Jérémy

Mathilde Fernando

Anne Marie

A big merci also to every member of the lab, past and present, who I had the pleasure of working with during my time in Paris

was later proven to be caused by the same bacteria [START_REF] Glick | Pontiac fever: an epidemic of unknown etiology in a health department[END_REF]. The name for this disease, Pontiac Fever, is nowadays mostly used for a milder progression of the infection with members of the genus Legionella, since no deaths occurred during this outbreak in 1968. The earliest case of Legionnaires' disease was identified in frozen sera samples from 1947 [START_REF] Mcdade | Legionnaires' Disease Bacterium Isolated in 1947[END_REF].

Soon after its discovery, the members of the genus Legionella started to grow rapidly, as more and more species were identified.

From lakes to lungs -Characteristics of the genus Legionella

All members of the genus Legionella are aerobic, rod-shaped, Gram-negative bacteria.

L. pneumophila expresses a polar flagellum, enabling bacterial motility (Figure 1A). It is a facultative intracellular bacterium that can grow in a planktonic form but is most frequently found parasitizing different free-living protozoa in aquatic environments [START_REF] Anand | Interaction of L. pneumophilia and a free living amoeba (Acanthamoeba palestinensis)[END_REF]Rowbotham, 1980). In the laboratory, L. pneumophila is grown under strictly aerobic conditions on specific medium called buffered charcoal yeast extract (BCYE), a nutrient rich medium supplemented with iron and L-cystein [START_REF] Fields | The molecular ecology of legionellae[END_REF]. On the black BCYE plates, the bacteria appear as opaque, round, white/gray colonies, with clear edges and a smooth shine (Figure 1B). The main energy and carbon source for the bacteria are amino acids and other organic acids [START_REF] George | Amino acid requirements of Legionella pneumophila[END_REF][START_REF] Sauer | The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages[END_REF][START_REF] Tesh | Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources[END_REF]. However, carbohydrates also play a role in in the bacterial metabolism [START_REF] Eylert | Isotopologue Profiling of Legionella pneumophila: ROLE OF SERINE AND GLUCOSE AS CARBON SUBSTRATES[END_REF][START_REF] Gillmaier | Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila[END_REF][START_REF] Herrmann | GamA is a eukaryotic-like glucoamylase responsible for glycogen-and starch-degrading activity of Legionella pneumophila[END_REF]. The genus Legionella comprises 65 species to date, which are mostly found in natural aquatic environments such as lakes, rivers, and ponds, but also human-made environments such as cooling towers, showers, and air conditioning systems (Gomez-Valero et al., 2019;Mondino et al., 2020a). Out of the 65 species known today, at least 20 have been connected to human disease, in particular pneumonia [START_REF] Muder | Infection Due to Legionella Species Other Than L. pneumophila[END_REF]. In human-made environments Legionella spp. are mainly present in biofilms, however, their replication within these biofilms seems dependent on the presence of a protozoan host [START_REF] Murga | Role of biofilms in the survival of Legionella pneumophila in a model potable-water system[END_REF][START_REF] Rogers | Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora[END_REF].

Many protozoa have been identified to enable replication of Legionella spp, including amoebae such as Acanthamoeba, Neaglaria, Dictyostelium, Balamuthia and Hartmannella as well as ciliates like Tetrahymena and Paramecium [START_REF] Barbaree | Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila[END_REF][START_REF] Fields | Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis[END_REF][START_REF] Hägele | Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella[END_REF][START_REF] Kuiper | Intracellular Proliferation of Legionella pneumophila in Hartmannella vermiformis in Aquatic Biofilms Grown on Plasticized Polyvinyl Chloride[END_REF][START_REF] Newsome | Interactions between Naegleria fowleri and Legionella pneumophila[END_REF][START_REF] Shadrach | Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria[END_REF][START_REF] Steinert | Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii[END_REF][START_REF] Watanabe | Ciliate Paramecium is a natural reservoir of Legionella pneumophila[END_REF]. Biofilms and the association with protozoa seem to be the main reservoirs of Legionella spp. in natural as well as human-made aquatic environments. In natural aquatic environments, the bacterial burden with Legionella spp. is generally very low due to non-optimal growth conditions (low temperatures and limited nutrients). Yet, in human-made environments such as air conditioning systems, the elevated water temperature can promote bacterial growth, especially if the system has longer periods of water flow stagnation. Indeed, many outbreaks of Legionnaires' disease have been linked to the dissemination of Legionellacontaminated aerosols by human-made aquatic systems [START_REF] García-Fulgueiras | Legionnaires' disease outbreak in Murcia, Spain[END_REF][START_REF] Nhu Nguyen | A Community-Wide Outbreak of Legionnaires Disease Linked to Industrial Cooling Towers-How Far Can Contaminated Aerosols Spread?[END_REF][START_REF] Shivaji | A large community outbreak of Legionnaires disease in Vila Franca de Xira, Portugal, October to[END_REF][START_REF] Weiss | A Large Community Outbreak of Legionnaires' Disease Associated With a Cooling Tower in New York City[END_REF]. Also the outbreak at the American Legion convention in 1976 was later linked to the hotel's air conditioning system [START_REF] Fraser | Legionnaires' Disease -Description of an Epidemic of Pneumonia[END_REF][START_REF] Terranova | 1974 outbreak of Legionnaires' Disease diagnosed in 1977: Clinical and Epidemiological Features[END_REF].

Thus, the emergence of Legionnaires' disease in the past decades is thought to be caused by the progressive industrialization as well as the ubiquity of artificial aquatic environments.

Legionellosis

"But whatever it was, it came out of the trees" -Epidemiology of legionellosis As mentioned before, the case numbers of legionellosis, the umbrella term for infections by Legionella spp., are steadily increasing over the past decades. There are large outbreaks occurring worldwide, with the biggest cluster reported to date being an outbreak in a hospital in Murcia, Spain with more than 800 suspected cases [START_REF] García-Fulgueiras | Legionnaires' disease outbreak in Murcia, Spain[END_REF]. However, the precise incidence rate of Legionnaires' disease worldwide is not known, due to vastly different surveillance methods, diagnostics and generally different awareness levels around the globe. This can lead to a strong discrepancy between diagnosed and undiagnosed cases.

Legionellosis is one of the most common causes of community-acquired pneumonia [START_REF] Von Baum | Community-Acquired Legionella Pneumonia: New Insights from the German Competence Network for Community Acquired Pneumonia[END_REF][START_REF] Muder | Community-acquired Legionnaires' disease[END_REF]. In the United Stated in 2018, around 10.000 cases of legionellosis were reported, which corresponds to an increase of almost nine times since the year 2000. The possible explanations for this steep rise in cases are manifold: increased presence of Legionella in the environment, increased susceptibility of the population to the infection, increased awareness and therefore, increased diagnosis, or a combination of factors.

In addition, seasonal variations in case numbers can be seen, with most cases occurring in summer and early fall (https://www.cdc.gov/legionella/about/history.html). Moreover, the 2016-2017 Legionnaires' disease Surveillance Summary Report revealed that the two most prevalent exposure categories for Legionnaires' disease in the United States are healthcare and travel, with 21.3% and 15.8% of cases being traced back to these settings, respectively (https://www.cdc.gov/legionella/health-depts/surv-reporting/2016 -17-surv-report-508.pdf). In a recent study from January 2021, it is estimated that the true burden of Legionnaires' disease in the United States is still vastly under-reported and that the true case number might be more than two times higher [START_REF] Collier | Estimate of Burden and Direct Healthcare Cost of Infectious Waterborne Disease in the United States[END_REF].

In Europe, the European Center for Disease Prevention and Control (ECDC) coordinates the European Legionnaires' Disease Surveillance Network (ELDSNet), which traces and records legionellosis cases in the 27 member states of the European Union, as well as Norway and Iceland (EEA). The ELDSNet reported more than 11.000 cases of Legionnaires' disease in 2019, an increase of almost 100% since 2009. In France, the case numbers rose by around 50% in the same time frame, from 1206 in 2009 to 1816 in 2019 (Figure 2).

As previously stated, at least 20 of the 65 Legionella species known to date have been linked to disease [START_REF] Muder | Infection Due to Legionella Species Other Than L. pneumophila[END_REF]. Yet, if we look at the distribution of the different Legionella species among legionellosis cases in Europe and the United States, we see that the vast majority (91,5%) can be attributed to one species, L. pneumophila. Within this species, members of the serogroup 1 (Sg1) are responsible for around 84% of infections [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]. Furthermore, it should be noted that five sequence types of Sg1 -out of a total of over 2000 sequence types -have emerged and are responsible for over 50% of legionellosis cases in northwest Europe today [START_REF] David | Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently[END_REF]. The most common causes of non-pneumophila legionellosis are Legionella longbeachae, Legionella bozemanii, and Legionella micdadei [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]. Interestingly, Legionella longbeachae only accounts for a small percentage of infections worldwide, however, this changes if we look at Australia and New Zealand. In Australia for example, L. longbeachae was responsible for 40% of legionellosis cases in 2016 (2021).

It should be noted that the human infection is usually a dead end for the bacteria. All infections -where the source has been identified -could be traced back to direct exposure to contaminated aerosols and there has been only one recorded case of putative person-to-person transmission [START_REF] Correia | Probable Person-to-Person Transmission of Legionnaires[END_REF]. "But whatever it was, it drove them to their knees" -Risk factors for legionellosis Legionella spp. are opportunistic pathogens, meaning that individuals with specific risk factors are more prone to develop disease after exposure to the bacteria.

Interestingly, legionellosis has a higher prevalence in men than in women. In 2017 in the United States and Europe, men accounted for around 62% and 70% of infections, respectively. The reason for this phenomenon is yet to be discovered. Another risk factor is age (Figure 3). In 2020, around 50% of reported legionellosis cases in Europe were observed in people over the age of 65, but the same group accounts for almost 80% of all fatalities (https://www.ecdc.europa.eu/en/legionnaires-disease/surveillance/atlas). Yet, it should be noted that in recent years several cases of fatal neonatal pneumonia were connected to Legionnaires' disease, in particular in the context of birthing pools [START_REF] Barton | Legionellosis following water birth in a hot tub in a Canadian neonate[END_REF][START_REF] Collins | Heated birthing pools as a source of Legionnaires' disease[END_REF][START_REF] Vanderlaan | Systematic Review of Case Reports of Poor Neonatal Outcomes With Water Immersion During Labor and Birth[END_REF].

Other risk factors include chronic lung disease, immunosuppression, and smoking [START_REF] Cameron | Comparison of Legionella longbeachae and Legionella pneumophila cases in Scotland; implications for diagnosis, treatment and public health response[END_REF][START_REF] Carratala | Risk factors for nosocomial Legionella pneumophila pneumonia[END_REF]. In addition, many cases of legionellosis occur as nosocomial infections, with transplant patients being a high risk group [START_REF] Bangsborg | Nosocomial legionellosis in three heart-lung transplant patients: Case reports and environmental observations[END_REF][START_REF] Fraser | Cavitary Legionella pneumonia in a liver transplant recipient[END_REF][START_REF] Wilmes | Bacterial pneumonia in kidney transplant recipients[END_REF]. "Now within my heart, it sure put on a squeeze" -Clinical manifestations of legionellosis As mentioned before, legionellosis is the umbrella term for illnesses caused by different Legionella species. Besides Legionnaires' disease, this term comprises the non-pneumonic Pontiac fever, as well as other rare infections, such as wound infections and arthritis.

• Legionnaires' disease Legionnaires' disease is an atypical pneumonia and the most severe form of legionellosis. The average incubation time from exposure to onset of the first symptoms is 2-10 days. However, incubation times of up to 24 days have been recorded [START_REF] Bargellini | A culture-proven case of community-acquired legionella pneumonia apparently classified as nosocomial: diagnostic and public health implications[END_REF]. The first symptoms to occur are usually non-productive cough, shortness of breath, and fever. This clinical presentation is similar to many other bacterial pneumonias, for example infections caused by Streptococcus pneumoniae [START_REF] Bradley | Emerging respiratory infections: The infectious disease pathology of SARS, MERS, pandemic influenza, and Legionella[END_REF]. However, one feature that helps to distinguish Legionnaires' disease from more common bacterial pneumonias is the frequent manifestation of extrapulmonary symptoms. These symptoms include gastrointestinal and renal symptoms as well as neurological abnormalities, such as mental confusion [START_REF] Cunha | Legionnaire's Disease: A Clinical Diagnostic Approach[END_REF][START_REF] Kao | Community-acquired Legionnaires' disease at a medical center in northern Taiwan[END_REF]. A rare but severe complication of Legionnaires' disease is the formation of cavitary lesions. The formation of these cavities has been most prevalent in immunocompromised individuals or patients with other underlying conditions [START_REF] Fraser | Cavitary Legionella pneumonia in a liver transplant recipient[END_REF][START_REF] Guy | Legionella pneumophila lung abscess associated with immune suppression[END_REF][START_REF] Khokher | Legionella pneumophila as a cause of cavitary lung disease in systemic lupus erythematous[END_REF][START_REF] Morales | Cavitary Pulmonary Nodules in an Immunocompromised Patient With Urothelial Carcinoma of the Bladder[END_REF].

The overall fatality rate of Legionnaires' disease is highly variable depending on the outbreak setting and other factors, but can reach from 5-40% [START_REF] Bradley | Emerging respiratory infections: The infectious disease pathology of SARS, MERS, pandemic influenza, and Legionella[END_REF].

• Pontiac fever

Pontiac fever is a non-pneumonic disease caused by the infection with different Legionella spp. It is a self-limited febrile illness without pneumonia. The most common symptoms include cough, fever, myalgia, headaches and malaise. Interestingly, the incubation period for Pontiac fever is with 1-2 days much shorter than the one of Legionnaires' disease.

No fatalities for Pontiac fever have been reported yet [START_REF] Kaufmann | Pontiac fever: isolation of the etiologic agent (Legionella pneumophila) and demonstration of its mode of transmission[END_REF][START_REF] Marrie | Legionellosis[END_REF].

• Other types of extrapulmonary legionellosis

Even though infections with Legionella spp. are usually connected to pulmonary infections, there have been reported cases of extrapulmonary infections with different members of this genus. Several reports of wound infections caused by different Legionella spp. (L. pneumophila, L.micdadei, L. dumoffii, L. longbeachae) have been reported [START_REF] Ampel | Cutaneous Abscess Caused by Legionella micdadei in an Immunosuppressed Patient[END_REF][START_REF] Brabender | Legionella pneumophila Wound Infection[END_REF][START_REF] Lowry | A Cluster of Legionella Sternal-Wound Infections Due to Postoperative Topical Exposure to Contaminated Tap Water[END_REF][START_REF] Mentula | Legionella longbeachae infection in a persistent hand-wound after a gardening accident[END_REF]. Another possible manifestation of extrapulmonary legionellosis is arthritis. This can mostly be seen in patients with underlying conditions or the elderly [START_REF] Flendrie | Septic Arthritis Caused by Legionella dumoffii in a Patient with Systemic Lupus Erythematosus-Like Disease[END_REF][START_REF] Ibranosyan | The clinical presentation of Legionella arthritis reveals the mode of infection and the bacterial species: case report and literature review[END_REF][START_REF] Naito | Reactive Legionella pneumophila arthritis diagnosed by polymerase chain reaction[END_REF][START_REF] Thurneysen | Legionella pneumophila Serogroup 1 Septic Arthritis With Probable Endocarditis in an Immunodeficient Patient[END_REF]. Other rare infections include sinusitis and peritonitis [START_REF] Dournon | Legionella pneumophila peritonitis[END_REF][START_REF] Schlanger | Sinusitis caused by Legionella pneumophila in a patient with the acquired immune deficiency syndrome[END_REF].

"Oh, that Legionnaire's disease" -diagnosis and treatment of legionellosis

The discovery and first description of L. pneumophila was performed by isolating the bacteria from lung tissue of four patients who have succumbed to the disease. This was a complicated and tedious process. First, guinea pigs were intraperitoneally inoculated with these samples. Afterwards, during the necropsy of these animals, tissue samples were taken and used to inoculate embryonated hen's eggs. However, to avoid contamination, antibiotics were added, thus no bacteria could be isolated. Later, the bacteria could be isolated from the yolk sac of the egg inoculated without antibiotics. This procedure was also applied for production of antigens, which subsequently were used to screen patient samples for the presence of Legionella-specific antibodies [START_REF] Mcdade | Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease[END_REF]. Another common method was culturing the bacteria on a, at that time, recently developed medium, buffered-charcoal yeast extract (BCYE) [START_REF] Edelstein | Laboratory diagnosis of Legionnaires disease[END_REF]. The serological tests and the culture-based identification were common practice for several years for the diagnosis of Legionnaires' disease and are still used today to investigate possible outbreaks in retrospect or to diagnose infections with less frequent species of Legionella. However, these tests are very time consuming and tedious, which makes them less than ideal for a rapid diagnosis of suspected cases of Legionnaires' disease. Within a few years after the initial discovery of L. pneumophila several new tests have been developed to diagnose a possible infection.

One of the first ones were urinary antigen tests, which took advantage of the fact that bacterial LPS is present in the patient's urine. First, a radioimmunoassay was developed, shortly followed by an enzyme-linked immunosorbent assay (ELISA)-based test to detect these urinary antigens [START_REF] Kohler | Rapid radioimmunoassay diagnosis of Legionnaires' disease: detection and partial characterization of urinary antigen[END_REF][START_REF] Sathapatayavongs | Rapid diagnosis of Legionnaires' disease by urinary antigen detection: Comparison of ELISA and radioimmunoassay[END_REF]. Today, lateral flow tests are widely used for the detection of soluble Legionella antigens in urine samples. However, these tests are only able to detect infection with L. pneumophila serogroup 1 [START_REF] Domínguez | Evaluation of a Rapid Immunochromatographic Assay for the Detection of Legionella Antigen in Urine Samples[END_REF]. In parallel to the development of urinary antigen tests, new methods were developed based on a now very frequently used technique, polymerase chain reaction (PCR). First only used for the detection of Legionella spp. in environmental samples [START_REF] Starnbach | Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization[END_REF], this technique was quickly refined to detect and differentiate between several pathogenic Legionella species in patients samples [START_REF] Jaulhac | Detection of Legionella spp. in bronchoalveolar lavage fluids by DNA amplification[END_REF]. In recent years, these PCR tests have been improved more and more to enable not only the differentiation between specific emerging L. pneumophila sequence types, but also to detect several non-pneumophila Legionella species in a multiplex approach [START_REF] Benitez | Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay[END_REF][START_REF] Cross | Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay[END_REF][START_REF] Mentasti | Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47[END_REF][START_REF] Mérault | Specific Real-Time PCR for Simultaneous Detection and Identification of Legionella pneumophila Serogroup 1 in Water and Clinical Samples[END_REF]. Yet, the "gold standard" for the identification of a specific Legionella strain and serogroup remains culture and isolation, which are widely used in outbreak investigation [START_REF] Mercante | Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations[END_REF]Mondino et al., 2020a). All these techniques enable a rapid diagnosis of Legionnaires' disease, which subsequently allows physicians to decide on effective treatment plans.

