1. Simplified model of action of trans-encoded and cis-encoded RNAs. (A1) trans-encoded sRNAs interact with their target RNA through imperfect base-pairing and hence promote RNase degradation of the double-stranded RNA molecules. Alternatively the sRNAs might affect the target translation positively (A2) and negatively (A3) by releasing or masking the ribosome-binding site, respectively. Conversely the cis-encoded sRNAs bind through full sequence complementarity the mRNA target, affecting translation, and end in the degradation of the sRNA-target RNA complex (B1). A small cis-encoded RNA, antisense between two genes, can lead to mRNA cleavage (B2) or the transcriptional termination through a putative loop formation and consequently the cessation of the RNA polymerase activity (B3).

Figure 2 .

 2 Figure 2. Model of CRISR/Cas system function. (A) Basic structure of the CRISPR/Cas system: several Cas proteins are encoded by the cas genes, located typically in the close proximity of the CRISPR arrays, composed of a variable number of DNA repeats interrupted by unique spacer regions which originate from foreign DNA acquisition. (B) The Cas proteins are in charge of the crRNAs processing and interference with the cDNA region, promoting DNA degradation or silencing (C).

Figure 3 .

 3 Figure 3. Schematic representation of the mechanisms of action employed by the RNA-binding protein, Hfq. (A) Inhibition at the translational level of the target mRNA by interaction of the sRNA-Hfq complex with the ribosome-binding site (RBS). (B) The complex sRNA-Hfq-mRNA favours the target translation by setting free the RBS, which alternatively would be locked by the formation of a secondary structure. (C) The RNA-binding protein Hfq modulates the mRNA and sRNA turnover rate by recruiting RNase E. (D) Hfq protects the sRNA cleavage site against RNase E attack. (E) Hfq facilitates the mRNA target polyadenylation by the poly(A)polymerase and degradation by exoribonucleases.

3 Figure 4 .

 34 Figure 4. Simplified schematics of the general function of the Csr system. (A) The dimeric RNA-binding protein CsrA interacts with one or more A(N)GGA motifs of the target RNA, typically represented in a hairpin loop structure, leading to an alteration of the accessibility of the translation machinery, transcription (anti-)termination and/or the stability of the RNA. (B) Production of regulatory sRNA under the control of a two-component system and the alternative sigma-factor RpoS antagonizes CsrA function by binding CsrA with high affinity and hence abolishing the interaction with their target RNAs. This results in a global metabolic shift within the pathogen and amongst others to the activation of virulence-related proteins.

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Figure 5 .

 5 Figure 5. Examples of RNA-mediated regulation implicated in adaptation to the host environment in intracellular bacteria. (A) The regulatory cascade comprising CsrA and two sRNAs (RsmY and RsmZ) regulates the expression of secreted effectors of L. pneumophila that are released into the host cell cytosol. These effectors manipulate different processes of the secretory pathway to help in establishing the Legionella-containing vacuole (LVC). (B) The trans-encoded regulatory sRNAs RfrA and RfrB of Salmonella enterica serovar Typhi play a central role in iron homeostasis regulation, and are implicated in protecting the bacteria against oxidative stress, bactericidal antibiotics and acid resistance when replicating in the host cell. (C) Listeria monocytogenes virulence genes are under the control or PrfA, the master regulator of virulence gene expression. A thermosensor present in the 5 ′ UTR of prfA senses the variation of the temperature when L. monocytogenes changes from the extracellular environment (lower temperature) to invade a mammalian host cell (37 • C). The increase in the temperature changes its conformation, freeing the ribosome-binding site, which is in turn allows the translation of PrfA and subsequent virulence gene expression necessary for invasion of eukaryotic cells and the rapid escape from the vacuole to the cytosol.

1

 1 General properties and structure of HfqAs mentioned before, Hfq was identified in the late 1960s in E. coli as host factor for the efficient replication of the RNA bacteriophage Qβ (Franze deFernandez, Eoyang, & August, 1968). It was demonstrated that Hfq binds at the cytosine-rich 3' end of the plus-strand of the viral RNA genome, thus allowing the initiation of the minus-strand RNA synthesis by the Qβreplicase[START_REF] Schuppli | Synergism of mutations in bacteriophage Qbeta RNA affecting host factor dependence of Qbeta replicase[END_REF]. Franze de Fernandez and collaborators purified and biochemically characterized a hexameric protein, first named HF I , capable of binding different AU-rich single-stranded RNAs (Franze de Fernandez, Hayward, & August,

Figure 7 .

 7 Figure 7. Schematic representation of the Sm fold and the hexameric structure of the Hfq protein. (A) Fold of the LSm domain of Salmonella typhimurium Hfq. The five β-strands (β1-5) are depicted in green and form an open barrel with the α-helix on top (α1 in red). (B) Hexameric association of the monomeric Hfq showing a typical doughnut-like structure. Intersubunit interactions are shown in green and are provided by backbone interactions between strands β4 and β5 to strands β4* and β5* in the neighbouring monomers, respectively. (C) Sequence alignment of Hfq proteins from diverse bacterial species. The secondary structure of Salmonella typhimurium Hfq (PDB-ID: 2YLB9) is superimposed on the primary sequence. The Sm consensus sequences are shown below the alignment (the nature of the amino acid side-chains is: s = small hydrophobic, I, L, V; h = hydrophilic, S, T; a = aromatic, Y, F). Highly conserved residues are red (> 70% conservation) or white in red boxes (100% conservation). While the Sm1 signature is conserved in all domains of life, Sm2 is divergent in bacteria. The species abbreviations and UniProt-IDs are: γ-Proteobacteria: SAL TY, Salmonella typhimurium (P0A1R0); ECOLI Escherichia coli (P0A6X3); YERPE, Yersinia pestis (A4TRN9); HAEI N, Hemophilus influenza, (P44437); LE GPA Legionella pneumophila (Q5X982). β-Proteobacteria: NEI ME, Neisseiria meningitides (B9VV 05); RAL SO, Ralstonia solanacearum (Q8Y025). α-Proteobacteria: GLU DI, Gluconacetobacter diazotrophicus (Q8RMG6); Acidobacteria: ACI BL, Acidobacteria bacterium (Q1II F9). Spirochaetales: LEPI N, Leptospira interogans (Q8F5Z7). Aquafecales: AQUAE Aquifex aeolicus, (O66512). Thermotogales: THEMA, Thermotoga maritime (Q9WYZ6). Fermicutes: BAC SU, Bacillus subtilis (O31796); STAA M, Staphylococcus aureus (Q99UG9). Adapted from[START_REF] Sauer | Structure and RNA-binding properties of the bacterial LSm protein Hfq[END_REF] .