To this date, antibiotic resistance is a very rare occurrence in L. pneumophila infection.

It should be noted that Legionella spp. are naturally resistant to b-lactam antibiotics [START_REF] Fu | Inactivation of beta-lactam antibiotics by Legionella pneumophila[END_REF], thus highlighting the importance of rapid diagnosis, since b-lactam antibiotics are a common first-line treatment for other bacterial pneumonias [START_REF] Ceccato | Safety considerations of current drug treatment strategies for nosocomial pneumonia[END_REF][START_REF] Garau | Role of beta-lactam agents in the treatment of community-acquired pneumonia[END_REF]. However, in recent years there have been several reports of antibiotic resistances in L. pneumophila [START_REF] Bruin | Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia[END_REF][START_REF] Jia | Antibiotic Resistance and Azithromycin Resistance Mechanism of Legionella pneumophila Serogroup 1 in China[END_REF][START_REF] Shadoud | Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans[END_REF]. To stay one step ahead of the possible emergence of antibiotic resistant L. pneumophila the Study Group for Legionella Infection (ESGLI) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) endorsed the routine use of antimicrobial susceptibility testing of Legionella isolated from patients [START_REF] Portal | Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?[END_REF]. Today, the recommended treatment for Legionella infections are fluoroquinolones (e.g. levofloxacin or ciprofloxacin) and macrolides (e.g. azithromycin) [START_REF] Yu | Legionella: macrolides or quinolones?[END_REF]. Over the past few years, new approaches for the treatment of Legionnaires' disease are being developed. These new approaches include drugs like lefamulin, a novel pleuromutilin antibiotic, or the usage of gene therapy to target and inhibit specific bacterial genes [START_REF] Mercuro | Clinical Utility of Lefamulin: If Not Now, When?[END_REF][START_REF] Pashaei-Asl | Legionella Pneumophila and Dendrimers-Mediated Antisense Therapy[END_REF][START_REF] Veve | Lefamulin: Review of a Promising Novel Pleuromutilin Antibiotic[END_REF]. These techniques could be on the forefront of Legionnaires' disease treatment in the near future.

Hijacking of the host by Legionella

As mentioned before, L. pneumophila can infect a wide range of protozoan hosts but also alveolar macrophages. The basic process is the same, independent of the host. After the entry of the bacteria into the host cell, the bacteria establish a protective vacuole, the so-called Legionella-containing vacuole (LCV), which allows the bacteria to replicate in a safe niche within this otherwise hostile environment. The LCV is remodeled during the infection by recruiting endoplasmic reticulum (ER) vesicles and ribosomes (Isberg et al., 2009). The similarities between the two host types underline the high adaptability of the bacteria. However, due to the lack of transmission between humans it was hypothesized that the co-evolution of L. pneumophila with its protozoan hosts has led to their capability to use these very same strategies to infect and replicate within mammalian macrophages [START_REF] Al-Quadan | Exploitation of evolutionarily conserved amoeba and mammalian processes by Legionella[END_REF][START_REF] Cianciotto | Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages[END_REF][START_REF] Escoll | From Amoeba to Macrophages: Exploring the Molecular Mechanisms of Legionella pneumophila Infection in Both Hosts[END_REF][START_REF] Molmeret | Amoebae as training grounds for intracellular bacterial pathogens[END_REF]. One indispensable factor for the infection is the bacterial type IV secretion system, Dot/Icm [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]. Through this system, more than 330 bacterial effectors are secreted into the host cell, manipulating countless signaling and metabolic pathways to the advantage of the microbial invaders (Figure 4) [START_REF] Burstein | Genome-scale identification of Legionella pneumophila effectors using a machine learning approach[END_REF][START_REF] Campodonico | A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system[END_REF]Heidtman After uptake of the bacteria, they establish the Legionella-containing vacuole (LCV). Through secretion of a wide variety of effectors, the bacteria promote the recruitment of mitochondria and ER-derived vesicles to the LCV. Within the LCV, the bacteria replicate and switch again to their more virulent, flagellated form, until they egress from the host cell. Adapted from Mondino et al. (2020). et al., 2009;[START_REF] Lifshitz | Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal[END_REF][START_REF] Zhu | Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila[END_REF]. The focus of this chapter will be the different strategies L. pneumophila deploys to replicate within a host cell. The general intracellular life cycle of L. pneumophila will be discussed, as well as its usage of the Dot/Icm secretion system and its effectors to manipulate the host.

It's the inner values that count -the intracellular life cycle of L. pneumophila

The first step of the infection is the attachment of the bacteria to the host cell. For macrophages, this process seems to be dependent on the complement receptors CR1 (CD35) and CR3 (CD18/CD11b). By blocking these receptors via monoclonal antibodies, bacterial uptake is strongly reduced [START_REF] Payne | Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors[END_REF]. However, this CR1/CR3-dependent attachment seems also to rely on the presence of specific antibodies, produced in response to the infection [START_REF] Husmann | Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors[END_REF]. There have also been several reports of noncomplement mediated adherence of L. pneumophila, which might play a role during the early stages of infection, when no specific antibody response has been mounted yet [START_REF] Elliott | Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila[END_REF][START_REF] Gibson | Adherence of Legionella pneumophila to U-937 cells, guinea-pig alveolar macrophages, and MRC-5 cells by a novel, complement-independent binding mechanism[END_REF][START_REF] Rodgers | Opsonin-independent adherence and intracellular development of Legionella pneumophila within U-937 cells[END_REF]. The bacterial factors that have been described so far to be involved in the attachment to the host are the two proteins MOMP and Lcl. MOMP, an outer membrane porin, was shown to bind complement components C3b and C3bi (Bellinger-Kawahara and [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]. In addition, it was reported to regulate bacterial attachment in a complement-independent manner [START_REF] Krinos | Role of the 25 kDa major outer membrane protein of Legionella pneumophila in attachment to U-937 cells and its potential as a virulence factor for chick embryos[END_REF]. Lcl binds a complement receptor, C1qR (CD93), to mediate attachment to the host [START_REF] Vandersmissen | A Legionella pneumophila collagenlike protein encoded by a gene with a variable number of tandem repeats is involved in the adherence and invasion of host cells[END_REF].

Another bacterial protein shown to influence bacterial entry is LpnE. It has been shown that an lpnE knock out leads to a decrease in bacterial entry in several different cell types [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]. In addition, LpnE interacts with the eukaryotic polyphosphate-5 phosphatase, OCRL (oculocerebrorenal syndrome of Lowe), leading to its recruitment to the LCV [START_REF] Voth | The structure of Legionella effector protein LpnE provides insights into its interaction with Oculocerebrorenal syndrome of Lowe ( OCRL ) protein[END_REF][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 exact mode of bacterial entry into the host cell is still a strongly discussed topic. A mechanism called coiled phagocytosis was described in several hosts, but only seems to represent a small portion of bacterial uptake (Figure 5) [START_REF] Bozue | Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion[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].

The conventional internalization of the bacteria is mediated by the MOMP-C3b interaction, mentioned before (Bellinger-Kawahara and [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]. Even though, many questions remain about the exact mechanisms of uptake, it is well-known that the uptake and the following internalization in mammalian cells is dependent on actin polymerization. By using cytochalasin-D -a known inhibitor of actin polymerization -the uptake of L. pneumophila in macrophages as well as lung epithelial cells is inhibited [START_REF] Elliott | Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila[END_REF][START_REF] King | Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells[END_REF][START_REF] Prashar | Mechanism of invasion of lung epithelial cells by filamentous L egionella pneumophila[END_REF].

Once the bacteria enter the host cell, they start to establish the LCV. Under normal conditions, after the cell engulfs different microbes in a phagosome, the phagosome matures by different fusion/fission steps with parts of the endosomal network, leading to the formation of the phagolysosome. Subsequently, this leads to the acidification of the phagolysosome triggering the degradation of its contents [START_REF] Vieira | Phagosome maturation: aging gracefully[END_REF]. However, Legionella is able to inhibit the fusion with the lysosome, thus manipulating the host cell and enabling the formation of a replicative vacuole where the bacteria can grow to high numbers (Isberg et al., 2009;Mondino et al., 2020a;[START_REF] Newton | Molecular Pathogenesis of Infections Caused by Legionella pneumophila[END_REF]. Interestingly, the process of acidification is delayed in the LCV but not completely inhibited. In the first few hours of infection, the pH within the phagosome stays neutral. This changes at later infection stages, when the LCV acquires more endosomal markers and the LCV acidifies. Moreover, the bacteria obtained from cell culture, but not the ones from broth, have a higher tolerance to low pH, indicating that maintaining the neutral pH within the LCV is only necessary at the beginning of the infection [START_REF] Sturgill-Koszycki | Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles[END_REF]. So far, one secreted effector protein of L. pneumophila, SidK, has been reported to inhibit vacuole acidification by interacting with the proton pump VatA (L et al., 2010;[START_REF] Zhao | Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein[END_REF]. However, many questions remain about the exact regulation of LCV acidification.

The remodeling of the LCV membrane during infection is an intricate and finely regulated process. Within the first few minutes, ER-derived vesicles are recruited to the LCV.

By intercepting secretory vesicles of the ER and Golgi, the bacteria transform the LCV into an ER-like organelle that permits their replication [START_REF] Derré | Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system[END_REF][START_REF] Kagan | Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle[END_REF][START_REF] Weber | Legionella-Containing Vacuoles Capture PtdIns(4)P-Rich Vesicles Derived from the Golgi Apparatus[END_REF]. Two of the earliest and most prominently recruited proteins are the small GTPase Rab1 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) Sec22b [START_REF] Kagan | Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle[END_REF]. Rab1 usually regulates the fusion of ER-derived vesicles with the Golgi and Sec22b is responsible for vesicle tethering and fusion between said vesicles and the Golgi [START_REF] Moyer | Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering[END_REF][START_REF] Xu | Subunit structure of a mammalian ER/Golgi SNARE complex[END_REF]. Moreover, the inhibition of Rab1 activity in infected cells can drastically reduce L. pneumophila replication, highlighting its importance in the process of establishing a functional LCV [START_REF] Kagan | Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle[END_REF]. The fusion of the ERderived vesicles also releases their contests into the LCV, functioning as a nutrient delivery system for the bacteria [START_REF] Robinson | Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila[END_REF]. At late infection times, the LVC associates with late-endosome markers, like LAMP-1 (lysosome-associated membrane protein 1), suggesting that the lysosomal compartments provide a nutrient-rich environment for bacterial replication [START_REF] Sturgill-Koszycki | Legionella pneumophila replication vacuoles mature into acidic, endocytic organelles[END_REF]. After 18-24 hours post-infection, the cells are filling with bacteria, ready to be released and infect new cells (Figure 6). The depletion of nutrients in the host cell triggers a transformation within the bacteria. Under these conditions, the bacteria change from their replicative form -characterized by low cytotoxicity and low motility -to their transmissive form, where the bacteria produce their flagellum, causing high motility, and strongly increase the expression of other virulence associated genes [START_REF] Molofsky | Differentiate to thrive: lessons from the Legionella pneumophila life cycle[END_REF][START_REF] Oliva | The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism[END_REF]. Following this phenotypic switch is the bacterial egress. At late infection stages the bacteria seem to disrupt the LCV and are freely present in the host cell cytoplasm. There is evidence that this disruption of the LCV membrane is caused by the ability of the bacteria to form pores, which consequently cause membrane lysis [START_REF] Alli | Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila[END_REF]. The bacterial egress from the cell itself also seems to be pore-related. However, it is suggested that this process is dependent on the host response to the cytoplasmic bacteria. The activation of the NLRC4 inflammasome by the cytoplasmic bacterial flagellin induces this pore formation. This phenotype can neither be seen when cells are infected with an L. pneumophila flagellin knockout strain nor when cells lacking caspase 1 -a crucial component for the activation of the inflammasome -are infected [START_REF] Silveira | Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis[END_REF].

Moreover, several studies have demonstrated that L. pneumophila actively inhibits host cell apoptosis during the early stages of infection [START_REF] Abu-Zant | Anti-apoptotic signalling by the Dot/Icm secretion system of L. pneumophila[END_REF][START_REF] Arasaki | Legionella blocks autophagy by cleaving STX17 (syntaxin 17)[END_REF][START_REF] Losick | NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila[END_REF]. It is only at late stages that macrophages exhibit increasingly apoptotic phenotypes. This regulation of apoptosis by the bacteria represents another possible strategy to control the bacterial egress [START_REF] Speir | Programmed cell death in Legionella infection[END_REF].

Weapon of choice -Legionella's Dot/Icm secretion system

In L. pneumophila, four secretion systems have been characterized: a type I secretion system (T1SS), a T2SS and two T4SS (T4ASS, T4BSS) [START_REF] De Buck | The role of protein secretion systems in the virulence of the intracellular pathogen Legionella pneumophila[END_REF]. In addition, the presence of a T5SS and a TAT secretion system has been predicted through genome sequence analysis in certain L. pneumophila strains (Cazalet et al., 2004). The knowledge about the T1SS and the T4ASS are quite limited, but both have been implicated in bacterial replication and virulence [START_REF] Fuche | Functional type 1 secretion system involved in Legionella pneumophila virulence[END_REF][START_REF] Ridenour | Identification of a gene that affects the efficiency of host cell infection by Legionella pneumophila in a temperature-dependent fashion[END_REF]. The T2SS has been shown to translocate about 25 effectors into the host cell and its activity has been linked to bacterial virulence [START_REF] Cianciotto | Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila[END_REF][START_REF] Cianciotto | Type II Secretion and Legionella Virulence[END_REF][START_REF] Hiller | Secreted phospholipases of the lung pathogen Legionella pneumophila[END_REF][START_REF] Lang | Disulfide loop cleavage of Legionella pneumophila PlaA boosts lysophospholipase A activity[END_REF]. However, the main virulence factor of L. pneumophila and the majority of other Legionella spp. is a type IVB secretion system (T4BSS), the so-called Dot/Icm (defective organelle trafficking/intra cellular multiplication) secretion system. The Dot/Icm system is essential for intracellular replication of the bacteria [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]. There are many other pathogens that possess a T4SS, yet what makes the Dot/Icm of Legionella exceptional is the large number of proteins that are translocated through this system. To this date, more than 330 Dot/Icm effector proteins have been shown to be translocated in L. pneumophila, which represents around 10% of the bacterial genome. Surprisingly, these effectors are highly variable between different Legionella species, with only 8 being conserved between 58 analyzed species (Gomez-Valero et al., 2019). However, if we look at the conservation level of the Dot/Icm system between species, we can see that all components except one (IcmR) are present in all Legionella species (Burstein et al., 2016;[START_REF] Feldman | Coevolution between nonhomologous but functionally similar proteins and their conserved partners in the Legionella pathogenesis system[END_REF]Gomez-Valero et al., 2019). In addition to its protein secretion function, the Dot/Icm system has also been described to transfer DNA, the main function of the first described T4SS, the VirB/D4 system in Agrobacterium tumefaciens [START_REF] Bundock | Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[END_REF][START_REF] Vogel | Conjugative transfer by the virulence system of Legionella pneumophila[END_REF].

The Dot/Icm system is a big complex, with around 27 proteins predicted to be involved in its assembly and function. The main components are an inner membrane complex and a core complex, spanning the periplasmic space. In addition, the Dot/Icm system shows polar localization, which is crucial for bacterial virulence, as well as tethering the bacteria to the LCV, facilitating protein translocation [START_REF] Böck | The Polar Legionella Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane[END_REF][START_REF] Jeong | Polar delivery of Legionella type IV secretion system substrates is essential for virulence[END_REF]. The core complex structure has been described as a "Wi-Fi symbol"-like particle, due to its distinct curved layers (Figure 7) [START_REF] Ghosal | In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography[END_REF]. Recently, new structural components of the Dot/Icm system have been described. A protein, previously not connected to the secretion complex, has been detected by isolating the complex directly from L. pneumophila and analyzing it by mass spectrometry [START_REF] Durie | Structural analysis of the Legionella pneumophila Dot/Icm type IV secretion system core complex[END_REF]. For more in-depth information about the Dot/Icm structure and T4SS in general, please refer to the review by [START_REF] Costa | Type IV secretion systems: Advances in structure, function, and activation[END_REF].

Another vast field of study connected to the Dot/Icm system is effector recognition and their translocation signals. One of the earliest discoveries was that many translocated effectors contain short C-terminal translocation signals consisting of polar and negatively charged amino acids [START_REF] Burstein | Genome-scale identification of Legionella pneumophila effectors using a machine learning approach[END_REF][START_REF] Nagai | A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells[END_REF]. A big step forward was the discovery of the socalled E block motif, a cluster of glutamine residues within 20 amino acids from the C-terminus present in more than 100 Dot/Icm effectors [START_REF] Huang | The E Block motif is associated with Legionella pneumophila translocated substrates[END_REF]. However, it was also seen that adaptor proteins, such as IcmS and IcmW can modulate the translocation of proteins with and without E block motifs, indicating that other mechanisms of translocation recognition are also involved in this process [START_REF] Lifshitz | Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal[END_REF]. Indeed, it was described that DotL, a known type IV coupling protein, together with the adaptor proteins IcmS and IcmW, as well as other components, forms a complex that can regulate effector translocation [START_REF] Sutherland | The Legionella IcmSW complex directly interacts with DotL to mediate translocation of adaptor-dependent substrates[END_REF].

Moreover, recent studies revealed that the composition of this complex seems to be variable and that this variability can determine which effectors are recognized. In addition, the interaction with the effectors seems more dependent on structural features, than exact amino acid motifs (Kim et al., 2020;[START_REF] Meir | Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila[END_REF]. Moreover, the expression of these Dot/Icm effectors is regulated in a life-cycle dependent manner, adding another layer of secretion regulation [START_REF] Aurass | Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors[END_REF]. Taken together, these examples show that effector recognition and translocation are a highly complex processes that enable the bacteria to manipulate the host in specific ways at specific times of the infection.

Legionella: a jack of many (eukaryotic-like) traits

The ability of Legionella species to invade amoebae and macrophages, to manipulate numerous host pathways, and to create an environment enabling bacterial replication, is key to its survival. One notch of this key is the fact that members of this genus, probably due to their close co-evolution with their protozoan hosts, have acquired numerous protein-coding genes from said hosts (Cazalet et al., 2004;Corpas-López et al., 2019;[START_REF] De | [END_REF][START_REF] Gomez-Valero | Comparative and functional genomics of legionella identified eukaryotic like proteins as key players in host-pathogen interactions[END_REF][START_REF] Lurie-Weinberger | The origins of eukaryotic-like proteins in Legionella pneumophila[END_REF]Mondino et al., 2020b). The high number of secreted eukaryotic-like proteins -even though their mode of acquisition is still unknown -represent a unique feature of this bacterial genus. As mentioned before, more than 18,000 effectors have recently been predicted in the genus Legionella, comprising 137 different eukaryotic domains and more than 250 eukaryotic-like proteins (Gomez-Valero et al., 2019).

In L. pneumophila, these eukaryotic-like proteins influence countless pathways in the host, from intracellular trafficking and signal transduction, to metabolism and gene transcription, just to name a few (Figure 8). Here we will discuss some examples of bacterial effectors targeting these pathways.

Influencing intracellular trafficking pathways is essential for L. pneumophila to establish and maintain the LCV. One class of key regulators of membrane trafficking are small GTPases, such as the Arf, Rho, Ras, and Rab families. Small GTPases can be found in an active GTP-bound state and an inactive, GDP-bound state. Switching between these two states is regulated by two classes of proteins, guanine nucleotide exchange factors (GEFs) and GTPaseactivating proteins (GAPs) [START_REF] Cherfils | Regulation of small GTPases by GEFs, GAPs, and GDIs[END_REF]. L. pneumophila secrets several effectors directly influencing small GTPases, one of which is RalF, the first L. pneumophila Dot/Icm secreted effector characterized. RalF acts as a GEF for Arf 1, leading to its recruitment to the LCV [START_REF] Nagai | A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes[END_REF]. It was shown that RalF indeed contains a Sec7 domain, a feature usually found in Arf GEFs and that this domain is highly conserved in the bacterial protein [START_REF] Amor | The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site[END_REF]. Another L. pneumophila effector imitating eukaryotic GEFs is MitF (LegG1). This effector causes activation of Ran proteins, thereby stimulating microtubule stabilization, cell migration, and LCV motility [START_REF] Rothmeier | Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization, Pathogen Vacuole Motility and Infection[END_REF]Simon et al., 2014).

Moreover, MitF (LegG1) was shown to promote mitochondrial fragmentation, consequently leading to a Warburg-like metabolism in the host cell, thus promoting bacterial replication [START_REF] Escoll | Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages[END_REF]. The example of MitF (LegG1) also shows that the same effector can be involved in manipulating several different pathways in the host.

Signal transduction is another potent target for pathogens. Protein kinases and phosphatases are crucial components of many signaling pathways in eukaryotic cells [START_REF] Graves | Protein phosphorylation and signal transduction[END_REF]. The L. pneumophila LegK1, a eukaryotic-like serine/threonine protein kinase, can activate the transcription factor NF-kB. NF-kB is a key regulator of innate and adaptive immunity, inflammation, and cell death [START_REF] Perkins | Integrating cell-signalling pathways with NF-kappaB and IKK function[END_REF]. The activation of NF-kB is achieved by LegK1 mimicking the activity of the IkB kinase (IKK), a protein promoting the degradation of the NF-kB inhibitor IkB. The activity of LegK1 is independent of the known upstream pathways of IKK, suggesting that it is a constitutively active kinase or that it is activated through an unknown mechanism [START_REF] Ge | A Legionella type IV effector activates the NF-kappaB pathway by phosphorylating the IkappaB family of inhibitors[END_REF]. Furthermore, L. pneumophila not only secrets its own protein kinases, but also phosphatases. The recently discovered Ceg4, with its haloacid dehalogenase (HAD)-like domain, is a phosphotyrosine phosphatase. In vitro, Ceg4 attenuates the activation of mitogen-activated protein kinases (MAPK) [START_REF] Quaile | The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways[END_REF].

MAPKs are key regulators of countless cellular processes such as gene expression, cell differentiation, and apoptosis, just to name a few [START_REF] Pearson | Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions[END_REF]. However, the exact consequence of this MAPK inhibition during infection is yet to be determined.

Another process targeted by L. pneumophila is host cell metabolism. There are several effectors connected to the manipulation of metabolic pathways, whether it is to scavenge nutrients and promote bacterial replication or to manipulate host cell pathways that are dependent on different metabolic products. In eukaryotes, amylases are responsible for the hydrolysis of starch and glycogen into glucose, a process usually not occurring in bacteria, since they do not synthesize these products. LamB, an amylase-like protein encoded by L. pneumophila, exhibits strong amylase activity in vitro and its loss severely impairs bacterial growth in human monocyte-derived macrophages and decreases pathogenicity in a mouse model [START_REF] Best | A Legionella pneumophila amylase is essential for intracellular replication in human macrophages and amoebae[END_REF]. However, L. pneumophila not only targets carbohydrates but also lipids, and in particular sphingolipids. One of the main enzymes regulating the intracellular concentration and degradation of sphingolipids, especially sphingosine-1 phosphate (S1P), is the sphingosine-1 phosphate lyase (SPL) [START_REF] Bourquin | Structure and function of sphingosine-1phosphate lyase, a key enzyme of sphingolipid metabolism[END_REF]. L. pneumophila encodes its own SPL-like protein called LpSpl (LegS2). During infection LpSpl promotes the degradation of host sphingolipids. This decrease in sphingolipid levels subsequently impairs the autophagic response of the cell, thus promoting the intracellular survival of the bacteria [START_REF] Rolando | Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy[END_REF].

Last, but certainly not least, is the manipulation of the host cell on the transcriptional level. Many pathogenic bacteria encode so-called nucleomodulins: bacterial effectors that specifically target the host cell nucleus to hijack the transcriptional machinery [START_REF] Bierne | When bacteria target the nucleus: the emerging family of nucleomodulins[END_REF][START_REF] Escoll | Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy[END_REF]. One of the 8 core effectors conserved between all sequenced Legionella species in AnkH (Gomez-Valero et al., 2019). AnkH contains four ankyrin repeats, which in eukaryotic proteins mediate protein-protein interaction. During infection, AnkH binds to the 7SK small nuclear ribonucleoprotein (snRNP) complex, specifically the La-related protein 7 (LARP7). The interaction of AnkH with LARP7 interferes with its binding to the 7SK snRNP complex, ultimately impeding the transcriptional elongation by RNA polymerase II. In addition, by mutating the ankyrin domains, the interaction of AnkH with LARP7 can be abolished, leading to an intracellular growth defect of L. pneumophila [START_REF] Von Dwingelo | Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex[END_REF]. One of the best described nucleomodulins in L. pneumophila strain Paris is the SET (su[var]3-9, enhancer-of-zeste and trithorax)-domain containing eukaryotic-like protein RomA.

In eukaryotes, SET-domain proteins function as lysine methyltransferases, that can target and modify histones, thus influencing chromatin condensation and transcriptional activity [START_REF] Dillon | The SET-domain protein superfamily: protein lysine methyltransferases[END_REF]. RomA is a Dot/Icm secreted effector that targets the host cell nucleus and specifically methylates lysine 14 of histone H3 (H3K14). The methylation of H3K14 -a residue that is usually acetylated -causes a transcriptional repression of specific host genes involved in the innate immune response of the cell (Rolando et al., 2013). Moreover, RomA was also shown to target non-histone proteins. AROS, a regulator of histone deacetylase SIRT1, is also methylated by RomA. However, the exact effect of this methylation is yet to be discovered (Schuhmacher et al., 2018). In addition to its SET-domain, RomA also contains several ankyrin repeats, but their role during infection remains to be investigated. In L. pneumophila strain Philadelphia-1 a homologue of RomA, LegAS4, was reported methylate lysine 4 on histone H3 (H3K4). It is proposed that this methylation promotes the transcription of host cell ribosomal DNA, which boosts bacterial replication [START_REF] Li | SET-domain bacterial effectors target heterochromatin protein 1 to activate host rDNA transcription[END_REF]. However, strain Philademphia-1 also methylates H3K14, thus LegsA4/RomA might fulfill both functions [START_REF] Rolando | Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell[END_REF].

In conclusion, these examples demonstrate that the acquisition of eukaryotic-like proteins has enabled Legionella spp. to manipulate a wide variety of host cell pathways, ensuring the bacterial survival and replication by molecular mimicry of eukaryotic proteins [START_REF] Gomez-Valero | Comparative and functional genomics of legionella identified eukaryotic like proteins as key players in host-pathogen interactions[END_REF]. Moreover, many of the 250+ predicted eukaryotic-like proteins are yet to be investigated and the number of possible strategies employed by the bacteria to subvert the host response seem sheer endless.

Chromatin and infection

Chromatin and Epigenetics

A string of information -Chromatin and its structure Each eukaryotic cell contains an enormous amount of genomic DNA. This DNAaround 2 m from end to end -has to be organized in a meticulously planned manner, to fit into the cell nucleus, with its size of 2-10 µm. To achieve that, genomic DNA interacts with small basic proteins, called histones. Histones form octamers that the DNA is wrapped around. This DNA-histone complex is called a nucleosome (Figure 9). With the help of so-called linker histones, nucleosomes can interact with each other and form chromatin fibers, which lastly, can be organized in higher structures, such as chromosomes [START_REF] Alberts | Chromosomal DNA and Its Packaging in the Chromatin Fiber[END_REF]. Each of these organization steps regulates DNA-accessibility and thereby all DNA-related processes, such as transcription and replication. The regulation of chromatin organization is highly dynamic and flexible, to enable a rapid response of the cell to numerous internal and external stimuli. The histone octamer consists of the highly conserved core histones: H2A, H2B, H3, and H4.

However, several different variants/isoforms of these core histones are known to date, representing another level of chromatin structure regulation [START_REF] Singh | Replication-dependent histone isoforms: a new source of complexity in chromatin structure and function[END_REF]. All of these core histones contain a so-called histone fold region as well as flexible N-terminal tails. The octamer is formed by the interaction of two H2A/H2B dimers with one H3/H4 tetramer. In addition, the linker histone, H1, acts as a clamp, tethering the DNA to the core histones [START_REF] Arents | Topography of the histone octamer surface: repeating structural motifs utilized in the docking of nucleosomal DNA[END_REF]. This interaction is stabilized by several binding sites, such as a fourhelix bundle and the H2A docking domain. The DNA is wrapped around this histone core around 1.7 times, -which equals to 147 base pairs -in a left-handed manner (Figure 10) [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF]. These nucleosome core particles (NCPs) are connected via 15-50 base pair long linker DNA, which, at low salt concentrations, adopts characteristic "beads on a string" appearance [START_REF] Baldi | Beads on a string-nucleosome array arrangements and folding of the chromatin fiber[END_REF][START_REF] Olins | Nucleosomes: The Structural Quantum in Chromosomes: Virtually all the DNA of eukaryotic cells is organized into a repeating array of nucleohistone particles called nucleosomes. These chromatin subunits are close-packed into higher-order fibers and are mod[END_REF]. At higher salt concentrations, chromatin compacts into fibers with a diameter of ∼30-nm [START_REF] Wedemann | Computer simulation of the 30-nanometer chromatin fiber[END_REF].

The exact structural mechanism of chromatin fiber formation is still an open question.

There are two models that have been proposed: the zigzag model and the solenoid model [START_REF] Chen | Structures and Functions of Chromatin Fibers[END_REF]. In the first one, as the name suggests, the nucleosomes are connected by straight linkers, leading to a ladder-like structure, where the nucleosomes zigzag back and forth, leading to a "two start model" [START_REF] Staynov | Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin[END_REF]. In the solenoid model, the nucleosomes are stacked on top of each other connected by bend linkers, forming a "one start" helix-like structure (Figure 11) [START_REF] Mcghee | Higher order structure of chromatin: Orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer length[END_REF]. The type of structure is determined by a variety of factors.

One factor it the average nucleosome repeat length (NRL). The NRL is the average distance between two nucleosomes. Shorter NRLs promote the formation of the zigzag topology, whereas longer NRLs lead to the formation of the more compact solenoid structure (Robinson et al., 2006). Another factor is the binding of the linker histones, H1. Interestingly, H1 is not has highly conserved as the other histone proteins. In humans 11 different H1 variants have been identified [START_REF] Izzo | The histone H1 family: specific members, specific functions[END_REF]. The different H1 variants bind to the NCPs slightly differently, subsequently promoting the formation of one chromatin configuration over the other [START_REF] Song | Cryo-EM Study of the Chromatin Fiber Reveals a Double Helix Twisted by Tetranucleosomal Units[END_REF][START_REF] Zhou | Structural Mechanisms of Nucleosome Recognition by Linker Histones[END_REF].

Besides the histones, there are also several non-histone proteins that are involved in chromatin architecture. One of these non-histone proteins is the FACT (facilitates chromatin transcription) complex. This complex acts as a chaperone for H2A/H2B dimers, and is crucial for nucleosome destabilization during transcription, enabling RNA polymerase II progression [START_REF] Belotserkovskaya | FACT Facilitates Transcription-Dependent Nucleosome Alteration[END_REF]. Other examples of chromatin decondensation by non-histone proteins are the high mobility group (HMG) proteins. HMG proteins regulate chromatin decompaction, which consequently enables the targeting of the chromatin by different regulatory factors. Their binding sites compete with the ones of linker histone H1, attenuating the H1-dependent compaction of chromatin [START_REF] Postnikov | Regulation of chromatin structure and function By HMGN proteins[END_REF]. Another protein competing with H1 for its binding site is the methyl CpG binding protein 2 (MeCP2). However, it has the opposite effect of the HMG proteins. Through its binding, MeCP2 drastically reduces the angles though which DNA enters and exits the nucleosomes, causing a dramatic increase in chromatin condensation [START_REF] Ghosh | MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites[END_REF].

The level of chromatin condensation can influence which genes are accessible for transcription and which genes are silenced. Less condensed chromatin with higher accessibility for the transcription machinery is called euchromatin. In euchromatin, the nucleosomes are wider spaced and are exactly positioned at promoters due to specific DNA sequence guided DNA-protein interactions. The counterpart of euchromatin, heterochromatin, lacks nucleosome positioning and is characterized by high levels of condensation and less transcriptional activity, which includes tissue-specific and developmental genes (the facultative heterochromatin) or gene-poor regions (the constitutive heterochromatin) [START_REF] Morrison | Molecular Complexes at Euchromatin, Heterochromatin and Centromeric Chromatin[END_REF].

As mentioned above, the condensation is dependent on a variety of different factors, including DNA-histone interaction, histone variants, and the binding of non-histone proteins.

All of these factors together represent transcriptional information on chromatin that is independent of the DNA sequence. This sequence-independent information is summarized under the term epigenetics [START_REF] Radford | Exploring the extent and scope of epigenetic inheritance[END_REF].

Change without change -the principle of epigenetics

The definition of epigenetics has changed over the past decades to reflect the newest advances in this ever-growing field of research. One of the best-known definitions is from Russo et al., stating epigenetics is "the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence" [START_REF] Russo | Overview of epigenetic mechanisms[END_REF].

Under this definition, epigenetics only encompasses phenomena that are stringently heritable from one cell to its daughters. However, the latest research in this field suggests that, yes, in some cases these changes can indeed be inherited by the progeny, but this is not always the case. Epigenetic changes are more and more seen as dynamic changes that can appear and disappear within the life time of a single cell. Therefore, newer and updated definitions are becoming more popular. One definition, by Adrian Bird, summarizes epigenetics as follows:

"the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states" (Bird, 2007). This definition does not limit epigenetics to changes stably maintained over several cell generations, but also includes transient modifications, which can still affect cells greatly.

There are several possibilities how epigenetic information can be classified: covalent modifications of the DNA (e.g. DNA-methylation) (Jones, 2012), activity of long non-coding RNAs [START_REF] Mercer | Structure and function of long noncoding RNAs in epigenetic regulation[END_REF], as well as covalent modifications of the histones (Kimura, 2013).

Epigenetic changes and infection

Reading between the genetic lines -classes of epigenetic information

As mentioned before, there are different classes of epigenetic information: activity of long non-coding RNAs, DNA methylation, and post-translational modification of histones.

Long non-coding RNAs (lncRNAs), as the name suggests, have a length of more than 200 base pairs and show no apparent potential to encode proteins. After their transcription, this type of RNA is similarly processed to mRNAs, which includes splicing, 5' capping, and 3' polyadenylation. However, these processes seem to be less efficient for lncRNAs [START_REF] Melé | Chromatin environment, transcriptional regulation, and splicing distinguish lincRNAs and mRNAs[END_REF]. Another difference between the two types of RNA is their localization: mRNAs are almost exclusively found in the cytoplasm, whereas lncRNAs are mainly found in the nucleus, where they are involved in a wide variety of processes, such as chromatin architecture and remodeling, transcriptional regulation, and formation of nuclear bodies [START_REF] Yao | Cellular functions of long noncoding RNAs[END_REF]. In fact, lncRNAs contain structural domains can sense or bind RNAs, via complementary base pair interactions, proteins, and possibly DNA that can induce allosteric conformational changes due to the other structures in the lncRNA [START_REF] Mercer | Structure and function of long noncoding RNAs in epigenetic regulation[END_REF]. The probably most studied lncRNA is the X-inactive-specific transcript (Xist), which controls the X-chromosome inactivation in female mammals [START_REF] Hall | XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome[END_REF]. After transcription, Xist accumulates at many sites across the X chromosome, where it can recruit heterochromatin protein HP1 to satellite repeats. The continued local accumulation of HP1 results in the spreading of facultative heterochromatin to demarcate broad repressive chromosomal domains [START_REF] Wutz | Gene silencing in X-chromosome inactivation: advances in understanding facultative heterochromatin formation[END_REF]. LncRNAs can also directly influence transcription. This can be achieved by, for example, recruiting transcription factors and other chromatin remodelers to specific chromatin regions, or by directly interfering with the activity of RNA polymerase II [START_REF] Mariner | Human Alu RNA is a modular transacting repressor of mRNA transcription during heat shock[END_REF][START_REF] Postepska-Igielska | LncRNA Khps1 Regulates Expression of the Proto-oncogene SPHK1 via Triplex-Mediated Changes in Chromatin Structure[END_REF]. Moreover, lncRNAs are involved in immune and inflammatory processes, which are crucial during viral and bacterial infections [START_REF] Prasad | Host-virus interactions mediated by long non-coding RNAs[END_REF][START_REF] Schmerer | Long noncoding RNAs in bacterial infection[END_REF].

Covalent modification of the DNA, by methylation, and post-translational modifications (PTMs) of the histone tails can be attributed to specific protein complexes that regulate their generation, interpretation, as well as removal. Depending on which step of this process the complexes regulate, they can be classified in three different groups: writers, reader, and erasers.