  , 2011a; Beich- Frandsen, Vecerek, Sjöblom, Bläsi, & Djinovic-Carugo, 2011b;[START_REF] Olsen | C-terminally truncated derivatives of Escherichia coli Hfq are proficient in riboregulation[END_REF][START_REF] Vecerek | The C-terminal domain of Escherichia coli Hfq is required for regulation[END_REF].

  structural element, the C-termial domain, modulates Hfq activity. This domain in E. coli is required for the dissociation of the double stranded RNA product and consequently stimulates the cycling of the sRNA and the duplex RNA on the ring[START_REF] Santiago-Frangos | C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA[END_REF]. Moreover, possible interaction sites could be associated with the flexible C-terminal extensions of Hfq and the groove of the sRNA helix, which would change the view of how sRNAs and Hfq interact(Beich-Frandsen, Vecerek, Konarev, Sjöblom, Kloiber, Hämmerle, et al., 2011a; Ishikawa, Otaka, Maki, Morita, & Aiba, 2012;[START_REF] Salim | Requirement of upstream Hfq-binding (ARN)x elements in glmS and the Hfq C-terminal region for GlmS upregulation by sRNAs GlmZ and GlmY[END_REF] Sauer, Schmidt, & Weichenrieder, 2012).

Figure 8 .

 8 Figure 8. Structure of the Hfq hexamers showing its main surfaces. The Hfq proximal face and distal face with the RNAs (orange) displayed on opposite sides of the hexamer. The proximal face with the exposed amino-terminal -helix includes residues in the Sm2 sequence motif. The single monomers are presented in different colours. Adapted from (Vogel & Ben F Luisi, 2011a).

  the RNA duplex formation[START_REF] Kawamoto | Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq[END_REF][START_REF] Soper | Positive regulation by small RNAs and the role of Hfq[END_REF]. Moreover, due to the fact that Hfq has been shown to bind in-vitro almost any single-stranded, unstructured RNA molecule with high affinity, an intriguing question to address is how Hfq can bind and affect the function of so many RNAs. The debate is still under way; however two main mechanisms how Hfqfacilitated base pairing may work have been established. The first proposed mechanism is a

  . The second proposed effect concerns the metabolic stability of the bound RNA molecules. Indeed, Hfq impacts multiple steps by changing the secondary structure of bound RNAs, placing the RNAs in the proximity, neutralizing the charge of the RNAs or promoting the annealing of the first base pairs(Updegrove, Zhang, & Storz, 2016;[START_REF] Wagner | Cycling of RNAs on Hfq[END_REF]. Moreover, several sRNAs are unstable in the absence of Hfq, due to the lack of protection from degradation by RNase E and other exoribonucleases(Chao, Papenfort, Reinhardt, Sharma, &

  downregulated in stationary phase by the two component response regulator LetA(McNealy, Forsbach-Birk, Shi, & Marre, 2005) In contrast in P. aeruginosa, Hfq is expressed twice as much during stationary phase as compared to earlier growth phases(Sonnleitner, Sorger- CHAPTER TWO ! Domenigg, & Bläsi, 2006). Similarly, in Listeria monocytogenes, hfq transcript levels are strongly upregulated by the alternative stress sigma factor in stationary phase of growth[START_REF] Christiansen | The RNA-binding protein Hfq of Listeria monocytogenes: role in stress tolerance and virulence[END_REF]. The expression of hfq has also been studied in the Gram-positive bacterium Staphylococcus aureus. The work of Liu and colleagues demonstrated that under their conditions, Hfq is a pleiotropic regulator of gene expression that is involved in pigmentation, one of the different facets influencing S.

Figure 9 .

 9 Figure 9. The L. pneumophila life cycle. Free-swimming, motile, transmissive bacteria reside in the extracellular environment before being engulfed by phagocytic cells, in which they replicative within a vacuole. In this phase the bacteria are not virulent and not motile. When the nutrients become limiting, the bacteria stop multiplying and the expression of traits for motility, cytotoxicity, survival in the environment and the transmission into a new host is promoted. After they left the host cell a new phagocyte will be infected and, the cycle starts again.Many aspects of the pathogen's life cycle like the growth phase-dependent regulation of numerous virulence traits are similar to what is seen during infection. Thus the biphasic life cycle of L. pneumophila can be mimicked in synchronous broth cultures[START_REF] Byrne | Expression of Legionella pneumophila virulence traits in response to growth conditions[END_REF]. In detail, at the beginning of the infectious cycle, nutrients are abundant and thus the conditions are favourable for the replication of L. pneumophila comparable to exponential

Figure 10 .

 10 Figure 10. Life cycle of L. pneumophila in broth culture. The exponential phase of L. pneumophila growth is coupled with intracellular multiplication and the induction of replicative traits (red line). The entry into the post-exponential, mostly stationary phase, is correlated with the expression of virulence and transmissive traits.