In eukaryotes, the two main enzyme classes involved in the process of DNA methylation are DNA methyltransferases (DNMTs) -which promote methylation -and proteins of the ten-eleven translocation (TET) methylcytosine dioxygenases family, which actively remove the methyl moieties through a complex cycle of oxidation (Wu and Zhang, 2017). In addition, DNA methylation can also be passively reduced through cell division [START_REF] Lyko | The DNA methyltransferase family: a versatile toolkit for epigenetic regulation[END_REF][START_REF] Rasmussen | Role of TET enzymes in DNA methylation, development, and cancer[END_REF]. DNA can be methylated at the C5 position of cytosine and occurs mainly in a context of CpG dinucleotides. CpG dinucleotides are symmetrically methylated on both strands, enabling the faithful inheritance of pre-existing CpG methylation marks on new daughter strands during semiconservative DNA replication [START_REF] Ming | Mitotic inheritance of DNA methylation: more than just copy and paste[END_REF]. In general, DNA methylation is thought to contribute to the formation of heterochromatic regions on the genome and connected to transcriptional silencing. In contrast, regions of the genome that are enriched for non-methylated CpGs are associated with gene promoters and contribute to transcriptionally permissive environments. However, this notion gets more and more challenged by new research showing that DNA methylation can inhibit but also promote transcription, depending on its position [START_REF] Lyko | The DNA methyltransferase family: a versatile toolkit for epigenetic regulation[END_REF]. DNA methylation is an intricately balanced process, whose deregulation has been linked to several diseases, e.g. Alzheimer's disease and epilepsy [START_REF] Mastroeni | Epigenetic changes in Alzheimer's disease: decrements in DNA methylation[END_REF][START_REF] Zhang | Differential DNA Methylation Profiles in Patients with Temporal Lobe Epilepsy and Hippocampal Sclerosis ILAE Type I[END_REF]. In recent years however, the role of DNA methylation during infection has moved more and more into the limelight. For more detailed information on this topic, we refer the reader to the recent review by Qin et al., in which the involvement of DNA methylation in infection as well as its potential as a target for pathogens is explained [START_REF] Qin | The Role of Host Cell DNA Methylation in the Immune Response to Bacterial Infection[END_REF].

Histone modifications represent the third group of epigenetic information. The modifications mainly target the histone tails, short peptide sequences reaching out of the core histone structure. The histone tails are prone to a wide variety of modifications: methylation, phosphorylation, ubiquitination, SUMOylation, and acetylation, the latter being the most common one (du Preez and Patterton, 2013). PTMs on histone proteins function in elaborate combinations to regulate the many activities associated with chromatin. Many of the enzymes that are responsible for the placement (writers) or the removal (erasers) of these modifications, as well as the multi-protein complexes they participate in, have been extensively characterized.

While the functional significance of some of these modifications remains to be determined, it is clear that they, whether at the single amino acid level or in combinatorial ways, can disturb contacts between neighboring histones as well as histones and DNA (Lawrence et al., 2016).

Acetylation of lysine residues, for example, neutralizes the basic charge of the residue on which it occurs, thereby disrupting electrostatic interactions between the histones and the phosphate group in the DNA, leading to a loser configuration and, in turn, to an open chromatin fiber [START_REF] Shahbazian | Functions of site-specific histone acetylation and deacetylation[END_REF].

Intercepted at the source -how pathogens manipulate epigenetic information

Epigenetic information is crucial for every cell and is not only deciding its function and fate, but also its response to numerous stimuli. This omnipresence of epigenetic information also makes it an excellent target for a range of pathogens, which can manipulate this information to their own advantage. In fact, one of the strategies employed by them is to hijack and alter the host's epigenetic information. The research in this field has been growing rapidly in recent years, highlighting the importance of epigenetic information, not only for the host, but also the pathogens. Both bacterial and viral pathogens have been shown to directly interfere with the different classes of epigenetic information listed before.

The Hepatitis C virus (HCV) is an RNA virus causing chronic hepatitis, which frequently can lead to hepatocellular carcinoma (hepatoma) [START_REF] Westbrook | Natural history of hepatitis C[END_REF].

Moreover, the virus can manipulate lncRNA transcription. In hepatoma patients, an lncRNA called HULC was identified to be involved in cancer progression and its levels in the blood are also used as a post-treatment prognostic tool [START_REF] Panzitt | Characterization of HULC, a novel gene with striking upregulation in hepatocellular carcinoma, as noncoding RNA[END_REF]. It was recently demonstrated that an increase in HULC levels promotes HCV replication and conversely, HULC suppression impedes viral replication. Moreover, the viral non-structural protein NS5A was shown to promote HULC transcription by directly increasing promotor activity, thus increasing viral replication [START_REF] Kitabayashi | Upregulation of the Long Noncoding RNA HULC by Hepatitis C Virus and Its Regulation of Viral Replication[END_REF].

One example of a pathogen directly targeting DNA methylation is Pseudomonas aeruginosa. P. aeruginosa is one of the most common sources for chronical airway infections in patients with cystic fibrosis and, in addition, one of the most common causes for nosocomial pneumonia [START_REF] Fujitani | Pneumonia due to Pseudomonas aeruginosa: part I: epidemiology, clinical diagnosis, and source[END_REF]. It was demonstrated that an infection with P. aeruginosa in bronchial epithelial cells (BECs) inhibits the expression of NODAL by manipulating the methylation levels of its promotor region. NODAL is a key regulator of BEC proliferation as well as BEC-induced T helper cell differentiation [START_REF] Wang | Expression of Nodal on Bronchial Epithelial Cells Influenced by Lung Microbes Through DNA Methylation Modulates the Differentiation of T-Helper Cells[END_REF]. In addition, lung macrophages exposed to extracellular vesicles of P. aeruginosa show a loss of DNA methylation in specific genetic regions, including a downregulation in immune response gene expression (Kyung [START_REF] Lee | Exposure to extracellular vesicles from Pseudomonas aeruginosa result in loss of DNA methylation at enhancer and DNase hypersensitive site regions in lung macrophages[END_REF]. However, the probably best-known pathogen for modulation of host DNA methylation is Helicobacter pylori. H. pylori is a Gram-negative extracellular bacterium that infects the stomach, where it induces excessive acid production, leading to severe inflammation, including ulcers, then chronic inflammation, and finally gastric cancers [START_REF] Touati | When bacteria become mutagenic and carcinogenic: lessons from H. pylori[END_REF]. The vast majority of gastric cancers are commonly associated with H. pylori infections, due to aberrant DNA methylation in gastric mucosae, in particular in promoter regions of genes encoding tumor-suppressor proteins and oncogenes [START_REF] Alvarez | Methylation pattern of THBS1, GATA-4, and HIC1 in pediatric and adult patients infected with Helicobacter pylori[END_REF].

The wide variety of histone modifications, as well as the army of proteins responsible for their regulation, makes this type of epigenetic information a cornucopia of possible manipulations by pathogens. Secreted bacterial proteins targeting the nucleus to hijack cellular pathways by manipulating gene transcription and other nuclear processes are collectively termed nucleomodulins (Figure 12) [START_REF] Bierne | When bacteria target the nucleus: the emerging family of nucleomodulins[END_REF] and their number, as well the histone proteins they target, are continuously increasing. For a recent review extensively describing nucleomodulins and their roles during infection, please refer to (Bierne and Pourpre, 2020).

One example for a bacterium targeting histone proteins with a secreted effector is Shigella flexneri. OspF, a secreted phosphothreonine lyase, modifies MAPK, rendering it permanently inactive in the host cell nucleus. This inactivation consequently inhibits H3S10 phosphorylation by MAPK and impedes the expression of NF-kB regulated proinflammatory genes [START_REF] Arbibe | An injected bacterial effector targets chromatin access for transcription factor NF-κB to alter transcription of host genes involved in immune responses[END_REF] (Figure 12A).

Histone methylation is another known target for pathogens. As mentioned before, L. pneumophila secrets a SET-domain methyltransferase into the host cell, targeting H3K14 (Rolando et al., 2013) (Figure 12B). For more information about this effector, please refer to the chapter "Legionella -a jack of many (eukaryotic-like) traits". But not only L. pneumophila encodes its own methyltransferases, also Mycobacterium tuberculosis secrets similar proteins.

The Rv1988 protein of M. tuberculosis is a methyltransferase targeting a non-canonical histone residue, arginine 42 on histone H3 (H3R42). This methylation, comparable to the methylation of H3K14 by RomA, causes a decrease in gene expression connected to the cell's response to the bacteria. In addition, the deletion of Rv1988 leads to a decrease of bacterial survival in the host [START_REF] Yaseen | Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3[END_REF] (Figure 12C). Chlamydia trachomatis is another bacterium possessing its own histone methyltransferase, named NUE. This T3SS-dependent effector was shown to methylate histones H2B, H3, and H4 in vitro, however its specific target and function in vivo remains to be determined [START_REF] Pennini | Histone methylation by NUE, a novel nuclear effector of the intracellular pathogen Chlamydia trachomatis[END_REF]. Bacillus anthracis, the causative agent of anthrax, has a rather uncommon target for histone methylation, namely histone H1. The bacterial BaSET protein is secreted through an unknown mechanism and localizes to the eukaryotic nucleus, where it methylates histone H1. This methylation seems to specifically target the promotor regions of NF-kB regulated genes, causing a decrease in the transcription of pro-inflammatory genes. Moreover, the deletion of BaSET renders the bacteria unable to cause disease in a mouse model [START_REF] Mujtaba | Anthrax SET protein: a potential virulence determinant that epigenetically represses NF-κB activation in infected macrophages[END_REF].

Histone acetylation, the most common modification of histones, is also subject to manipulation by a variety of pathogens, due to its importance in many immune and inflammatory processes. Listeria monocytogenes, a foodborne pathogen, targets histone acetylation during infection by secreting an effector called LntA into the host cell. LntA translocates to the nucleus where it interacts with BAHD1, a promotor of chromatin compaction and histone deacetylation (Lebreton et al., 2011). BAHD1 is part of a chromatin remodelingcomplex which includes histone methyltransferases, histone deacetylases, and heterochromatin proteins, that promotes heterochromatin formation and gene silencing [START_REF] Bierne | Human BAHD1 promotes heterochromatic gene silencing[END_REF]. The interaction of LntA with BAHD1 impedes its recruitment to the promotor regions of interferon stimulated genes (ISGs), subsequently enhancing acetylation and activating ISGs, a process crucial for infection in a mouse model (Lebreton et al., 2011) (Figure 12D). Another example of manipulation of histone acetylation by a pathogen is the secreted metalloprotease NleC of enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC, respectively). NleC degrades histone acetyltransferase p300 in the host cell nucleus, causing a decrease in histone acetylation, followed by a decrease in the expression of pro-inflammatory IL-8 [START_REF] Shames | The pathogenic Escherichia coli type III secreted protease NleC degrades the host acetyltransferase p300[END_REF] (Figure 12E). Mycobacterium tuberculosis goes one step further by expressing its own histone acetyltransferase, Rv3423.1. This protein was shown to directly interact with histone H3 and acetylating K9/14. In addition, the recombinant expression of Rv3424.1 in Mycobacterium smegmatis increased intracellular replication of the bacteria. However, the exact consequence of this hyperacetylation remains an open question [START_REF] Jose | Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase[END_REF] (Figure 12C). These examples show that the balance between histone acetylation and deacetylation is a finely tuned process crucial for cellular processes and thus, presents a potent target for pathogens to promote their own survival. This topic, with a strong focus on histone deacetylases, will be discussed in more detail in the next chapter. [START_REF] Goll | Eukaryotic cytosine methyltransferases[END_REF]. This repressive 67 effect is mostly connected to promotor methylation (Jones, 2012) [START_REF] Breen | Modulating the masters: chemical tools to dissect CBP and p300 708 function[END_REF]. The balanced interplay between writers, readers, and erasers 121 is paramount to a variety of cellular processes [START_REF] Biswas | Epigenetic tools (The Writers, The Readers and The Erasers) and 701 their implications in cancer therapy[END_REF]. [START_REF] Ito | p300/CBP-820 mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by 821 MDM2[END_REF][START_REF] Ito | MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation[END_REF]Menden et al., 143 2019). MyD88 is a crucial component of toll-like-receptor (TLR) signaling, leading to the 144 activation of NF-B, consequently promoting the production of pro-inflammatory cytokines 145 [START_REF] New | TLR Adaptor Protein MYD88 Mediates Sensitivity to HDAC Inhibitors via a Cytokine-922 Dependent Mechanism[END_REF]. MyD88 is deacetylated by HDAC6, thereby increasing both its activity 146 and half-life time (Menden et al., 2019). Thus, acetylation of MyD88 is paramount in the TLR 147 signaling cascade because it supports the interaction of MyD88 with downstream effectors 148 such as TNF receptor associated factor-6 (TRAF6) [START_REF] Kawai | Page 27 of 38 https://mc.manuscriptcentral.com/microlife Manuscripts submitted to microLife MyD88 and TRAF6[END_REF].

F o r R e v i

149

The regulation of HDAC activity is a complex and multilayered process with four 150 main regulation mechanisms: gene expression, subcellular localization, protein complex 151 formation, and post-translational modifications (PTMs). The expression of histone 152 deacetylase-coding genes has been studied in detail, but exceeds the scope of this review [START_REF] Sengupta | Regulation of histone deacetylase activities[END_REF]. However, an interesting characteristic of HDACs is that some can 154 regulate their own expression by interacting with their own promoter regions. For example in 155 mice, HDAC1 autoregulates its own expression by deacetylating the promoter region and, 156 thereby, repressing its own gene expression (Schuettengruber, Simboeck, Khier, & Seiser, 157 2003). In addition, HDAC1 seems to be involved in regulating the gene expression of 158 HDAC2 and HDAC3 [START_REF] Lagger | 853 Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression[END_REF].

159

The regulation dependent on the subcellular localization is seen mostly with class IIa 160 HDACs since these proteins shuttle between the nucleus and the cytoplasm. A striking 161 example for the regulation by subcellular localization is the interaction of HDAC4 and 162 HDAC5 with the 14-3-3 family adaptor proteins. In mammals, the seven different isoforms of 163 14-3-3 proteins are highly conserved and they assist in various processes such as protein-164 protein interactions, protein folding, and protein localization [START_REF] Stevers | Modulators of 14-3-3 Protein-Protein Interactions[END_REF]. HDAC4

165 and HDAC5 enzymes directly interact with 14-3-3 proteins, leading to an accumulation of 166 these HDACs in the cytoplasm. This cytoplasmic retention of HDAC4 and HDAC5 dampens 167 the transcriptional repression of a subset of genes [START_REF] Grozinger | Regulation of histone deacetylase 4 and 5 and 790 transcriptional activity by 14-3-3-dependent cellular localization[END_REF]. For 168 HDAC7, a similar mechanism of regulation is known [START_REF] Kao | Mechanism for 834 nucleocytoplasmic shuttling of histone deacetylase 7[END_REF]. A different 169 regulation mechanism, which is best studied in class I HDACs, is the control of HDAC 174 2013;[START_REF] Lakisic | Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and 859 Regulation of Steroid Metabolism[END_REF][START_REF] Millard | Targeting Class I Histone 904 Deacetylases in a "Complex" Environment[END_REF]. As part of these 175 complexes, the HDACs become maximally activated and are targeted to specific regions of 176 chromatin. Moreover, through scaffolding proteins, HDACs are connected with other 177 important epigenetic regulators [START_REF] Lakisic | Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and 859 Regulation of Steroid Metabolism[END_REF][START_REF] Millard | Targeting Class I Histone 904 Deacetylases in a "Complex" Environment[END_REF]. The NuRD The recruitment of these complexes to specific chromatin regions is mediated by their 189 interaction with different transcription factors or directly by histone-recognition motifs

178
F o r R e v i e w O n l y F o r R e v i e w O n l y F o r R e v i e w O n l y 11 285
In B cells, a SIRT1 knockout shows a decrease in viability and IgM production (Han 286 et al., 2019). Another histone deacetylase, HDAC3, is involved in regulating B cell 287 maturation and function. The loss of HDAC3 impairs B cell maturation and changes the 288 expression of numerous B cell genes. In addition, the absence of HDAC3 reduces productive 289 VDJ rearrangement, a process essential for generating suitable B cell antigen receptors 290 [START_REF] Stengel | 994 Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and 995 B-cell development[END_REF].

291

These examples summarize a small fraction of what is known about the involvement 292 of HDACs in the activation and regulation of immune cells. However, they underline the 293 abundance of HDACs in these processes. For a more detailed discussion on this specific topic 294 we refer the readers to excellent reviews about this topic [START_REF] Busslinger | Epigenetic control of immunity[END_REF]295 Shakespear, Halili, Irvine, Fairlie, & Sweet, 2011). In the following sections of this review, 296 we will focus on examples of how HDACs may be manipulated by pathogens and discuss the 297 possibility to use HDAC inhibitors as treatment options against infectious diseases. In T. gondii, the secreted serine-threonine kinase ROP18 was identified as a major 400 virulence factor [START_REF] Taylor | A secreted 1004 serine-threonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. 1005 Science[END_REF]. Recently, it was shown that ROP18 targets the host 401 protein RTN1-C, a key regulator of the stress response of the endoplasmic reticulum.

416

The histone deacetylase SIRT1 shuttles between the nucleus and the cytoplasm, a 417 process regulated by the PI3K-AKT signaling pathway (Tanno, Sakamoto, Miura, 418 Shimamoto, & Horio, 2007). Infection with Salmonella typhimurium, which causes 419 nontyphoidal salmonellosis, a severe enteritis leading to diarrhea and fever, induces the 420 activations of mTOR and AKT. This activation leads to the accumulation of SIRT1 in the 421 cytoplasm, where it is degraded in a lysosome-dependent manner. This relocalization of 422 SIRT1 allows the bacteria to evade the host autophagy response and it promotes their 423 intracellular survival. The type III-secretion system is crucial for this process, however no 424 specific effector connected to this mechanism has been identified (Ganesan et al., 2017).

425 Another bacterium, L. monocytogenes has its own method to manipulate the PI3K-AKT 426 signaling pathway. One of its surface proteins, InlB, interacts with the host c-Met receptor, 427 which subsequently leads to the uptake of the bacteria by non-phagocytic cells. In addition, 467 growth [START_REF] Cheng | Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic 733 target against tuberculosis[END_REF].

468

Changes in HDAC expression have also been observed during viral infections. The 469 hepatitis C virus (HCV), causing chronic hepatitis, upregulates HDAC9 expression, which 470 linked to metabolic changes, that can lead to hyperglycemia and type 2 diabetes mellitus.

471 HDAC9 was shown to play a major role in regulating hepatic gluconeogenesis by 472 deacetylating Forkhead box O1 (FoxO1), a transcription factor central to the regulation of 473 gluconeogenic genes in hepatocytes. These results suggest that HDAC9 is a key regulator in 474 gluconeogenesis connected to type 2 diabetes (Jizheng Chen et al., 2015).

475

Influenza A virus (IAV) infection decreases the protein levels of HDAC4, a histone 476 deacetylase known to regulate antiviral responses (Q. [START_REF] Yang | Host HDAC4 regulates the 1037 antiviral response by inhibiting the phosphorylation of IRF3[END_REF]. HDAC4

477 downregulation is caused both, at the gene expression level, where the viral RNA-478 endonuclease PA-X strongly interferes with HDAC4 mRNA levels, and at the protein level, 479 due to the proteolytic activity of caspase 3. However, the exact mechanism of caspase 3 480 activation by IAV remains to be elucidated. Furthermore, this decrease cannot be rescued by 481 adding caspase inhibitors. This demonstrates that the virus employs two independent 482 mechanisms to impede HDAC4 and its antiviral activity (Galvin & Husain, 2019).

483

The hepatitis B virus (HBV) induces autophagy in infected hepatocytes, a step critical 484 to promote its own DNA replication [START_REF] Sir | The early autophagic 990 pathway is activated by hepatitis B virus and required for viral DNA replication[END_REF]. One of the key activators of autophagy 485 is the viral protein X (HBx), which was shown to inhibit binding of transcription factor SP1 to 486 the promoter region of HDAC1, hence repressing HDAC1 expression. The lack of HDAC1

487 subsequently causes the accumulation of acetylated high mobility group box 1 (HMGB1), 488 causing its translocation from the nucleus to the cytoplasm, where it acts as an enhancer of 489 autophagy by directly binding Beclin1, a major regulator of autophagy. In addition, HBx is 490 directly involved in the regulation of autophagy, by binding HMGB1 and promoting the 491 complex formation with Beclin1 [START_REF] Fu | Crosstalk between 770 hepatitis B virus X and high-mobility group box 1 facilitates autophagy in hepatocytes[END_REF]. This shows that a single viral protein not 492 only changes expression levels of a specific HDAC, but it can also influence processes 493 downstream of this histone deacetylase.

495 Pathogens may mimic HDAC enzymes

496 Pathogens can influence many steps in the regulation of HDAC activity, but some go even a 497 step further by encoding HDAC-like proteins to manipulate the host cell.

498

HDAC-like proteins have been structurally described in different bacterial species, but 499 were never assessed for their possible involvement in virulence (Finnin et al., 1999;Hildmann

536

Interestingly, the two HDAC domains are matching differently with the HMM profile 537 as the match of the group 2 HDAC domain against the HMM profile is complete, whereas the 538 match of the group 1 HDAC domain is only partial. To understand their evolution within the 539 genus Legionella, we mapped their distribution on the phylogenetic tree of the genus that we 540 had constructed previously (Gomez-Valero et al., 2019). We found that the distribution of the 541 two HDAC groups followed the phylogeny of the species. Their distribution on the Legionella 542 phylogeny suggests that both HDAC types have followed an independent vertical evolution 543 for a long time, with several loss events in specific strains/clusters. According to the most 544 parsimonic scenario, proteins of group 1 were probably present in the ancestor of Legionella, 545 as they are also present in the outgroup species L. geestiana and L. osnabrueckensis. The 546 second group might have been acquired at the same time but was lost in the outgroup species 547 or it was acquired in a more recent ancestor. However, additional analyses are necessary to 548 determine the exact evolution of the two different HDAC domains. It will be particularly 549 exciting to functionally analyze whether these HDAC domains confer histone deacetylase 550 activity in the host cell and help Legionella to establish their intracellular infection cycle.

551

The examples discussed above further show that histone deacetylases are auspicious 552 targets for pathogens, whether it is by using their activity for their own advantage, obstructing 553 their intracellular localization or directly interfering with their expression on the genetic level.

554 As promising as HDACs are for pathogens, the same can be said about their potential as

555

HDAC inhibitors as promising therapeutics for infections

558 In recent years, it became clear that HDAC functions are not only exploited, or inhibited by 559 pathogens, but may also be used as novel targets to fight infectious diseases (Table 2). Their 560 influence on immune cells and the host immune response is multifaceted, underlining the 561 importance they might have as therapeutic targets.

562

The pre-treatment of macrophages with broad spectrum histone deacetylase inhibitors 563 (HDACi), such as suberanilohydroxamic acid (SAHA, also known as vorinostat) and 604 parasites in a cell culture model [START_REF] Guo | The Existing Drug Vorinostat as a 794 New Lead Against Cryptosporidiosis by Targeting the Parasite Histone Deacetylases[END_REF].

564

605

The efficacy of a vorinostat derivative was also tested using a mouse model of visceral 606 leishmaniasis, a severe infection with members of the genus Leishmania. Visceral 616 Another mouse model, one for fungal keratitis caused by Fusarium solani, illustrated that 617 HDAC1 is upregulated during infection, causing an overexpression of proinflammatory 618 cytokines. This overexpression and the resulting keratitis can be counteracted using vorinostat 619 (X. Li et al., 2019).

620

Taken together, the studies presented here show that the inhibition of histone 621 deacetylases may be a potent mean to fight infectious diseases. However, further research is

622

Aim of the PhD thesis

The influence of bacterial effectors on the host's epigenetic landscape has moved into the limelight in the past decade. Numerous strategies have been identified how pathogens manipulate gene transcription, but the goal is always the same, ensure their own survival and promote their replication.

For L. pneumophila it was shown that many secreted effectors interfere with cell signaling to disable the defenses that are aimed at controlling and eliminating invaders (Mondino et al., 2020a). As mentioned before, RomA is directly secreted by L. pneumophila to modify the host chromatin by methylating lysine 14 of histone H3 (H3K14), a usually acetylated residue, consequently leading to a down regulation of host response genes (Rolando et al., 2013).

The finding that RomA methylates a usually acetylated histone mark led to the question how deacetylation of this mark might happen to allow its subsequent methylation, and whether this is also induced by the bacteria directly or via recruiting host histone deacetylases (HDACs).

An in-depth bioinformatics search for additional putative chromatin modifying effectors in the L. pneumophila genome, led to the identification of a protein predicted to code for a eukaryotic histone deacetylase, later named LphD (Legionella pneumophila histone deacetylase).

The main objective of this work was to understand if and how LphD targets host

Results

Project Progress

As mentioned before, the project was split into three main objectives:

I.
Decipher the functional role of the LphD in the eukaryotic cell

To determine the influence of LphD during L. pneumophila infection, we constructed a lphD knock out strain (∆lphD). We then compared the ability to replicate of this strain with the wild type strain, using different infection models (A. castellanii and human THP-1 cells),

showing that the ∆lphD strain exhibits a replication defect. In addition, we proofed that LphD is secreted into the host cell during infection and that this secretion is dependent on the Dot/Icm secretion system. Furthermore, we followed the intracellular localization of LphD in the host cell, first in transfected cells and later during infection. In both models we observed a clear nuclear localization of LphD, further supporting the hypothesis of its chromatin modifying function.

II. Solve the structure and the biochemical function of LphD

To determine the biochemical function, we first decided to construct a catalytic inactive version of LphD. Through sequence analysis and comparison to eukaryotic HDACs we predicted the catalytic center of the enzyme (Y392). By a single base pair mutation, we replaced this tyrosine residue with a phenylalanine, a mutation previously used to generate catalytically dead eukaryotic HDACs. We then set up a purification protocol to isolate LphD from E. coli.

This purified LphD was then used for several different assays. We first showed that the enzyme indeed has lysine deacetylase activity in an in vitro assay. Moreover, the Y392F mutation completely abolished the enzymatic activity. Interestingly, we also observed that Trichostatin A (TSA) -a known inhibitor of eukaryotic Zn 2+ -dependent HDACs -impedes the activity of LphD, highlighting its homology to this class of enzymes. To further investigate the enzyme kinetics, we set up a collaboration with Jérémy Berthelet and Fernando Rodrigues-Lima (Université de Paris). By comparing the catalytic efficiency of LphD on different acetylated histone H3 peptides, they revealed that LphD shows the highest efficiency for H3K14, the same residue targeted by RomA. In our lab we confirmed these results in the context of infection,

showing that cells infected with L. pneumophila have lower levels of H3K14ac, and that this decrease is indeed dependent on the presence of LphD. This effect on acetylation seems to be specific for H3K14, since other tested residues show no difference between the wild-type strain and the lphD knock out.

For the last part of this project, deciphering the structure of LphD, we set up a collaboration with Mathilde Ben Assaya and Anne Marie Wehenkel (Institut Pasteur). Through numerous tests they were able to improve the purification process, as well as optimize the crystallization conditions. They generated several crystals so far, however, the resolution obtained when analyzed in the synchrotron did not allow an in-depth analysis. This project is still on-going and they work tirelessly to further optimize the crystallization conditions. To get a first glimpse of the putative structure, they used the AlphaFold 2 artificial intelligence program.

III. Understand the mechanisms of function of the LphD in a context of multi-effector synergy as well as its implication on transcriptional changes during infection

To understand if LphD directly influences the activity of RomA, we analyzed the H3K14 methylation levels in cells infected with the ∆lphD strain. We observed a drastic decrease of H3K14me in the absence of LphD, further supporting the hypothesis of a synergy between LphD and RomA. In addition, we showed that LphD, as well as RomA, interacts with KAT7, a eukaryotic histone acetyl transferase known to target H3K14. This interaction confirmed in transfected cells as well as in the context of infection. To get a better understanding of the influence of both bacterial effectors in the transcriptional landscape of the cells, we performed RNA-seq of infected THP-1 cells in a time course experiment. The analyses of these data revealed dynamic changes in the transcription of the host cell within the first few hours of infection, mainly focused on the response to the bacterial intruders. Furthermore, we showed by using RT-qPCR, that LphD and RomA can influence gene transcription synergistically, but also independently from each other. These results highlight the fine-tuned regulation of the host's transcriptional response by L. pneumophila via the secretion of LphD and RomA.

This work is summarized in my main publication (in preparation for submission).

LphD crystal structure

As mentioned before, to determine the structure of LphD, we set up a collaboration with Mathilde Ben Assaya and Anne Marie Wehenkel (Structural Microbiology, Institut Pasteur).

Together with the Production and Purification of Recombinant Proteins Technological

Platform at Institut Pasteur, they developed the purification protocol for HIS6-tagged LphD.

Crystallization screens were performed using LphD protein alone or in complex with the inhibitor Trichostatin A (TSA) (molar ratio 1:4) with the sitting-drop vapour diffusion method and a Mosquito nanolitre-dispensing crystallization robot (TTP Labtech). The apoprotein resulted in small needle-like crystals that could not be optimized and did not diffract.

Small crystals arose for the protein-inhibitor complex in the following conditions crystallization condition: I) 0.2 M MgCl2, 0.1 M MES pH 6.5, 25% PEG 4K, II) 22% (w/v) PEG 8k, 250 mM MgCl2, 0.1 M MES pH 6.5. The crystals were cryo-protected in mother liquor containing 33%

(v/v) ethylene glycol or glycerol33% (v/v) (Figure 13).

The crystals were tested at the Soleil synchrotron (PX1 and 2), but diffracted poorly (only several spots at low resolution). We are currently optimizing the crystallization conditions to improve the diffraction properties. In parallel we have generated a structural model using AlphaFold 2 [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF]. This model suggests that the N-and C-terminal flanking regions of the protein are disordered, and these limits could be used to design new constructs.

In addition, when highlighting the catalytic tyrosine (Y392) and other conserved residues implicated in substrate binding in eukaryotic HDACs (H177/178, D218/330, N218, Y248) (Uba and Yelekçi, 2017), we can see that it is clearly localized in the probable binding pocket of the enzyme (Figure 14). Apart from the flanking regions that do not present a secondary structure, the core of the protein is predicted with high confidence and will be used to derive structurebased hypotheses for future experiments.

Moreover, we used the Phyre2 structure prediction tool to look for structurally similar proteins. This led to the identification of HDAC10 (of Danio rerio) as a possibly highly homologous protein [START_REF] Kelley | The Phyre2 web portal for protein modeling, prediction and analysis[END_REF]. When superposing the HDAC domain of LphD with the HDAC domain of a catalytic inactive HDAC10 (Y307F) [START_REF] Hai | Histone deacetylase 10 structure and molecular function as a polyamine deacetylase[END_REF], we can see a clear overlap in the position of the binding pockets as well as the position of the catalytic tyrosine (Figure 15).

These results further highlight the similarity of the bacterial HDAC domain with its eukaryotic counterparts. In addition, future experiments can be designed to test the involvement of different amino acid residues in the activity of LphD. E-mail: cbuch@pasteur.fr, mrolando@pasteur.fr !

INTRODUCTION

The accessibility of chromatin to transcription factors and the subsequent changes in gene expression are a key regulatory mechanism in cells. The process of changing this accessibility is known as chromatin remodeling. The dynamic modification of histones, the small basic proteins that DNA is wrapped around to form chromatin, is one of the most studied mechanisms of chromatin remodeling. The so-called histone tails -peptide sequences reaching out of the core histone structure -are subjected to a variety of modifications (du Preez and Patterton, 2013). In fact, histones undergo various forms of post-translational modifications (PTMs), such as acetylation, methylation, ubiquitination and phosphorylation (Kouzarides, 2007). However, methylation and acetylation of the amino-terminal tail of histone proteins are the most studied and best characterized PTMs. They have been the first post-translational modifications discovered and were linked to altered rates of DNA transcription almost six decades ago [START_REF] Allfrey | ACETYLATION AND METHYLATION OF HISTONES AND THEIR POSSIBLE ROLE IN THE REGULATION OF RNA SYNTHESIS[END_REF][START_REF] Murray | The Occurrence of iε-N-Methyl Lysine in Histones[END_REF]. Importantly, combinations of acetylation and methylation of lysine residues in histone tails can function in a concerted manner with both cooperative or antagonistic functions. Methylation is associated to compaction of chromatin and reduced transcription [START_REF] Rice | Histone methylation versus histone acetylation: new insights into epigenetic regulation[END_REF]. Acetylation impairs the affinity of histones to DNA thus, loosening chromatin compaction, promoting the recruitment of transcription factors [START_REF] Mathis | Effect of histone acetylation on structure and in vitro transcription of chromatin[END_REF] and increasing the mobility of histones along the DNA [START_REF] Cosgrove | Regulated nucleosome mobility and the histone code[END_REF].

Therefore, the balanced activity of enzyme classes involved in attaching (histone methyltransferases and histone acetyltransferases (HAT)) and removing (histone demethylases and histone deacetylases (HDAC)) these groups ensures the correct expression of specific genes at specific times.

Numerous different stimuli have been shown to influence the PTM levels of histones, an emerging topic is the manipulation of histone modifications by different pathogens. Histone acetylation has been shown to be a potent target for a variety of pathogens to promote their replication and the research on these processes has gained momentum in recent years (An et al., 2018;Galvin and Husain, 2019;Ganesan et al., 2017).

Legionella pneumophila, a facultative intracellular, Gram-negative bacterium that parasitizes free-living protozoa, is the causative agent of a severe atypical pneumonia in humans, called Legionnaires' disease (Mondino et al., 2020a). L. pneumophila has closely co-evolved with its eukaryotic hosts, giving rise to numerous mechanisms to manipulate the host and to thrive in this otherwise hostile intracellular environment. One consequence of Legionella co-evolution with its hosts is the presence of a high number of genes encoding eukaryotic-like proteins in the Legionella genome (Cazalet et al., 2004;[START_REF] De | [END_REF]Gomez-Valero et al., 2019).

4

A key factor for the manipulation of the host cell is the Dot/Icm type-IV secretion system (T4SS) which is highly conserved in the entire genus and is used by the bacteria to translocate more than 300 proteins into the host cell (Burstein et al., 2016;Ensminger, 2016;Gomez-Valero et al., 2019). Several of these secreted effectors represent eukaryotic-like proteins and have been functionally characterized in order to understand how Legionella spp. manipulate the host cell response thus, facilitating bacteria survival and replication (Mondino et al., 2020b). One of these secreted effectors, RomA, is a SET-domain histone methyltransferase that directly modifies host chromatin by tri-methylating lysine 14 on histone H3 (H3K14) (Rolando et al., 2013). This histone modification is present at very low levels in human cells (Zhao et al., 2018), however, its distribution is increased under specific conditions (e.g. stress response) (Zhu et al., 2021). The drastic increase in H3K14 methylation -due to RomA activity -revealed a new strategy L. pneumophila employs to subvert the host response. By secreting these so-called nucleomodulins, effectors targeting the host cell nucleus, the bacteria are able to vastly change the chromatin landscape (Bierne and Pourpre, 2020). In fact, RomA activity strongly affects the expression of several genes involved in the host's response to the infection (Rolando et al., 2013). However, it should be noted that K14 is a major acetylation site on histone H3, together with K9, K18, K23 and K27. Although histone marks are not necessarily explicit codes, they may still serve as epigenetic indicators of chromatin state associated with gene activation and repression (Kimura, 2013). In general, histone acetylation is correlated with transcriptional activation, being localized to transcription start sites and/or enhancers of potentially actively transcribed genes [START_REF] Karmodiya | H3K9 and H3K14 acetylation cooccur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells[END_REF]. The fact that RomA targets a usually acetylated histone residue gave rise to the question of how the bacteria might influence the acetylation of H3K14 to enable its subsequent methylation. Due to the plethora of eukaryotic-like proteins in L. pneumophila we hypothesized that the bacteria might encode their own eukaryotic-like histone deacetylase (HDAC). Here we report that L. pneumophila encodes an HDAC-like protein that specifically targets H3K14 and works in synergy with RomA to ensure intracellular replication of the bacteria and finetune the host's gene expression to its own advantage.

!

5

RESULTS

L. pneumophila secretes the effector LphD, an active HDAC

Bioinformatic searches for L. pneumophila strain Paris genes encoding histone deacetylase-like domains, identified the gene lpp2163 as a possible candidate. This gene encodes a 425 amino acid long protein -with residues 54-409 predicted to be a Zn 2+ -dependent histone deacetylase domain -that we named LphD (Legionella pneumophila histone Deacetylase) (Schator et al., 2021). Indeed, the histone deacetylase domain contains conserved binding pocket residues and the active-site tyrosine (Y392), features found as part of the so-called charge relay system in eukaryotic histone deacetylases (Figure S1) (Uba and Yelekçi, 2017).