  coli and S. flexneri to act as cofactor for the (p)ppGpp-dependent transcriptional regulation, activating the transcription of critical virulence regulators and inhibiting some others such as the flagellar regulon[START_REF] Aberg | Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo[END_REF][START_REF] Lemke | DksA and ppGpp directly regulate transcription of the Escherichia coli flagellar cascade[END_REF][START_REF] Nakanishi | ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes[END_REF][START_REF] Sharma | Induction of expression of hfq by DksA is essential for Shigella flexneri virulence[END_REF]. In E.coli, DksA can additionally compensate the lack of the alarmone, indicating that DksA works independently of (p)ppGpp[START_REF] Potrykus | p)ppGpp: still magical?[END_REF]. In vitro evidences have shown that DksA and (p)ppGpp oppositely regulate some process and promoters[START_REF] Aberg | Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo[END_REF] Aberg, Fernández-Vázquez, Cabrer-Panes, Sánchez, & Balsalobre, 2009;[START_REF] Magnusson | Identical, independent, and opposing roles of ppGpp and DksA in Escherichia coli[END_REF][START_REF] Łyzen | Transcription from bacteriophage lambda pR promoter is regulated independently and antagonistically by DksA and ppGpp[END_REF]. Similarly to E. coli it has been shown that in L. pneumophila, the effect of the second messenger (p)ppGpp and DksA depends on the context. In particular, DksA seems to respond to fatty acid stress to promote the bacterial differentiation in a (p)ppGpp-independent manner, as judged by the expression of flagellin and the evasion of the lysosomal degradation by macrophages(Dalebroux, Yagi, Sahr, Buchrieser, & Swanson, 2010b). However, when the (p)ppGpp levels increase, DksA and (p)ppGpp itself coordinately regulate the hierarchical cascade for flagellar expression (see below). Thus L. pneumophila employs both (p)ppGpp and DksA in order to act independently or cooperatively during the bacterial differentiation(Dalebroux, Svensson, Gaynor, & Swanson, 2010a).One of the most important features of L. pneumophila in the late phase of its life cycle is the formation of a flagellum, whose coordinated expression is crucial for efficient and maximal virulence of the bacterium(Molofsky & Swanson, 2004). The flagellar regulon of L. pneumophila is composed of four different classes of genes, regulated in a cascade like manner. First the flagellar master regulator and σ 54 activator protein FleQ together with the alternative sigma factor RpoN (σ 54 ) (class I genes) simultaneously enhance the expression of the class II genes that code for proteins forming the flagellar basal body, hook and regulatory proteins (Albert-Weissenberger et al., 2010). Finaly, the flagellar sigma factor FliA (σ 28 ) REGULATION OF L. PNEUMOPHILA VIRULENCE ! ! (encoded by a class III gene and regulated by DksA) is directly controlling the flagellar class IV genes such as flaA, encoding the flagellin, and fliDS, encoding the filament cap, leading to the complete formation of the flagellum (Albert

  , as demonstrated by the direct activation of the CsrA encoding gene by PmrA. Thus, PmrA activates CsrA and consequently post-transcriptional repression of the CsrA-regulated effectors. As such, it is likely that a regulatory switch between two sets of REGULATION OF L. PNEUMOPHILA VIRULENCE ! ! effectors occurs: one set of effectors, activated by PmrA/B and expressed in the replicative/exponential phase of growth and the second group of effectors which is regulated by LetA/S TCS during the entry into the transmissive/stationary phase of L. pneumophila.

Figure 11 .

 11 Figure 11. Schematic representaion of the regulatory network controlling the L. pneumophila life cycle. During transmissive phase, amino acid starvation triggers the ppGpp synthetase RelA and fatty acid starvation triggers the ppGpp synthetase/hydrolase SpoT to produce the alarmone (p)ppGpp. (p)ppGpp may be sensed by the sensor kinase LetS which then phosphorylates LetA. Phosphorylated LetA binds upstream of the small ncRNAs RsmX, RsmY and RsmZ and activates their transcription.CsrA that is bound near the ribosomal binding site of its mRNA targets inhibits their translation. RsmX, RsmY and RsmZ titrate CsrA away from its targets, enabling translation of the mRNAs, leading to expression of transmissive phenotypes. Hfq also influences the different phenotypes, however its exact role is not known.

Figure 12 .

 12 Figure 12. Schematic representation of the L. pneumophila hfq and anti-hfq locus. (A) Schematic representation of the transcriptional starting site (TSS) of the hfq and anti-hfq gene of L. pneumophila strain Paris. (B) Representation of the deep sequencing data obtained from bacteria cultured at the replicative phase of growth (OD=2; left panel) and transmissive phase of growth (OD=4; right panel), indicating when the two transcripts are expressed. Red colour, transcription of the sRNA anti-hfq during the replicative phase; green colour, expression of the hfq transcript during the transmissive phase.

FIG 1

 1 FIG 1 Legionella Hfq is conserved across the genus and other bacterial species. (A) Schematic organization of the L. pneumophila hfq locus. TSS, transcription start site; aa, amino acids. (B) Alignment of the L. pneumophila Hfq protein sequence with other bacterial Hfq protein sequences reveals high sequence and RNA binding site conservation. (C) Alignment of the L. pneumophila Paris Hfq protein sequence with the Hfq protein sequences from different L. pneumophila strains and other Legionella or Legionella-like species. Amino acids involved in RNA binding are boxed. Conserved amino acid residues (asterisks) and semiconservative substitutions (dots) and conservative substitutions (colons) are indicated. The bars above the sequence alignment indicate the sequence percentage of sequence conservation.

FIG 2

 2 FIG 2 Transcript and protein expression of hfq are growth phase dependently regulated. (A) Northern blot and Western blot analyses of bacterial lysates from wild-type L. pneumophila Paris strain during growth (OD 600 s of 1, 2, 3, and 4) using an hfq probe and anti-Hfq antiserum, respectively. 16S RNA and the stained membrane (Mb) signals are shown as loading controls. (B) Schematic representation of the insertion of the apramycin resistance cassette (apraR) in the ⌬hfq mutant. (C) Detection of Hfq by Western blotting in the wild-type (wt) and ⌬hfq mutant strains grown to an OD 600 of 4. (D) Detection of Hfq by Western blotting in the wild-type, ⌬hfq mutant, and complemented strain ⌬hfq pBChfq (Wt and ⌬hfq carrying the empty plasmid pBC-KS) grown to an OD 600 of 4.

FIG 3 5 mbio

 35 FIG 3 Efficient intracellular replication of L. pneumophila in A. castellanii and THP-1 macrophages is dependent on functional Hfq. (A) THP-1 cells were infected with wt and ⌬hfq mutant strains at an MOI of 10 at 37°C. The number of intracellular bacteria was monitored for 72 h, revealing a slightly diminished replication of the ⌬hfq mutant compared to the wt. (B and C) Monolayers of A. castellanii were infected with wt and ⌬hfq strains at an MOI of 0.1 at 37°C (B) and at an MOI of 1 at 20°C (C), showing a slight growth defect of the ⌬hfq mutant at 37°C but a clear defect at 20°C. (D) Infection of A. castellanii with the complemented ⌬hfq pBChfq strain at an MOI of 1 at 20°C, showing complementation of the growth phenotype. The wt strain carrying plasmid pBC-KS, the ⌬hfq strain carrying the empty plasmid, and complemented strain ⌬hfq pBChfq were examined. The number of intracellular bacteria was determined by recording the number of CFU per milliliter. Results are expressed at log 10 ratio of CFU at T n /T0. Each time point represents the mean Ϯ standard deviation (SD) (error bar) from at least three independent experiments.

FIG 4

 4 FIG 4 hfq transcript and protein expression are influenced by LetA and RpoS and impact flagellar expression. (A) Northern blot analyses of hfq transcripts in wt L. pneumophila and the ⌬hfq, ⌬letA, and ⌬rpoS regulatory mutants grown until they reached an OD 600 of 4 show that the hfq transcript is under the control of LetA and RpoS and is abolished in the ⌬hfq mutant. (B) Western blot analysis of Hfq protein levels in wt L. pneumophila and ⌬hfq, ⌬letA, and ⌬rpoS mutants grown until an OD 600 of 4 revealed a significantly decreased expression of Hfq in the regulatory mutants, indicating that RpoS and LetA influence Hfq expression. (C) Western blot analysis of FlaA protein levels in wt L. pneumophila and ⌬hfq, ⌬letA, and ⌬rpoS mutants grown until an OD 600 of 4 revealed that expression of FlaA is strongly decreased in the ⌬hfq mutant and as expected missing in the ⌬letA and ⌬rpoS mutants, suggesting that Hfq also influences flagellar expression. Mb, stained membrane signal as a loading control. (D) Schematic overview of the major regulatory elements governing L. pneumophila virulence expression in transmissive/postexponential phase and the place and role of Hfq in this network.

FIG 5 A

 5 FIG 5 A small noncoding RNA named Anti-hfq is expressed antisense to hfq and influences Hfq expression and intracellular replication. (A) Schematic organization of the chromosomal organization of the L. pneumophila hfq and anti-hfq locus. (B) 3= RACE PCR product in a 2% agarose gel obtained from exponentially grown wt L. pneumophila confirms the presence of an sRNA of 101 bp, named Anti-hfq. (C) Structure of the Anti-hfq sRNA of L. pneumophila as predicted by the program FindTerm. (D) qPCR analyses of the expression of Anti-hfq in the wt strain grown to exponential (E) phase and to postexponential (PE) phase, showing that Anti-hfq is expressed about 1.5 times in the E phase and 0.05 in the PE phase normalized to an OD 600 of 1. gyrB and tldD were used as internal controls for normalization. Each time point represents the mean plus standard deviation from three independent experiments. The means for the wt strain at the E and PE phases were statistically significantly different (P Ͻ 0.05) by the t test as indicated by the bar and asterisk. (E) The anti-hfq sRNA influences Hfq and FlaA protein expression as evaluated by Western blotting analysis using the anti-Hfq or anti-FlaA antisera and lysates of wt and Anti-hfq-overexpressing (pMMBanti-hfqOE) strains grown to an OD 600 of 4. Membrane (Mb) signals are shown as loading controls. (F) Infection of A. castellanii with the pMMBanti-hfqOE strain shows a similar growth defect as the hfq mutant strain, indicating a role in intracellular replication. Monolayers of A. castellanii were infected with wt and the pMMBanti-hfqOE strain at an MOI of 1 at 20°C. Intracellular replication was determined by recording the number of CFU per milliliter. Results are expressed in log 10 ratio CFU T n /T0. Each time point represents the mean Ϯ SD from three independent experiments.

FIG 6

 6 FIG 6 In an anti-hfq mutant, Hfq is already expressed during exponential growth. (A) Schematic presentation of the ⌬hfq ⌬anti-hfq mutant and sequence changes introduced in the anti-hfq promoter region to construct the ⌬anti-hfq(-10) mutant without disrupting the Hfq amino acid sequence. (B) The Anti-hfq sRNA influences Hfq protein expression as evaluated by Western blot analysis of Hfq in the ⌬anti-hfq(-10) mutant strain. Stained membrane (Mb) signals are shown as a loading control. (C) Western blot analysis of Hfq protein levels in the ⌬hfq ⌬anti-hfq mutant complemented with hfq and anti-hfq (⌬hfq⌬anti-hfq pBChfq) shows that the growth phase-dependent Hfq expression pattern is restored. In contrast, the control strain carrying the empty plasmid (⌬hfq⌬anti-hfqpBC) does not express Hfq. Stained membrane (Mb) signals are shown as loading control. M, molecular weight marker.

FIG 7

 7 FIG 7 Anti-hfq regulates hfq expression through binding to its complementary region which is facilitated by Hfq. (A) EMSA using 25 nM radioactively labeled Anti-hfq and 0, 10, 15, 30, or 50 nM cold full hfq transcript or 0, 15, 30, or 50 nM hfqOUT as control RNA probes shows that Anti-hfq binds hfq mRNA. The amount of RNA probe is indicated by the height of the black triangle above the lane. (B and C) EMSAs using 25 nM radioactively labeled Anti-hfq (B) and hfq (C) RNA alone or with the indicated increasing molar amounts of Hfq protein, revealing that Anti-hfq and hfq bind Hfq. (D) A radioactively labeled Anti-hfq RNA probe and the cold hfq mRNA probe were incubated (lanes 1, 3, and 5), showing the formation of a duplex complex or with 1 M Hfq protein (lanes 2, 4, and 6) showing the formation of a ternary complex. The ability of the protein to bind separately was evaluated by incubating radioactively labeled hfq (lanes 7 to 9) or Anti-hfq (lanes 10 to 12) RNA probes for 10 min. The duplex and ternary complexes were incubated for 0.5 min (lanes 1 and 2), 1.5 min (lanes 3 and 4), and 2.5 min (lanes 5 and 6) at room temperature. The resulting complexes were analyzed on an 8% native polyacrylamide gel as described in Materials and Methods. Abbreviations: (A), radioactively labeled Anti-hfq RNA probe; (h), radioactively labeled hfq mRNA probe; (H), Hfq6XHis; (I) and (II), formation of complexes. Symbols: *, radioactively labeled RNA probes; ϩ, cold RNA probes; Ϫ, no RNA probes. (E and F) RNA stability assays reveal the RNase III dependence of the hfq transcript mRNA in vivo. Wt and RNase III deletion strains were grown in BYE medium before rifampin treatment, showing that RNase III-dependent hfq mRNA decay was favored. The graphs show the relative amount of hfq (E) and GAPDH (F) mRNA remaining at each time point in the wt and RNase III gene deletion strains. 16S was used as internal control for normalization. Each time point represents the mean plus standard deviation from three independent experiments. The quantitative data were analyzed using two-way analysis of variance (ANOVA) test with Bonferroni posttest. A P of Ͻ0.05 was considered to be statistically significant. The values that are significantly different are indicated by a bar and asterisk as follows: **, P Ͻ 0.01; *, P Ͻ 0.05.