To determine if LphD indeed holds lysine deacetylase activity, we purified the full-length protein in Escherichia coli and performed a fluorometric enzymatic assay that allows the quantification of lysine deacetylation on an acetylated lysin side chain. Figure 1A shows that LphD exhibits strong in vitro lysine deacetylase activity in a dose-dependent manner.

Furthermore, the single amino acid substitution (Y392F) on the predicted active site completely inactivates the enzyme. Adding Trichostatin A (TSA), a broad range inhibitor of Zn 2+dependent histone deacetylases, LphD activity was drastically reduced (Figure 1A), further suggesting that LphD is a Zn 2+ -dependent histone deacetylase.

To identify the substrate recognized by LphD, we performed an enzymatic activity assay and measured the catalytic efficiency of LphD on histone H3 peptides acetylated on different lysine residues (H3K4, H3K9, H3K14, H3K18, H3K23 and H3K27). We measured the activity of both LphD and LphD Y392F against these different peptides while varying the peptide concentration. We clearly observed the highest catalytic efficiency of LphD on H3K14, compared to the other tested residues (Figure 1B). The deacetylase activity of LphD on H3K14 was also confirmed on histone octamers isolated from human cells (Figure 1C, Figure S2A).

In addition, the H3K14ac antibody was tested to ensure its specificity (Figure S2B).

Given that LphD is an active histone deacetylase targeting H3K14 in vitro, we characterized its function in an intracellular context. To assess whether LphD is indeed an effector secreted by the T4SS we infected THP-1 cells with an L. pneumophila strain expressing a b-lactamase-LphD fusion construct and measured the effector translocation by quantifying the number of cells exhibiting b-lactamase activity against a fluorescent substrate (CFF4) in their cytosol. A b-lactamase-RomA fusion protein was used as positive control. This clearly showed that LphD is a secreted effector (Figure 2A). By knocking out a central component of the Dot/Icm 6 secretion system (dotA), we could completely abolish the secretion of the LphD fusion protein, confirming that the secretion process is T4SS-dependent (Figure 2A). To assess the subcellular localization of LphD, we transiently transfected HeLa cells with an EGFP-LphD fusion product. The LphD fusion protein accumulates in the nucleus in transfected cells, compared to the cytosolic localization typically found for EGFP (Figure 2B). To note, the LphD-Y392F catalytic inactive mutant is still located in the nucleus of transfected cells (Figure S2C). To determine the nuclear localization of LphD during infection, we generated a specific anti-LphD antibody from rabbits (Figure S2D) to follow the translocation of LphD. This showed that, at late stages of the infection cycle, LphD accumulates in the host cell nucleus (Figure 2C).

Furthermore, the nuclear accumulation of LphD in transfected cells, correlates with a drastic decrease in H3K14ac signal, an effect not seen in cells transfected with the Y392F mutant (Figure 2D).

To better assess the influence of LphD on the epigenetic status of H3K14 during infection, we isolated histones from cells infected either with L. pneumophila wild type or the ∆lphD strain and followed H3K14 acetylation as a function of time. Figure 2E shows that L. pneumophila wild type leads to a decrease in H3K14ac within 7 hours of infection, dependent on the presence of LphD, as the infection with the ∆lphD strain led to an increase in H3K14ac in the same timeframe (Figure 2E). Moreover, this is specific for H3K14, since other tested residues (H3K18 and H3K23) did not show a difference between the wild type and the ∆lphD infection (Figure S3A,S3B).

To evaluate the role of LphD in infection, we analyzed the replication of the wild-type strain compared to the ∆lphD strain in Acanthamoeba castellanii, a natural host of L. pneumophila, as well as in macrophages derived from THP-1 cells. This revealed that the ∆lphD strain has a slight but consistent growth defect compared to the wild-type strain (Figure S3C and2F). In contrast, when grown in liquid medium, both strains show the same growth rate (data not shown). To determine if the deficiency in intracellular replication is dependent on the presence of LphD, we trans-complemented the ∆lphD strain with the full length lphD under the control of its native promotor, which led to a complete reversion of the defective phenotype (Figure 2G). The over expression of the gene, due to the plasmid copy number, even induced a clear increase in the replication of the mutant, further supporting the conclusion that LphD plays a role in virulence and intracellular replication.

7

LphD and RomA modify H3K14 in synergy

As previously reported, the secreted effector RomA is able to specifically target and methylate H3K14 (Rolando et al., 2013). We observed a clear decrease in H3K14 acetylation in presence of RomA, thus, we wondered if this was due to a genome wide H3K14me accumulation, or a specific and targeted deacetylase activity, possibly driven by the bacteria. Given the activity of LphD, we hypothesized that the two effectors act in synergy to manipulate the host transcriptional response. We first investigated the influence of LphD on the activity of RomA by analyzing the H3K14 methylation level of extracted histones from cells infected with the ∆lphD strain. Indeed, this showed that the absence of LphD significantly reduces the level of H3K14me already in the early stages of infection (1-3 hours) (Figure 3A). This strongly suggests that the two effectors act in synergy to modify the host chromatin landscape.

LphD and RomA hijack the HBO1 complex

To investigate the mechanism by which LphD and RomA target host chromatin, we sought to identify host complexes that the two bacterial effectors may interact with. To isolate potential targets of LphD, we performed affinity chromatography by GFP-trap pull down, followed by protein identification by mass spectrometry. Pull-down experiments of HEK293T cells transfected with an EGFP-LphD construct followed by MS/MS analysis identified a total of 687 significantly enriched proteins, compared to the control (EGFP) (Figure 3B). To identify candidate binding proteins with increased confidence, we set a threshold for proteins with at least two unique peptides detected and that displayed a significant (false discovery rate <0.1) >4-fold change compared to control condition (GFP). Interestingly, among the set of potential LphD binding proteins, many of the identified peptides were derived from proteins that are known to be involved in epigenetic regulation of the cell (Table S1). In particular, the histone acetyl transferase (HAT) KAT7 (HBO1) was one of the most promising candidates (Figure 3B). KAT7 is the enzymatic subunit of the so-called HBO1 complex, comprised of KAT7, BRPF1-3, ING4/5 and MEAF6. This complex is well-known to bind histone H3, regulating the acetylation of K14 (Figure 3C) (Xiao et al., 2021). To validate the interaction between LphD and KAT7, we performed co-immunoprecipitations (Co-IP) using GFP-trap to pull down EGFP-LphD (or EGFP) and blotted for the binding of endogenous KAT7. Figure 3D shows that EGFP-LphD indeed interacts with endogenous KAT7, compared to EGFP alone. Reverse immunoprecipitation also verified KAT7/LphD complex formation in transfected cells (Figure S4A). Importantly we observed that the binding of LphD to KAT7 is independent of its enzymatic activity, as the catalytically inactive LphD-Y392F still binds KAT7. Furthermore, 8 histone H3 is immunoprecipitated as well, confirming that the complex occurs at the chromatin level (Figure 3D, for IP control see Figure S4B). To determine if LphD targets the entire HBO1 complex and if RomA also participates in it, we performed co-IP of EGFP-LphD and EGFP-RomA and checked for the presence of the different components of the HBO1 complex (Figure 3E, for IP control see Figure S4C). We observed that LphD immunoprecipitates all components of the HBO1 complex (BRPF1, KAT7, MEAF6 and ING5), whereas RomA seems to preferentially bind ING5 and partially KAT7. Importantly, we could concomitantly show that RomA and LphD both bind the HBO1 complex, as well as the target histone H3.

To corroborate these interactions in the context of infection, we decided to use a previously established method, based on the infection of HEK293T cells that stably express the macrophage Fcg-RII receptor [START_REF] Arasaki | Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b[END_REF]. The expression of this receptor enables HEK293T cells to engulf bacteria that have been opsonized with antibodies, allowing an efficient infection of HEK293T cells in combination with high transfection efficiency. We transfected these cells with either EGFP or EGFP-KAT7 and infected them using L. pneumophila wild type strain overexpressing a tagged form (V5) of LphD or RomA. When pulling down the EGFP proteins we clearly detected an enrichment of both bacterial proteins in the EGFP-KAT7 sample (Figure 3F). This confirms that LphD and RomA target the same endogenous complex (HBO1) to specifically modify H3K14 during infection.

RNA-seq reveals dynamic changes during infection with contribution of nucleomodulins

Here we show that L. pneumophila secretes at least two effectors that target K14 of histone H3 to modify the host chromatin. In order to investigate how K14 remodeling changes the response of the cell to infection, we performed RNA-seq experiments. THP-1 cells were infected with either L. pneumophila wild type (WT), ∆lphD, ∆romA or the double knockout (∆lphD ∆romA) and the transcriptional response was analyzed 1, 3, 5, and 7 hours post-infection compared to non-infected cells.

First, we evaluated the transcriptional response of the cell to the L. pneumophila infection as a function of time (Figure 4A-4C) by assessing the overall changes of differently expressed genes (DEGs) with an adjusted p<0.05 and a shrunken log2 fold change >2 or <-2. The variance between the different time points shows that changes are quite drastic 1 h and 3 h post-infection, compared to the control condition (non-infected). However, the later time points -5 h and 7 h 9 post-infection -show a lower level of variance. independent of the bacterial strain (Figure 4A, S5A, S5B, S5C).

The dynamics of the expression changes were then explored by comparing the 100 most upand down-regulated genes (independent of their fold-change) per time point and their overlaps between time points (Figure 4B). Interestingly, for both up-and down-regulated genes, the genes that are unique for 1 h post-infection represent the biggest group (up: 56, down: 83), indicating that the expression changes very rapidly during the first hours of infection. However, after 1 h post-infection the two conditions show very different results. For the up-regulated genes we can see that almost half of the genes [START_REF] Hauslein | Pathway analysis using (13) Cglycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila[END_REF] are shared by 3 h, 5 h and 7 h post-infection and almost a quarter [START_REF] Liu | Viewing Legionella pneumophila Pathogenesis through an Immunological Lens[END_REF] are shared by all four time points. This suggests that the up-regulation of gene expression adapts quickly within the first three hours, leading to a core set of genes that is continuously up-regulated during the course of infection. In contrast, the down-regulation seems to be a more dynamic process with no clear pattern. Each time point has its own set of uniquely expressed genes, with very little overlap to the other time points, especially when compared to the pattern seen with the up-regulated genes (Figure 4B).

Analyses of functional changes by using gene ontology (GO) profiling (clusterProfiler [START_REF] Yu | clusterProfiler: an R package for comparing biological themes among gene clusters[END_REF])) of L. pneumophila wild-type infected cells, compared to uninfected cells, show a clear pattern within several ontology groups conserved between the four time points, which are all connected to the host cell response to infection (Figure 4C). GO groups such as "response to lipopolysaccharide", "cellular response to chemokines" and "positive regulation of defense response" are strongly conserved over the course of infection, highlighting their importance in host cell defense. Interestingly, 1 h post-infection we observed the highest number of unique GO-terms. One term that stands out is connected to promoting T cell differentiation, a probable first step of the immune response to an intracellular pathogen like L. pneumophila. Starting at 3 h post-infection we see GO groups associated with production and regulation of interferongamma, followed shortly by leukocyte migration starting at 5 h post-infection. Lastly, 7 h postinfection reveals the regulation of cytokine production, whereas early time points focused on the production and regulation of chemokines (Figure 4C). The down-regulated genes do not share this clear step-wise pattern seen with the up-regulated genes. For the early time points (1 h and 3 h) the program is not able to determine specifically enriched GO-terms. Very few enriched GO-terms are observed for 5 h and 7 h post-infection, but none of them are clearly 10 connected to the infection process. Especially 7 h post-infection focuses on sensory perception and pain, a quite unexpected topic (Figure S5D).

We then compared the transcriptional response of the cell of the L. pneumophila wild-type infection to the ones infected with the ∆lphD, ∆romA and ∆lphD∆romA knockout strain. Figure 4C shows the number of DEGs identified at different points after infection, for both wild-type and mutant strains. Again, we observed that a very small number of DEGs was identified at 1h post-infection, whereas at 3 hours a sudden increase for all the studied strains can be seen (Figure 4D). By looking at the total number of genes up-or down-regulated in L. pneumophila wild-type infected cells, compared to mutants, we did not observe any significant changes; we therefore analyzed the functional changes by comparing the GO-term results of the mutants with the wild type infected cells (Figure 4E). This revealed no direct overlap of the GO-terms between the two single knockouts, however, they are part of the same immunological processes, but belonging to different steps. Furthermore, the double knockout shows a mixture of GOterm of the two single knockouts, indicating that the presence of the two effectors causes a cumulative effect on the transcriptional response of the cell.

To confirm these results, a subset of genes was chosen for validation by RT-qPCR. The choice of these genes was based on their high fold change in the RNA-seq of the different knockout mutants and their possible involvement in the immune response (ADAM19, CCR2 and AJUBA). Furthermore, recently a list of DEGs in KAT7 knockout mutants of HEK293T and HeLa cells was published [START_REF] Kueh | HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells[END_REF]. We cross-referenced these lists with the results of our RNA-seq and chose three additional candidate genes for validation by RT-qPCR (CCL2, CLEC2D and TXNIP). Figure 4F shows that LphD and RomA can act together, as the double knockout exhibits a cumulative effect (in transcriptional repression or induction), compared to single mutants for ADAM19, CCL2, CCR2 and AJUBA. However, for TXNIP and CLEC2D the double knockout has an effect that can be recapitulated by a single mutant (∆lphD for TXNIP and ∆romA for CLED2D). This strongly suggests that the two proteins can also work independently from each other.

DISCUSSION

We identified here a new secreted nucleomodulins of L. pneumophila, named LphD, that encodes histone deacetylase activity. Importantly, LphD shows a high catalytic efficiency for H3K14 in vitro and targets the host cell nucleus during infection. In the nucleus, it deacetylates H3K14, thereby facilitating the methylation of this residue by another bacterial effector, the SET-domain methyltransferase RomA. Nuclear targeting of LphD to the nucleus does not depend on its enzymatic activity, as a catalytic inactive mutant (Y392F) shows the same localization (Figure S2C). However, the processes by which LphD targets the nucleus remains to be determined, as the deletion of a nuclear localization signal (NLS) -predicted at the Nterminus of the protein -did not affect its translocation to the nucleus (data not shown).

In vitro activities on short H3 peptides acetylated on different lysine residues showed a clear preference of LphD for H3K14 (Figure 1A), however, we cannot exclude the possibility that LphD has other targets than H3K14. In particular, non-histone targets should not be dismissed as many eukaryotic HDACs have been shown to target non-histone proteins. For example the tumor protein p53 and the immune response regulator MyD88 (Ito et al., 2002;Menden et al., 2019). Considering that acetylation is a very prevalent post-translational modification of proteins [START_REF] Philp | Understanding the acetylome: translating targeted proteomics into meaningful physiology[END_REF], its manipulations would be as a promising target for a pathogen like L. pneumophila to influence its host cell. Moreover, it has been previously shown that RomA indeed methylates also non-histone proteins, although the functional role of these modification is not established yet (Schuhmacher et al., 2018).

Importantly, we showed that LphD and RomA modify the same cellular target in synergy during infection. Other L. pneumophila effectors have been described to target the same cellular component to subsequently manipulate it. One example is the host small GTPase Rab1, which is sequentially modified by at least three effectors [START_REF] Rolando | Post-translational modifications of host proteins by Legionella pneumophila: a sophisticated survival strategy[END_REF]. Furthermore, we revealed that LphD and RomA have a common target in the host cell, namely H3K14. Moreover, we showed that both bacterial effectors bind the same cellular complex, HBO1, to target host chromatin (Figure 3). Indeed, they both co-immunoprecipitate with KAT7, a component of the HBO1 complex, a known regulator of H3K14 acetylation.

Our pulldown results show that LphD interacts with all components of the HBO1 complex, including its target, histone H3. RomA on the other hands seems to preferentially bind only to KAT7, ING5 and H3 (Figure 3E). However, as mentioned before, the analysis of possible interaction partners led not only to the identification of HBO1 but also suggested interaction 12 with several other complexes related to chromatin remodeling. Some of which, like the NuRDand the Sin3-complex, are known to comprise eukaryotic histone deacetylases and thus, might be additional interaction partners for LphD (Adams et al., 2018;Kelly and Cowley, 2013).

Further studies will elucidate whether additional possible interaction partners of LphD and RomA exist.

Many pathogens are known to indirectly manipulate histone acetylation to subvert the host immune response and promote their survival (Eskandarian et al., 2013;Rennoll-Bankert et al., 2015;[START_REF] Wang | Mycobacterium tuberculosis infection induces IL-10 gene expression by disturbing histone deacetylase 6 and histonedeacetylase 11 equilibrium in macrophages[END_REF]. The analysis of the activity of LphD and RomA -with respect to the transcriptional response of the host cell to the infection -by RNA-seq experiments revealed dynamic changes in the mRNA landscape of the host within the first few hours of infection, thus showing a rapid response to the bacterial intruders.

The enriched GO-terms for the L. pneumophila mutant strains do not show overlap between the two single knockout mutants, however, this is due to the definition of GO terms, where single genes that are present in both conditions might be attributed to different GO terms, depending on the rest of the genes in these conditions. We therefore used RT-qPCR to confirm the RNAseq results by choosing several genes related to immune response processes, three of which (CCL2, CLEC2D, and TXNIP) are known to be differently expressed in KAT7 knockout cells [START_REF] Kueh | HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells[END_REF].

The expression levels of ADAM19, AJUBA, CCR2 and CCL2 are up-regulated in the different knockout mutants, indicating that LphD and RomA are responsible for their down-regulation.

ADAM19 is a metalloprotease and a known sheddase for TNF-a, responsible for its release from the cellular membrane into the extracellular space [START_REF] Zheng | Evaluation of the contribution of different ADAMs to tumor necrosis factor alpha (TNFalpha) shedding and of the function of the TNFalpha ectodomain in ensuring selective stimulated shedding by the TNFalpha convertase (TACE/ADAM17)[END_REF]. This shows that LphD and RomA might not only directly influence the infected cell, but also manipulate bystander cells. Furthermore, it was shown that ADAM19 regulates CCL2 production as well as macrophage infiltration in the kidney [START_REF] Wang | MicroRNA-874-3p/ADAM (A Disintegrin and Metalloprotease) 19 Mediates Macrophage Activation and Renal Fibrosis After Acute Kidney Injury[END_REF].

AJUBA, a LIM-domain containing protein, has been described as a key regulator of NF-kB activation by modulating the assembly of the Czeta/p62/TRAF6 signaling complex [START_REF] Feng | The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex[END_REF]. NF-kB activation is a key characteristic of L. pneumophila infection and is positively correlated with bacterial virulence (Wang et al., 2018a).