  Figure S2

!Figure 13 .

 13 Figure 13. Consensus LetA binding site and putative LetA binding site of anti-hfq. A 18 bp consensus LetA binding site using the sequence of the already known LetA targets RsmY, RsmZ and RsmY was established (http://mergealign.appspot.com/) and compared to the putative LetA binding site found within the anti-hfq gene. Orange letters in the anti-hfq sequence indicate divergences with the highly conserved nucleotides in the LetA Legionella consensus.

Figure 15 .

 15 Figure 15. Activity assay to measure the level of anti-hfq expression (A) Schematic representation of the plasmid panti-hfq::βlac, in which the anti-hfq gene was cloned under the control of the inducible Ptac promoter and in fusion with the βlactamase gene. In order to evaluate the expression of the βlactamase the ribosome-binding site of the flaA gene was cloned upstream the βlac gene. The resulting plasmid was transformed in the L. pneumophila wild-type strain and in the L. pneumophila letA deletion mutant strains (B) The expression of the anti-hfq::βlac fusion was examined at the exponential phase, bacteria grown until OD=2 (white bars) and post-exponential phase, bacteria grown until OD=4 (grey bars), in the L. pneumophila wild-type strain (WT) and in the L. pneumophila letA deletion mutant strain (ΔletA). The βlactamase activity assay shown represents one of the four replicates.

Figure 16 .

 16 Figure 16. ChIP experiment in LetA2X FLAG clone using antibodies against IgG and FLAG. anti-hfq, rsmZ and flaA DNA enrichments were analyzed by qPCR (normalized as percent of input). ChIP experiment represents one of the three experimental replicates.

  ensures that global changes as well as temporal demands throughout the intracellular life cycle are rapidly answered. Although several of the key regulators and signals triggering the regulatory cascade controlling the transition from the replicative to the infectious form of CONCLUSIONS AND PERSPECTIVES! ! ! 96 L. pneumophila have been identified our understanding of the regulatory circuits and elements involved is far from being complete. During my PhD thesis I focused on the characterization of the global regulator and sRNA chaperone Hfq, which is implicated in the regulatory circuit controlling L. pneumophila virulence. Nearly 45 years ago, Hfq was discovered in E. coli as the host factor for the replication of a coliphage (Franze de Fernandez et al., 1968) but nowadays Hfq represents one of the most extensively studied RNA-binding protein in prokaryotes. Hfq is recognized as a RNA chaperone, capable of mediating riboregulatory mechanisms. Comprehensive reviews and different works have reported the biochemical binding properties and preferences of Hfq

Table 1 .

 1 Examples for the functional diversity of sRNAs in selected intracellular bacteria.

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  ). The AsdA sRNA is located in the complementary strand to the dnaA

		CHAPTER TWO
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Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner

	CHAPTER FOUR
	!
	!
	A Giulia Oliva 1,2 , Tobias Sahr 1,2 , Monica Rolando 1,2 , Maike Knoth 1,2 and Carmen Buchrieser 1,2
	1 Biologie des Bactéries Intracellulaires, Institut Pasteur, Paris, France and 2 CNRS UMR
	3525, Paris, France
	Running Head: An antisense RNA regulates Hfq in L. pneumophila
	Address correspondence to: Carmen Buchrieser
	Institut Pasteur
	Biologie des Bactéries Intracellulaires
	28 rue du Dr. Roux
	75724 PARIS CEDEX 15
	FRANCE
	Email: cbuch@pasteur.fr
	!
	!
	!
	!
	!
	!

!

A Unique

cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner Giulia Oliva, Tobias Sahr, Monica Rolando, Maike Knoth, Carmen Buchrieser

Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, Paris, France

  . sRNAs are classified as cis-or trans-

	RESEARCH ARTICLE
	crossm
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  Oliva et al.

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TABLE 1

 1 Bacterial strains and plasmids used in the study

Strain or plasmid Description a

  Amp r , ampicillin resistance; Apr r , apramycin resistance; Cm r , chloramphenicol resistance; Km r , kanamycin resistance.An Antisense RNA Regulates Hfq in L. pneumophila

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				Reference
				or source
	Strains
	L. pneumophila CIP 107629	L. pneumophila serogroup 1 strain Paris (LpP)	19
	L. pneumophila pBC	LpP carrying pBC-KS	54
	L. pneumophila pMMB207C	LpP carrying pMMB207C	28
	L. pneumophila ∆hfq	LpP hfq::Apr r		This study
	L. pneumophila ∆hfq pBC	LpP hfq::Apr r carrying pBC-KS	This study
	L. pneumophila ∆hfq pBChfq	LpP hfq::Apr r carrying pBChfq	This study
	L. pneumophila ∆hfq ∆anti-hfq	LpP hfq anti-hfq::Apr r	This study
	L. pneumophila ∆hfq ∆anti-hfq pBC	LpP hfq anti-hfq::Apr r carrying pBChfq	This study
	L. pneumophila ∆hfq ∆anti-hfq pBChfq	LpP hfq anti-hfq::Apr r carrying pBChfq	This study
	L. pneumophila ∆anti-hfq (-10)	LpP hfq anti-hfq::Apr r carrying pBChfqanti-hfq(-10)	This study
	L. pneumophila pMMBanti-hfqOE	LpP carrying pMMBanti-hfqOE	This study
	L. pneumophila ∆letA	LpP letA::Km r		28
	L. pneumophila ∆rpoS	LpP rpoS::Km r		13
	L. pneumophila ∆rnaseIII	LpP carrying the RNase III gene fused to the Apr r cassette	This study
	E. coli DH5␣	F Ϫ 80dlacZ∆M15 ∆(lacZYA-argF)U169 deoR recA1 endA1	Invitrogen
		hsdR17(r K Ϫ m K	ϩ phoA supE44 Ϫ thi-1 gyrA96 relA1
	Plasmids
	pGEM-T Easy	Cloning of PCR products; Amp r	Promega
	pBC-KS	Expression vector; Cm r	Stratagene
	pMMB207C	Legionella expression vector; ∆mobA; Cm r	55
	pBChfq	pBC-KS containing hfq and anti-hfq genes; Cm r	This study
	pMMBanti-hfqOE	pMMB207C containing anti-hfq gene under the ptac promoter; Cm r	This study
	pBCanti-hfq(-10)	pBChfq mutated in the Ϫ10 upstream region of anti-hfq; Cm r	This study
	January/February 2017 Volume 8 Issue 1 e02182-16			mbio.asm.org 13

a Abbreviations:

TABLE 2

 2 Primers used in this study

	Oliva et al.
	Primer	Primer sequence (5= ؊3=)	Purpose	Reference
	hfq-Mut_F	AAGAATTGATCAGGCCTGTC	Deletion of the hfq gene	This study
	hfq-Mut_R	CCGACGATGCGTAAATTGGA	Deletion of the hfq gene	This study
	apra_F	TTCATGTGCAGCTCCATCAGC	Deletion of the hfq gene	This study
	apra_R	GAGCGGATCGGGGATTGTCTT	Deletion of the hfq gene	This study
	hfq-apra_R	GCTGATGGAGCTGCACATGAATGCAATTTAATACCATTGACCAGG	Deletion of the hfq gene	This study
	hfq-apra_F	GAGCGGATCGGGGATTGTCTTTCTGGTGAGGAAGAAGGAACTG	Deletion of the hfq gene	This study
	hfqanti-hfq1_F	ACACTCCAAAACGAGGCGGCTG	Deletion of the hfq and anti-hfq genes	This study
	hfqanti-hfq2_R	GCTGATGGAGCTGCACATGAACGGGTATCTAACTATTTATTCGA	Deletion of the hfq and anti-hfq genes	This study
	hfqanti-hfq2_F	GAGCGGATCGGGGATTGTCTTACTGTGGCAGACTAATCAATTTA	Deletion of the hfq and anti-hfq genes	This study
	hfqanti-hfq1_R	CGACATCCAAATAATCGCTCG	Deletion of the hfq and anti-hfq genes	This study
	Hfq_comple_F	AAGCTTGCCAGTCTCAATGCAATTGCG	Complementation of hfq and	This study
			hfqanti-hfq
	Hfq_comple_R	GTCGACTTGATTAGTCTGCCACAGTTCC	Complementation of hfq and	This study
			hfqanti-hfq
	M-10anti-hfq_F	ATTGACCAGGAACACTGAAACCGGGACCTTTTCCTTGCGCAATTCATT	Mutation of the Ϫ10 promoter of	This study
			anti-hfq
	M-10anti-hfq_R	AATGAATTGCGCAAGGAAAAGGTCCCGGTTTCAGTGTTCCTGGTCAAT	Mutation of the Ϫ10 promoter of	This study
			anti-hfq
	anti-hfq_OE_F	TCTAGAGCGCAATTCATTTAGGAAAGG	Overexpression of anti-hfq	This study
	anti-hfq_OE_R	CTGCAGAAACCACGCTGTCATGAAAATATAC	Overexpression of anti-hfq	This study
	anti-hfq_3= RACE_F	TTTAGGAAAGGGTCTTGTAGTAAATG	3= RACE anti-hfq	This study
	anti-hfq_3= RACE_R	AATAGTTAGATACCCGTTTTTGCC	3= RACE anti-hfq	This study
	rnaseIII_Mut_F	ATGCGCTCAGCAATTGAATTAGC	Deletion of the RNase III gene	This study
	rnaseIII_Mut_R	TCTGGTCTGGATGAGTTGGAATG	Deletion of the RNase III gene	This study
	rnaseIII_Inv_F	GAGCGGATCGGGGATTGTCTTTATCGCTACCAGCACTGCAATG	Deletion of the RNase III gene	This study
	rnaseIII_Inv_R	GCTGATGGAGCTGCACATGAATGTAACATGCACAATTGAGGGAG	Deletion of the RNase III gene	This study
	anti-hfq RNA_T7_F	TAATACGACTCACTATAGGCGCAATTCATTTAGGAAAGGG	In vitro transcription of anti-hfq	This study
	anti-hfq RNA_T7_R	T AGTTAGATACCCGTTTTTGCC	In vitro transcription of anti-hfq	This study
	hfqmRNA_T7_F	TAATACGACTCACTATAGGGATAGGGTGTCGAATAAATAG	In vitro transcription of hfq mRNA	This study
	hfqmRNA_T7_R	TTGATTAGTCTGCCACAGTTCC	In vitro transcription of hfq mRNA	This study
	lpp0644 _T7_F	TAATACGACTCACTATAGGGGAATGTTATGAGTGACTTG	In vitro transcription of lpp0644	This study
	lpp0644_T7_R	TCCAGTCGTCTGCGCGCATCC	In vitro transcription of lpp0644	This study
	hfqOUT_T7_F	TAATACGACTCACTATAGGGTCAATGGTATTAAATTGCATGGG	In vitro transcription of hfq mRNA	This study
			missing the 5= UTR
	anti-hfq_qPCR_F	TTTAGGAAAGGGTCTTGTAGTAA	qPCR analysis of the anti-hfq region	This study
			overlapping hfq mRNA
	anti-hfq_qPCR_R	AATAGTTAGATACCCGTTTTTGCC	qPCR analysis of the anti-hfq region	This study
			overlapping hfq mRNA
	tldD_qPCR_F	AATCGGAACGTCGATGATGCTG	qPCR analysis of the tldD mRNA	This study
	tldD_qPCR_R	ATCCCTACCCCCTTATCCAGAG	qPCR analysis of the tldD mRNA	This study
	gyrB_qPCR_F	GAGCGTAGACGCCAGTTATGA	qPCR analysis of the gyrB mRNA	This study
	gyrB_qPCR_R	TGATGCAAACCGGTTCCATCA	qPCR analysis of the gyrB mRNA	This study
	hfqmRNA_NB_F	TAATACGACTCACTATAGGGAACAACTGTTGAAATGGCGTG	Northern blot analysis of hfq mRNA	This study
	hfqmRNA_NB_R	GTTTCAGTGTTCCTGGTCAATGG	Northern blot analysis of hfq mRNA	This study
	hfq_qPCR_F	TCAGTGTTCCTGGTCAATGG	Determination of hfq mRNA half-life	This study
	hfq_qPCR_R	AACAACTGTTGAAATGGCGTG	Determination of hfq mRNA half-life	This study
	gapdH_qPCR_F	TTGATACGACAGTGGTCTATGG	Determination of GAPDH mRNA	This study
			half-life
	gapdH_qPCR_R	CATGGACAGTGTTGACTAAGCC	Determination of GAPDH RNA half-life	This study
	16S_qPCR_F	TTGTCTAGCTTGCTAGACAGATGG	Determination of 16S half-life	This study
	16S_qPCR_R	AGCTTTCGTCCTCAGACATTATGC	Determination of 16S half-life	This study