CCR2 and CCL2 are a chemokine receptor/ligand pair with a multitude of immune related functions. They have been shown to be essential in the recruitment of NK cells to viral infection sites as well as the attraction of myeloid progenitors to the lung in a flagellin-dependent manner 13 [START_REF] Lei | Flagellin/TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages[END_REF][START_REF] Shou | PARP-1 controls NK cell recruitment to the site of viral infection[END_REF]. In addition, CCR2 deficiency has been linked to increased susceptibility of mice to another intracellular pathogen, Francisella tularensis LVS [START_REF] Kurtz | Deficiency in CCR2 increases susceptibility of mice to infection with an intracellular pathogen, Francisella tularensis LVS, but does not impair development of protective immunity[END_REF].

On the other hand, CLEC2D and TXNIP show decreased expression in the different knockout mutants, indicating that LphD and RomA positively influence their expression, albeit indirectly. CLEC2D (C-type lectin domain family member D), also known as LLT1 (Lectin like transcript 1), is a surface ligand mainly expressed on activated immune cells [START_REF] Germain | Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-γ contributes to modulate immune responses[END_REF]. It is well established that the interaction of LLT1 with NKRP1A -a receptor expressed on NK cells -inhibits NK cell mediated cytotoxic effects [START_REF] Rosen | Functional consequences of interactions between human NKR-P1A and its ligand LLT1 expressed on activated dendritic cells and B cells[END_REF]. This phenotype was mostly described on different types of cancer [START_REF] Malaer | Role of LLT1 and PCNA as Natural Killer Cell Immune Evasion Strategies of HCT 116 Cells[END_REF][START_REF] Mathew | Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction[END_REF], but might also play a major role in the clearance of an intracellular pathogen like L. pneumophila.

Indeed, it has been shown that NK cell activation during L. pneumophila infection leads to increased IFN-g production, which subsequently promotes bactericidal activity of monocytederived cells [START_REF] Brown | Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen[END_REF].

Finally, TXNIP (thioredoxin-interacting protein) is a key regulator of oxidative stress by inhibiting the activity of thioredoxin, a strong antioxidant [START_REF] Nishiyama | Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression[END_REF].

Interestingly, TXNIP has been shown to be down-regulated during Brucella abortus infection and thereby promoting the intracellular survival of the bacteria [START_REF] Hu | Brucella Infection Regulates Thioredoxin-Interacting Protein Expression to Facilitate Intracellular Survival by Reducing the Production of Nitric Oxide and Reactive Oxygen Species[END_REF]. In addition, TXNIP has been linked to phagosome acidification during infection with Escherichia coli in an NLRP3-dependent manner [START_REF] Yoon | Thioredoxin-Interacting Protein Promotes Phagosomal Acidification Upon Exposure to Escherichia coli Through Inflammasome-Mediated Caspase-1 Activation in Macrophages[END_REF]. This is different to what observed, as the knockout strains actually showed lower expression of TXNIP, indicating that the two effectors promote its upregulation. However, it has also been shown that in NK cells, TXNIP blocks IFN-g production during bacterial infection in a TAK-1 (transforming growth factor b-activated kinase 1)-dependent manner (Kim et al., 2020). It has been demonstrated that the production of IFN-g by NK cells plays a key role in L. pneumophila clearance in a mouse model [START_REF] Spörri | MyD88-dependent IFN-gamma production by NK cells is key for control of Legionella pneumophila infection[END_REF]. Interestingly, the ∆romA strain shows a stronger change in TXNIP expression than the ∆lphD and the ∆lphD∆romA strain, indicating that the lphD knockout somehow hides the ∆romA phenotype. These results suggest that LphD and RomA not only directly influence the reaction of the infected cell, but can also manipulate the response of bystander cells.

In conclusion, this study provides exciting insight on how L. pneumophila modifies host chromatin by using two distinct chromatin remodelers. Both, LphD and RomA, are deployed by the bacteria to strategically influence the response of the host cell to the infection and promote bacterial replication in this otherwise hostile environment.!

METHODS

Bacterial strains, growth conditions and cell culture

Legionella pneumophila strain Paris and mutants were cultured in N-(2-acetamido)-2aminoethanesulfonic acid (ACES)-buffered yeast extract broth (BYE) or on ACES-buffered charcoal-yeast (BCYE) extract agar [START_REF] Feeley | Charcoalyeast extract agar: primary isolation medium for Legionella pneumophila[END_REF]. For Escherichia coli Luria-Bertani broth (LB) was used. When needed antibiotics were added: for L. pneumophila (E. coli): kanamycin 12.5 μg/ml (50 μg/ml), gentamycin 12.5 μg/ml, apramycin 15 μg/ml, chloramphenicol 10 μg/ml (10 μg/ml), and ampicillin (only for E. coli) 100 μg/ml.

The knockout of lphD in the wild type background to generate a single mutant followed by the knockout of romA to generate the double mutant, was performed as previously described [START_REF] Sahr | Deep sequencing defines the transcriptional map of L. pneumophila and identifies growth phase-dependent regulated ncRNAs implicated in virulence[END_REF] and detailed in Supp. Methods. For the complementation construction, the fulllength lphD with its own promotor was cloned into pBC-KS (Stratagene). Bacteria expressing EGFP were obtained by introducing EGFP under the control of the flaA promotor of L. pneumophila into pBC-KS backbone. To generate the catalytic inactive Y392F mutant of LphD, a single base pair mutation was performed using mismatched primers (217H and 217B, Table S2).

THP-1 cells were maintained in RPMI-1640 (Gibco), HeLa and HEK293T in DMEM GlutaMAX (Gibco), both containing 10% FBS (Eurobio Scientific) in a humid environment with 5% CO2 at 37°C. Acanthamoeba castellanii (ATCC50738) were maintained in PYG 712 medium at 20˚C. For the virulence replication assays, THP-and A. castellanii were infected as previously described [START_REF] Lomma | The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication[END_REF] and detailed in Supp. Methods. Cell transfections were performed by using FuGENE (Promega) following the recommendations of the manufacturer.

b-lactamase translocation assay and immunofluorescence analysis b-lactamase assays were performed in THP1 infected cells as previously described [START_REF] Charpentier | Chemical genetics reveals bacterial and host cell functions critical for type IV effector translocation by Legionella pneumophila[END_REF] and as detailed in Supp Methods. For immunofluorescence analyses, cells are fixed with 4% para-formaldehyde in PBS for 15 minutes at room temperature, followed by quenching (PBS-50 mM NH4Cl for 10 minutes). Cells are permeabilized with 0.1% Triton X-100 and blocked for 30 minutes with 5% BSA in PBS. The cells are incubated with the respective primary antibodies overnight at 4˚C. They are washed three times using PBS and then stained with DAPI and secondary antibodies for 30 minutes at room temperature, followed by mounting to glass slides using Mowiol (SIGMA). Immunosignals were analyzed with a 15 Leica SP8 Microscope at 63× magnification. Images were processed using ImageJ software.

Antibodies used in this study are listed in Table S3.

LphD purification

N-terminal HIS6-tagged LphD was expressed in E. coli BL21 C41 following an auto-induction protocol [START_REF] Studier | Protein production by auto-induction in high density shaking cultures[END_REF]. After 4 hours at 37°C cells were grown for 20 hours at 20°C in 2YT complemented autoinduction medium containing 50 µg/ml kanamycin. Cells were harvested and flash frozen in liquid nitrogen. Cell pellets were resuspended in 50ml lysis buffer (50 mM HEPES pH8, 500 mM KCl, 5% glycerol, 1 mM MgCl2, benzonase, lysozyme, 1 mM DTT and supplemented with EDTA-free protease inhibitor cocktails (ROCHE)) at 4°C and disrupted by sonication (6 x 60 seconds). The lysate was centrifuged for 60 min at 10.000 x g at 4°C. The cleared lysate was loaded onto a Ni-NTA affinity chromatography column (HisTrap FF crude, GE Healthcare) equilibrated in Buffer A (50 mM Hepes pH8, 500 mM KCl, 5% glycerol, 10 mM imidazole, 1 mM DTT). HIS6-tagged proteins were eluted with a linear gradient of buffer B (50 mM Hepes pH8, 500 mM KCl, 5% glycerol, 1 M imidazole, 1 mM DTT). The eluted fractions containing the protein of interest were pooled and dialysed at 4°C overnight in SEC buffer (20 mM HEPES pH8, 300 mM KCl, 5% glycerol, 2 mM TCEP). The HIS6-tag was not removed as this led to precipitation of the protein. After dialysis, the protein was concentrated and loaded onto a Superdex 75 16/60 size exclusion (SEC) column (GE Healthcare). The peak corresponding to the protein was concentrated to about 12 mg/ml and flash frozen in liquid nitrogen and stored at -80°C.

In vitro enzymatic assays

Purified HIS6-LphD was used to perform in vitro enzymatic assays against FAM-conjugated H3-derived acetylated peptides, followed by RP-UFLC (see Supp Methods) or with Fluor de Lys Ò deacetylase assay (Enzo Life Sciences). Briefly, different amount of purified LphD and catalytically dead LphD Y392F were incubated with the substrate for 30 minutes at 37˚C and the signal was read using a plate reader (TECAN). Trichostatin-A (TSA) was added at 5 µM.

The kinetic parameters of LphD on H3-derived peptides were determined by UFL in a 96-wells ELISA. Briefly, LphD (7.7 nM) was mixed with different concentrations of acetylated H3 peptides (ranging from 12.5 to 200 µM final) for 15 minutes at 30°C and the reaction was stopped by adding 50 µL of HClO4 (15% v/v in water). Finally, 10 µl of the reaction mix were automatically injected into the RP-UFLC column and initial velocities (Vi, µM.min -1 ) were determined as described above. Vi were then plotted against substrate peptide concentrations 16 and curves were non-linearly fitted using Michaelis-Menten equation

! " * [%]
' " ([%] (OriginPro 8.0). Km (enzyme Michaelis's constant), Vm (enzyme maximal initial velocity) and kcat (enzyme turnover) values were extrapolated from these fits. A catalytic dead version of the enzyme was used as a negative deacetylation control [START_REF] Duval | An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides[END_REF].

In vitro histone deacetylating assays were performed on 250 ng of highly-acetylated purified histones (extracted as described in Supp Methods) and 10 ng LphD (WT or catalytic dead) in LphD purification buffer at 30°C. At different time points (0, 1, 2, 3, 4 and 5 minutes), the reaction was stopped with the addition of 10 µL Laemmli sample buffer. Samples were analyzed by western blot and the α-H3K14ac signal quantified.

Histone modification analysis

For the analysis of histone modifications during infection, THP-1 cells in suspension were infected with Legionella pneumophila wild type and a ∆lphD strain, both containing a plasmid for the expression of EGFP under the control of the flaA promotor at an MOI of 50. After 30 minutes, Gentamicin is added (100 µg/ml) to kill extracellular bacteria. Cells are then sorted by FACS (S3e, BIORAD) as previously described [START_REF] Rolando | Sorting of Phagocytic Cells Infected with Legionella pneumophila[END_REF]. Histones of infected cells were isolated as previously described with some modifications [START_REF] Luense | Comprehensive analysis of histone post-translational modifications in mouse and human male germ cells[END_REF]. Briefly, THP-1 cells (3x10 6 ) were incubated at 4°C with hypotonic lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2, with protease inhibitors) for two hours while rotating. Subsequently, nuclei were pelleted and resuspended in 0.4 M sulfuric acid -incubate overnight at 4°C. The supernatant was precipitated with 33% trichloroacetic acid (TCA) on ice.

Pelleted histones were washed twice with ice-cold acetone and were then resuspended in DNase/RNase free water. Sample quality of acid extraction was visualized on a Coomassiestained 12% SDS-PAGE. Histone modification signal (H3K14ac, H3K14me, H3K18ac, H3K23ac) is assessed by western blot and normalized to signal of histone H1. Samples are then compared to non-infected controls.

Co-immunoprecipitation experiments

For the GFP-pulldown HEK293T cells were seeded in 10 cm dishes and transfected 24 or 48 hours with 3 µg of the different EGFP construct expression plasmids. Transfected cells were washed three times with PBS before lysis in RIPA buffer (20 mM HEPES-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate). For the verification of protein interaction during infection we modified the previously established protocol by [START_REF] Arasaki | Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b[END_REF]. Briefly, we transiently transfected HEK293-17 FcgRII cells with either EGFP or a EGFP-KAT7 fusion product. After 48 hours of transfection, the cells were washed and fresh DMEM with IPTG (1 mM) was added. The bacteria -L. pneumophila over-expressing either V5-LphD or V5-RomA -are pre-opsonized by incubating them with an anti-FlaA antibody for 30 minutes at 37˚C. Then the cells are infected with MOI 50. After one hour, the cells are washed and fresh DMEM (with 1 mM IPTG) is added. After 7 hours of infection, the cells are collected and lysed in RIPA buffer.

To facilitate the lysis, cells were sonicated using a Bioruptor ® Pico sonication device (Diagenode) for 15 cycles of 30 seconds ON/OFF. Lysates were precleared and the pulldown was performed using GFP-trap magnetic agarose beads (Chromotek) following the manufacturer's instructions at 4˚C overnight. Proteins were eluted in 30 µl Laemmli buffer and then analyzed by western blot or the beads directly processed for MS/MS analyses (see Supp Methods).

Western blotting

Sample proteins are prepared in Laemmli sample buffer containing 400 mM β-mercaptoethanol and loaded on SDS PAGE gels, followed by a transfer onto a nitrocellulose membrane (0.2 µm). Ponceau S staining was carried out to ensure equal protein loading. Membranes were blocked with 5% non-fat milk in TBS-Tween 0.5% for 1 hour and incubated with the respective primary antibody overnight at 4˚C. Antibodies used are listed in Table S3. Membranes are washed and probed with horseradish peroxidase-coupled antibody against either mouse IgG or rabbit IgG (1:2500 in 5% non-fat milk TBS-Tween) for 1 hour. The proteins were then visualized by chemiluminescence detection using HRP Substrate spray reagent (Advansta) on the G:BOX instrument (Syngene). Images were processed and quantified using MultiGauge V3.0 and ImageJ softwares.

RNA-sequencing, GO-term analyses and RT-qPCR

For the RNA-seq and RT-qPCR, THP-1 monocytes were infected with either L. pneumophila wild type, ∆lphD, ∆romA or ∆lphD-∆romA, all of which were expressing EGFP. After 1 h, 3 h, 5 h, and 7 h post-infection, the infected cells were enriched by FACS and stored at -80˚C. RNAseq and data analysis was performed by Active Motif. In short, total RNA was isolated from samples using the Qiagen RNeasy Mini Kit (Qiagen). For each sample, 1 µg of total RNA was then used in the TruSeq Stranded mRNA Library kit (Illumina). Libraries were sequenced on Illumina NextSeq 500 as paired-end 42-nt reads. Sequence reads were analyzed with the STAR alignment -DESeq2 software pipeline. Genes that were differently regulated in the RNA-seq 18 were analyzed for possible GO term groups using the clusterProfiler package in R [START_REF] Yu | clusterProfiler: an R package for comparing biological themes among gene clusters[END_REF]. The function compareCluster with its standard setting was used to assess enriched biological processes within those differently regulated genes under the different experimental conditions. To remove possible redundant GO terms, the simplify function was used, again with standard settings. The resulting lists were visualized in dot plots or upset plots, also using R [START_REF] Lex | UpSet: Visualization of Intersecting Sets[END_REF]. For the RT-qPCR the RNA was isolated using the Arcturus ® PicoPure ® RNA isolation kit (Thermo Fisher Scientific). RT-PCR was performed using 2.5 µg of RNA. qPCR primers (Table S2) were designed to span an exon-exon junction to minimize amplification of genomic DNA. qPCR was performed using SYBR-Green Master Mix (Thermo Fisher Scientific) and mRNA expression was calculated using the 2 -∆∆Ct method [START_REF] Livak | Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method[END_REF]. Results were normalized to human GAPDH, ACTB and 36B4.

SUPPLEMENTAL METHODS

Mutant and Complementation Constructions

To construct the ΔlphD mutant strain the chromosomal gene lphD of the wild-type strain was replaced by introducing a gentamycin (GenR) resistance cassette. The mutant allele was constructed using a 3-steps PCR. Briefly, three overlapping fragments (lphD upstream regionprimers 195H and 196H, antibiotic cassette-primers 52H and 52B, lphD downstream regionprimers 195B and 196B; Table S2) were amplified independently and purified on agarose gels.

The three resulting PCR products were mixed at the same molar concentration (15nM) and a second PCR with flanking primer pairs (primers 195H and 195B; Table S2) was performed.

The resulting PCR product, the gentamycin resistance cassette flanked by 500 bp regions homologous to lphD was introduced into strain L. pneumophila Paris by natural competence for chromosomal recombination. Strains that had undergone allelic exchange were selected by plating on BCYE containing gentamycin and the mutant was verified by PCR and sequencing.

To resulting mutant was then used to generate the ∆lphD ∆romA double knockout strain, using a new set of primers (romA upstream region-primers 11H and 66H, kanamycin antibiotic resistance cassette-primers 60H and 60B, romA downstream region-primers 11B and 66B; Table S2).

Cell Culture and Infection Assay

Acanthamoeba castellanii ATCC50739 was cultured at 20°C in PYG 712 medium [2% proteose peptone, 0.1% yeast extract, 0.1 M glucose, 4 mM MgSO4, 0.4 M CaCl2, 0.1% sodium citrate dihydrate, 0.05 mM Fe(NH4)2(SO4)2•6H2O, 2.5 mM NaH2PO3, 2.5 mM K2HPO3]. A. castellanii were washed once with infection buffer (PYG 712 medium without proteose peptone, glucose and yeast extract) and seeded at a concentration of 4x10 6 cells per T25 flask.

L. pneumophila wild-type and mutant strains were grown on BCYE agar to stationary phase, diluted in infection buffer and mixed with A. castellanii at an MOI of 0.1. Intracellular multiplication was monitored plating a sample at different time points on BCYE plates and the number of intracellular bacteria was counted. The human monocyte (THP-1) were maintained in 5% CO2 at 37°C in RPMI 1640 medium GlutaMAX medium (Invitrogen) supplemented with 10% fetal bovine serum (Biowest). In THP-1 cell infection assays cells were seeded into 12well tissue culture trays (Falcon, BD lab ware) at a density of 2x10 5 cells/well. THP-1 cells were pretreated with 0.8 µM phorbol 12-myristate 13-acetate (PMA, Sigma) for 72 h to induce differentiation into macrophage-like adherent cells. Stationary phase L. pneumphila were resuspended in serum free medium and added to cells at an MOI of 10. After 2 hours of 20 incubation, infected cells were washed with PBS before incubation with serum-free medium.