®

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  An Antisense RNA Regulates Hfq in L. pneumophila Fold changes were calculated using the ∆∆C T method. Values represent mean values of three biological replicate experiments Ϯ standard deviations (SD), normalized to the 16S loading controls.

	RESULTS
	!
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TABLE S1 ,

 S1 DOCX file, 0.1 MB.

TABLE S2 ,

 S2 DOCX file, 0.1 MB.

unique cis-encoded RNA is regulating Legionella pneumophila Hfq expression in a life cycle dependent manner

  Giulia Oliva1,2 , Tobias Sahr 1,2 , Monica Rolando 1,2 , Maike Knoth 1,2 and Carmen Buchrieser1,2

	Oliva et al. ! RESULTS ! ! CHAPTER FOUR CHAPTER FOUR ! Supplemental Material A

FIG S1. In vitro growth of the Δhfq mutant at 37°C and 20°C is similar to, that of the wild type strain FIG S2. Sequence of anti-hfq and control EMSA showing that no ternary complex is formed when an unrelated RNA is used. Table S1: Differentially expressed genes in in vitro analyses of wt and Δhfq grown in AYE broth at 37°C until OD 4 (PE phase) Table S2: Differentially expressed genes according to transcriptome in vivo analyses of wt and Δhfq grown for 96h at 20°C within A. castelanii RESULTS ! ! Figure Legends: FIG S1. In vitro growth of the Δhfq mutant at 37°C and 20°C is similar to, that of the wild type strain. (A)

  Growth of the Δhfq deletion mutant at 37°C. (B) Growth of the Δhfq

	deletion mutant at 20°C. Optical density values at OD 600 of triplicate cultures in BCYE
	medium were determined for 24 hours (A) and for 96 hours (B) (Black, wild-type; red, Δhfq)
	FIG S2.

Sequence of anti-hfq and control EMSA showing that no ternary complex is formed when an unrelated RNA is used. A

  Sequence of the small RNA and the flanking regions, blue letters, the sequence of the small RNA, grey letters, promoter region. The arrow indicates the flanking lpp0008 starting codon. Red letters, Hfq start codon. B. EMSA using 25 nM radioactively labelled Anti-hfq together with 0, 10, 15, 30, 50, nM cold hfq full transcript or 0, 15, 30, 50 nM of the cold lpp0644 RNA probes.

Symbols: (A) indicates radioactively labelled Anti-hfq RNA probe, (h) indicates radioactively labelled hfq mRNA probe. Asterisks indicate radioactively labelled RNA probes, -the absence of the RNA probes, triangles indicate increased amount of cold RNA probes.

Table S1 : Differentially expressed genes in in vitro analyses of wt and Δhfq grown in AYE broth at 37°C until OD 4 (PE phase)

 S1 Up-regulated in the Δhfq strain (p<0.05, 8 up-regulated also in vivo analyses)

	RESULTS
	!
	gene.ID description	FC
	lpp0460 hypothetical protein	2,04
	lpp0973 pantothenate kinase type III (putative Bvg accessory factor family protein)	2,32
	lpp1229 flagellar biosynthesis protein FlgG	3,29
	lpp1230 flagellar L-ring protein precursor FlgH	3,36
	lpp1231 flagellar P-ring protein precursor FlgI	2,01
	lpp1291 flagellar protein FliS	2,49
	lpp1292 flagellar hook-associated protein 2 (flagellar capping protein)	2,49
	lpp1974	uncharacterized bacterial polysaccharide deacetylases, catalytic NodB homology domain	2,26
	lpp2350 chemiosmotic efflux system C protein A	3,09
	lpp2351 chemiosmotic efflux system protein A-like protein	2,21
	lpp2353 chemiosmotic efflux system C protein C	5,49
	lpp2354 domain of unknown function (DUF4156)	4,63
	lpp2693 enhanced entry protein EnhB -Sel1-like repeats protein	2,99
	lpp2694 enhanced entry protein EnhA -L,D-transpeptidase catalytic domain,	2,42
	lpp2988 lytic murein transglycosylase, SLT domain	3,43
	gene.ID description	FC
	lpp0009 host factor-1 protein Hfq	0,07
	lpp0679 StaR-like protein, TPR-domain -eukaryotic-like protein	0,48
	sRNA	RsmX	0,48
	!
	!

Down-regulated in the ∆hfq strain (p<0.05, 1 down-regulated also in vivo analyses)

Table S2 : Differentially expressed genes according to transcriptome in vivo analyses of wt and Δhfq grown for 96h at 20°C within A. castelanii

 S2

	RESULTS	CHAPTER FOUR
	!
	Up-regulated in the Δhfq strain (p<0.05) gene.ID description lpp0008 substrate of the Dot/Icm secretion system RavA lpp0122 protein of unknown function lpp0257 chitin-binding protein CbpD lpp0381 preprotein translocase secE subunit lpp0384 50S ribosomal protein L1 lpp0385 50S ribosomal subunit protein L1 lpp0386 50S ribosomal subunit protein L7/L12 lpp0389 30S ribosomal protein S12 lpp0392 translation elongation factor Tu lpp0393 30S ribosomal subunit protein S1 lpp0394 50S ribosomal subunit protein L3 lpp0396 50S ribosomal subunit protein L23 lpp0398 30S ribosomal subunit protein S19 lpp0399 50S ribosomal subunit protein L22 lpp0400 30S ribosomal protein S3 lpp0401 50S ribosomal protein L16 lpp0402 50S ribosomal subunit protein L29 lpp0403 30S ribosomal protein S17 lpp0404 50S ribosomal protein L14 lpp0406 50S ribosomal protein L5 lpp0407 30S ribosomal protein S14 lpp0408 30S ribosomal protein S8 lpp0410 50S ribosomal subunit protein L18 lpp0411 30S ribosomal subunit protein S5 lpp0412 50S ribosomal subunit protein L3 lpp0418 30S ribosomal subunit protein S4 lpp0419 DNA-directed RNA polymerase alpha chain lpp0460 hypothetical protein lpp0466 30S ribosomal protein S16 lpp0493 cold shock-like protein CspD lpp0544 50S ribosomal protein L28 lpp0570 outer membrane protein (OmpH-like) lpp0605 bacterial protein of unknown function (DUF945) lpp0606 global DNA-binding transcriptional regulator Fis1 lpp0688 substrate of the Dot/Icm secretion system lpp0725 predicted integral membrane protein (DUF2282) lpp0742 chaperonin 10 subunit, Cpn10 or GroES lpp0743 chaperonin 60, Cpn60 or GroEL lpp0855 macrophage infectivity potentiator Mip lpp0972 enhanced entry protein EnhA (L,D-transpeptidase catalytic domain) lpp1146 substrate of the Dot/Icm secretion system lpp1207 cold-shock protein (CSP) lpp1229 flagellar biosynthesis protein FlgG lpp1230 flagellar L-ring protein precursor FlgH lpp1324 global DNA-binding transcriptional regulator Fis2 lpp1772 hypothetical protein lpp1805 Com1-like membrane-associated immunoreactive protein, DsbA family lpp1958 Legionella major outer membrane protein lpp1974 uncharacterized polysaccharide deacetylases, catalytic NodB homology domain lpp2026 peptidoglycan-associated lipoprotein, OmpA-like domain lpp2164 heme-binding protein Hbp lpp2209 hypothetical membrane protein lpp2276 substrate of the Dot/Icm secretion system lpp2354 domain of unknown function (DUF4156) lpp2675 papain-like C1 peptidase lpp2689 30S ribosomal subunit protein S2 lpp2692 enhanced entry protein EnhC -Sel1-like repeats protein lpp2693 enhanced entry protein EnhB -Sel1-like repeats protein lpp2694 enhanced entry protein EnhA -L,D-transpeptidase catalytic domain, lpp2768 50S ribosomal protein L35 lpp2817 30S ribosomal protein S15 lpp2866 leucine aminopeptidase, Zn-peptidase M28 family lpp2968 hypothetical protein lpp2988 lytic murein transglycosylase, SLT domain lpp3021 hypothetical protein lpp3031 major outer membrane protein precursor lpp3032 major outer membrane protein precursor lpp3033 major outer membrane protein precursor ! Down-regulated in the Δhfq strain (p<0.05) gene.ID description lpp0009 host factor-1 protein Hfq lpp0034 hypothetical protein lpp0045 fatty acid hydroxylase lpp0845 global regulator CsrA lpp1823 hypothetical protein ! ! ! lpp1826 DNA-binding protein HU-beta ! 90	FC 1,91 1,88 1,87 1,93 2,07 2,19 2,40 2,03 2,39 2,05 1,88 2,02 2,08 1,88 2,19 2,26 1,96 1,98 2,14 1,96 2,46 2,12 2,20 2,17 2,08 2,03 1,98 2,65 1,87 2,47 1,95 2,10 1,99 2,35 2,21 2,08 1,96 1,94 2,52 2,56 2,46 2,24 1,86 1,82 2,48 2,07 1,88 2,82 2,45 1,86 2,07 1,92 2,48 2,09 1,91 1,93 2,08 1,83 1,97 1,68 2,05 1,94 1,89 1,95 1,72 2,09 2,47 3,24 3,07 FC 0,19 0,52 0,52 0,43 0,48

Hfq was identified in Escherichia coli as a host factor required for replication of the RNA phage Qβ > 40 years ago (Franze de by guest on August 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from

With the advent of next-generation sequencing (NGS) and comprehensive transcriptome analysis techniques, increasing numbers of new sRNAs have been detected in bacteria[START_REF] Albrecht | The transcriptional landscape of Chlamydia pneumoniae[END_REF] Wurtzel et al. 2012;[START_REF] Bilusic | Revisiting the coding potential of the E. coli genome through Hfq coimmunoprecipitation[END_REF] Boudry et al. 2014). Using custom-made microarrays and transcriptome analyses of the intracellular pathogens N. gonorrhoeae[START_REF] Dietrich | The effect of hfq on global gene expression and virulence in Neisseria gonorrhoeae[END_REF] or F. tularensis(Meibom et al. 2009), it was reported that by guest onAugust 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from

by guest on August 25, 2016 http://femsre.oxfordjournals.org/ Downloaded from

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ACKNOWLEDGEMENTS

Work in the laboratory of CB is financed by the Institut Pasteur, the Institut Carnot-Pasteur MI, the French Region Ile de France (DIM Malinf), grant no. ANR-10-LABX-62-IBEID, the Fondation pour la Recherche M édicale (FRM), grant no. DEQ20120323697, and the Pasteur-Weizmann consortium 'The roles of non-coding RNAs in regulation of microbial life styles and virulence'. GO was supported by a stipend from the Pasteur-Paris University (PPU) International PhD Program. ACKNOWLEDGMENTS Work in the C.B. laboratory is financed by the Institut Pasteur, the Institut Carnot-Pasteur MI, the French Region Île de France (DIM Malinf), grant ANR-10-LABX-62-IBEID, the Fondation pour la Recherche Médicale (FRM) grant DEQ20120323697, and the Pasteur-Weizmann consortium "The roles of noncoding RNAs in regulation of microbial life styles and virulence." G.O. was supported by a stipend from the Pasteur -Paris University (PPU) International Ph.D. Program.

Conflict of interest. None declared.