At 2 h, 24 h, 48 h and 72 hours the supernatant was collected and the cells were lysed with 0.1% TritonX-100. The infection efficiency was monitored by determining the number of colonyforming units (cfu) of the different L. pneumophila strains after plating on BCYE agar.

UFLC-mediated LphD deacetylase activity assay

In order to quantify LphD deacetylase activity, we synthetized six 5-fluorescein amidite (5-FAM)-conjugated acetylated peptide substrates based on the human H3.1 sequence and centered on various lysine residues of interest:

ARTKacQTARRSK-(5-FAM), referred to as H3K4ac peptide

(5-FAM)-QTARKacSTGG-NH2, referred to as H3K9ac peptide

(5-FAM)-STGGKacAPRR-NH2, referred to as H3K14ac peptide

(5-FAM)-RAPRKacQLAT-NH2, referred to as H3K18ac peptide

(5-FAM)-QLATKacAARR-NH2, referred to as H3K23ac peptide

(5-FAM)-TRAARKacSAPAT-NH2, referred to as H3K27ac peptide Non-acetylated versions of these peptides were also synthetized as detection standards.

Samples containing H3 peptides and their acetylated forms were separated by RP-UFLC (Shimadzu) using Shim-pack XR-ODS column 2.0 x 100 mm 12 nm pores at 40°C. The mobile phase used for the separation consisted of the mix of 2 solvents: A was water with 0.12% 21 trifluoroacetic acid (TFA) and B was acetonitrile with 0.12% TFA. Separation was performed by an isocratic flow depending on the peptide: ® 83 % A/17 % B, rate of 1 ml/min, 6 min run for H3K4ac peptide ® 80 % A/20 % B, rate of 1 ml/min, 6 min run for H3K9ac, H3K14ac, H3K18ac, H3K27ac peptides ® 79 % A/21 % B, rate of 1 ml/min, 8 min run for H3K23ac peptide H3 acetylated peptides (substrates) and their non-acetylated forms (products) were monitored by fluorescence emission (λ = 530 nm) after excitation at λ = 485 nm and quantified by integration of the peak absorbance area, employing a calibration curve established with various known concentrations of peptides.

Highly-acetylated histone extraction

HEK293T cells were cultivated in RPMI 1640 medium with 10 % heat-inactivated fetal bovine serum (FBS) and 1 mM L-glutamine at 37°C under 5 % CO2. For endogenous histone extraction, cells were seeded at 30 000 cells/cm² in a 100 cm² Petri dish (VWR). The next day, cells were treated with 20 mM sodium butyrate and 6 µM Trichostatin A (TSA). Cells were then put back in the incubator at 37°C and 5 % CO2 for 30 min before being harvested. Cells were lysed with cell lysis buffer (PBS 1x, 1% Triton X-100, 20 mM sodium butyrate, 6 µM TSA, protease inhibitors) for 30 min at 4°C, sonicated (2 sec, 10 % power) and centrifuged (15 min, 15500 g, 4°C). 500 µL of 0.2 N HCl was then put on remaining pellets. The mixture was sonicated 3 times (3 sec, 10 % power) and incubated overnight at 4°C. The next day, samples were centrifuged (15 min, 15500 g, 4°C) and the supernatant (containing extracted histones) was buffer exchanged three times into Tris 50 mM, 50 mM NaCl, pH 8 using MiniTrap G-25 desalting columns (GE Healthcare) and stored with protease inhibitor at -20°C until use.

Mass spectrometry analysis of GFP co-IP

MS grade Acetonitrile (ACN), MS grade H2O and MS grade formic acid (FA) was acquired from Thermo Chemical.

Proteins on magnetic beads were digested overnight at 37°C with 1µl (0.2 µg/µL) of trypsin (Promega) in a 25-mM NH4HCO3 buffer per sample. The resulting peptides were desalted using ZipTip μ-C18 Pipette Tips (Pierce Biotechnology).

Samples were analyzed using an Orbitrap Fusion equipped with an easy spray ion source and coupled to a nano-LC Proxeon 1200 (Thermo Fisher Scientific). Peptides were loaded with an online preconcentration method and separated by chromatography using a Pepmap-RSLC C18 22 column (0.75 x 750 mm, 2 μm, 100 Å) from Thermo Fisher Scientific, equilibrated at 50°C and operating at a flow rate of 300 nl/min. Peptides were eluted by a gradient of solvent A (H2O, 0.1 % FA) and solvent B (ACN/H2O 80/20, 0.1% FA), the column was first equilibrated 5 min with 95 % of A, then B was raised to 28 % in 105 min and to 40% in 15 min. Finally, the column was washed with 95% B during 20 min and re-equilibrated at 95% A during 10 min. Peptide masses were analyzed in the Orbitrap cell in full ion scan mode, at a resolution of 120,000, a mass range of m/z 350-1550 and an AGC target of 4.10 5 . MS/MS were performed in the top speed 3s mode. Peptides were selected for fragmentation by Higher-energy C-trap Dissociation (HCD) with a Normalized Collisional Energy of 27% and a dynamic exclusion of 60 seconds.

Fragment masses were measured in an Ion trap in the rapid mode, with and an AGC target of 1x10 4 . Monocharged peptides and unassigned charge states were excluded from the MS/MS acquisition. The maximum ion accumulation times were set to 100 ms for MS and 35 ms for MS/MS acquisitions, respectively. Label-free quantification was done on Progenesis QI for Proteomics (Waters) in Hi-3 mode for protein abundance calculation. MGF peak files from Progenesis were processed by Proteome Discoverer 2.4 with the Sequest search engine. A custom database was created using the Swissprot/TrEMBL protein database release 2019_08 with the Homo sapiens taxonomy and including LphD, both from the Legionella pneumophila taxonomy. A maximum of 2 missed cleavages was authorized. Precursor and fragment mass tolerances were set to respectively 7 ppm and 0.5 Da. Spectra were filtered using a 1% FDR using the percolator node.

Production of anti-LphD antibodies in rabbit

Rabbit polyclonal antibodies to LphD where generated for this study (Thermo Fisher). Briefly, the purified HIS6-LphD was injected in a rabbit for a protocol of 90 days. The produced antibody was qualitatively evaluated by affinity purified ELISA: the purified antibody is tittered by indirect ELISA against the protein bound to the solid-phase to measure the reactivity of the antibodies after elution.

Results

-106 - LphD Y392F

H3K14ac

Ponceau S

Results

-107 - PC1: 90% variance PC2: 6% variance group PC1: 90% variance PC2: 5% variance group

• • • • • non-infected dLphD-1h dLphD-3h dLphD-5h dLphD-7h • • • • • • • • • • • • • • • • -10 0
• • • • • non-infected dLphD-dRomA-1h dLphD-dRomA-3h dLphD-dRomA-5h dLphD-dRomA-7h • • • • • • • • • • • • • • • • -10 0

Conclusion and perspectives

Legionella pneumophila has closely co-evolved with its eukaryotic hosts. This coevolution gave rise to the countless strategies employed by the bacteria to invade their host cells, establish a protected niche -the Legionella containing vacuole -and enable bacterial replication within this self-made environment [START_REF] Escoll | From Amoeba to Macrophages: Exploring the Molecular Mechanisms of Legionella pneumophila Infection in Both Hosts[END_REF]Isberg et al., 2009). One of the main virulence factors in L. pneumophila is the type-IVB secretion system Dot/Icm. To date, more than 330 proteins have been predicted to be secreted by the Dot/Icm system, which corresponds to around 10% of the L. pneumophila genome [START_REF] Burstein | Genome-scale identification of Legionella pneumophila effectors using a machine learning approach[END_REF]Ensminger, 2016;[START_REF] Escoll | Targeting of host organelles by pathogenic bacteria: a sophisticated subversion strategy[END_REF][START_REF] Finsel | Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones[END_REF][START_REF] Lifshitz | Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal[END_REF][START_REF] Qiu | Legionella and Coxiella effectors: strength in diversity and activity[END_REF][START_REF] Zhu | Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila[END_REF]. The broad range of secreted proteins, as well as their redundancy in function, highlight the ability of the bacteria to survive in a wide variety of eukaryotic hosts. This is achieved by targeting numerous different host cell pathways and manipulating them to impede the host cell response as well as promote bacterial growth. A unique feature of members of the genus Legionella is the large number of eukaryotic-like proteins encoded in their genome. So far, 137 eukaryotic domains have been described in the genus Legionella, encompassing more than 250 different proteins (Gomez-Valero et al., 2019). The functions of these secreted eukaryotic-like proteins range from influencing intracellular trafficking and manipulating signal transduction, to salvaging host metabolites and directly interfering with gene transcription [START_REF] Hubber | Modulation of Host Cell Function by Legionella pneumophila Type IV Effectors[END_REF]Isberg et al., 2009;Mondino et al., 2020b). One of these effectors, a SET-domain methyltransferase called RomA, was described to target and the host cell nucleus and induce chromatin changes. RomA specifically methylates lysine 14 of histone 3 (H3K14), causing transcriptional repression of a subset of genes involved in the host cell response to the bacterial invasion (Rolando et al., 2013). H3K14 methylation usually occurs at very low levels in the host but accumulates very quickly during infection, which might be caused by low levels of the corresponding demethylase (Zhao et al., 2018;Zhu et al., 2021).

However, the fact that H3K14 is usually acetylated in eukaryotic cells -a modification connected to active transcription -led to the question how the bacteria efficiently methylate this residue. In eukaryotic cells, the process of removing acetyl moieties from histones is regulated by histone deacetylases (HDAC). One possibility was that the bacteria intercept the natural turnover of this residue, due to highly dynamic changes in these modifications. The other option was that L. pneumophila might encode its own secreted HDAC to facilitate the deacetylation of H3K14 at specific genome regions, consequently enabling their methylation by RomA.

Indeed, a bioinformatical search of the L. pneumophila genome revealed that the bacteria encode a protein with a predicted HDAC domain, which we called LphD. Furthermore, this HDAC domain shows high similarity to eukaryotic HDACs, especially the Zn +2 -dependent classes. In addition, phylogenetic analysis revealed that this HDAC-like protein is widely distributed in the whole Legionella genus. Interestingly, further analysis revealed that there are two distinct groups of HDAC-like proteins in the genus Legionella, which, due to their low homology, seem to have been acquired on two separate occasions during evolution (Schator et al., 2021). Group 2, which includes LphD, shows a lower level of homology to possible eukaryotic ancestor proteins, whereas group 1 seems to be more conserved and shows high homology to members of HDAC class II. The fact that HDAC-like proteins are widely distributed within the genus, strongly suggests that they have an important role in the life cycle of the bacteria.

The aim of my PhD thesis was to characterize the role and function of LphD during infection and to assess whether it acts in synergy with RomA to modify histone and alter gene transcription. HDACs have been described before as targets of a variety of pathogens to manipulate their host and ensure their own survival (An et al., 2018;Ganesan et al., 2017;[START_REF] Grabiec | Epigenetic regulation in bacterial infections: targeting histone deacetylases[END_REF][START_REF] He | A Decrease of Histone Deacetylase 6 Expression Caused by Helicobacter Pylori Infection is Associated with Oncogenic Transformation in Gastric Cancer[END_REF][START_REF] Kong | A Phytophthora Effector Manipulates Host Histone Acetylation and Reprograms Defense Gene Expression to Promote Infection[END_REF][START_REF] Murata | Epigenetic Histone Modification of Epstein-Barr Virus BZLF1 Promoter during Latency and Reactivation in Raji Cells[END_REF]Rennoll-Bankert et al., 2015;Terhune et al., 2010;Walters et al., 2010;Wang et al., 2005;[START_REF] Zhou | The CoREST/REST Repressor Is both Necessary and Inimical for Expression of Herpes Simplex Virus Genes[END_REF]. However, very rarely it has been shown that pathogens encode their own HDAC-like proteins to directly manipulate the host. One recently published example is Gc-HDAC, an HDAC-like protein encoded by Neisseria gonorrhoeae that interferes with epigenetic modifications at the promoters of certain proinflammatory genes, thereby promoting bacterial immune evasion (Zughaier et al., 2020). In addition, the synergy of LphD and RomA represents the first documented case of two bacterial effectors directly targeting host chromatin and modifying it step-by-step to impede the host cell response to bacterial infection.

First, we assessed the intracellular localization of LphD during infection. We demonstrated that LphD is indeed secreted during infection in a Dot/Icm-dependent manner and it localizes to the host cell nucleus. However, the exact mechanism of nuclear localization is not known yet. The protein encodes a putative nuclear localization signal (NLS) predicted in the N-terminal part (amino acids 2-22), however we confirmed that the deletion of this signal does not influence the localization of the protein. We cannot exclude the presence of another NLS not detected by classical NLS-finding algorithms, and additional experiments on truncated version of the protein must be performed to clearly define how LphD might actively reach the nucleus. In fact, to exert their functions, almost all eukaryotic class I HDACs need to be in the nucleus, but this localization not always occurs via an NLS: some of them reach the nucleus together with other proteins/HDACs. One example is HDAC3 that is recruited by HDACs 4, 5 and 7 through complex formation via SMRT and No-CoR [START_REF] Fischle | Human HDAC7 Histone Deacetylase Activity Is Associated with HDAC3in Vivo[END_REF][START_REF] Yang | Functional domains of histone deacetylase-3[END_REF].

HDAC translocation, especially HDAC class IIa, is also regulated by serine phosphorylation. It has been shown for several eukaryotic HDACs that their intracellular localization is dependent on protein kinases, such as CaMKII (Ca 2+ /calmodulin-dependent protein kinase), and phosphatases like PP2A (protein phosphatase 2) [START_REF] Martin | Protein phosphatase 2A controls the activity of histone deacetylase 7 during T cell apoptosis and angiogenesis[END_REF][START_REF] Mckinsey | Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation[END_REF][START_REF] Paroni | PP2A Regulates HDAC4 Nuclear Import[END_REF]. LphD contains several possible phosphorylation sites and their involvement in nuclear transport could be assessed by using phosphoablation mutants. Another regulator of HDAC nuclear import is CSE1L (chromosome segregation 1 like): CSE1L has been reported to export importin a from the nucleus to the cytoplasm, where it can bind NLS-containing proteins, enabling a new cycle of nuclear transport [START_REF] Cook | The structure of the nuclear export receptor Cse1 in its cytosolic state reveals a closed conformation incompatible with cargo binding[END_REF][START_REF] Kutay | Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor[END_REF][START_REF] Stewart | Molecular mechanism of the nuclear protein import cycle[END_REF]. It was shown that if CSE1L expression is downregulated, HDAC1/2/8 mislocalized to the cytoplasm. However, other HDACs (HDAC3/5/10) -also containing NLS -do not change their localization during CSE1L depletion [START_REF] Dong | Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing[END_REF]. The exact mechanism of this phenotype is yet to be understood.

Nonetheless, determining the possible involvement of CSE1L on the intracellular localization of LphD might give us a better understanding how the protein targets the nucleus and elucidate the possible involvement of importin a in this process.

Next, we analyzed the HDAC domain sequence of LphD. Several amino acids are conserved in LphD, when compared to its eukaryotic counterparts. One of these conserved amino acids is the catalytic site tyrosine (Y392). In addition, several other amino acid residues, all part of the so-called charge relay system (Uba and Yelekçi, 2017) -a crucial component of HDAC catalysis -are conserved in LphD. Moreover, when using the AlphaFold 2 model of LphD, we can see that all these conserved residues (H177/178, D216/330, N218, Y248/392) localize in what is predicted to be the binding pocket of the enzyme. These results will be confirmed as soon as we obtain the crystal structure of LphD. For the moment we are focusing on crystalizing LphD in presence of TSA, however, it would be interesting -as a next step -to co-crystalize the catalytic inactive form of LphD (Y392F) with an H3 peptide, corresponding to the N-terminal histone tail.

We then could show that purified LphD has lysine deacetylase activity in an in vitro assay and that this activity is indeed dependent on the predicted catalytic tyrosine, Y392. To determine which histone residue is targeted by LphD, we set up a collaboration with Jérémy Berthelet and Fernando Rodrigues-Lima. Using fluorescently-tagged acetylated histone peptides and incubating them with purified LphD or a catalytic inactive mutant (LphD Y392F), followed by HPLC analysis, they could determine that LphD shows the highest efficiency for H3K14, when compared to other known acetylation marks of H3 (K4, K9, K18, K23, K27).

However, this experiment does not exclude the possibility of LphD targeting residues on other histone proteins. This aspect of LphD might raise new questions, since this activity would be completely independent of the activity of RomA, which is limited in its activity on histones to H3K14 (Rolando et al., 2013). Also, non-histone targets should be investigated, since it is known that HDAC activity, contrary to their name, is not limited to histones. The best understood example of HDACs targeting non-histone proteins is HDAC6, which deacetylates a-tubulin, thereby regulating chemotactic cell movement and cell motility [START_REF] Hubbert | HDAC6 is a microtubule-associated deacetylase[END_REF]. We also see an example for non-histone targeting with RomA, which was shown to methylate AROS, a regulator of histone deacetylase SIRT1 (Schuhmacher et al., 2018).

We next assessed the influence of LphD on bacterial virulence and determine its possible synergy with RomA. For this, we demonstrated that a ∆lphD strain shows a slight reduction in virulence as seen in replication assays in A. castellanii as well as human THP-1 cells. In addition, the complementation of this knockout actually led to an increase in virulence, further supporting the idea of the importance of LphD in L. pneumophila replication. To get a better understanding of the synergy between the two bacterial effectors we compared the deacetylation and methylation of H3K14 in cells infected with wild type L. pneumophila or the ∆lphD strain. In wild type infected cells, we could see a LphD-dependent decrease in acetylation, specifically of H3K14. Moreover, the ∆lphD strain shows a drastic impediment not only in H3K14 acetylation, but also in its methylation, which further supports the theory of synergistic effects between LphD and RomA. However, it is not known how the secretion of these two effectors is regulated and timed. A recent publication shows that effector secretion might be regulated by c-di-GMP levels and that the secretion is following the functional consequence of the effectors and that it is independent of effector concentration [START_REF] Allombert | Deciphering Legionella effector delivery by Icm/Dot secretion system reveals a new role for c-diGMP signaling[END_REF]. Nonetheless, there are still many questions that remain unanswered concerning effector secretion regulation and little is known about this process (Kim et al., 2020;[START_REF] Meir | Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila[END_REF]. Two-component systems like LetAS and PmrAB have been implicated to regulate the expression of specific effectors [START_REF] Segal | The Legionella pneumophila two-component regulatory systems that participate in the regulation of Icm/Dot effectors[END_REF], and Dot/Icm components like IcmS and IcmW have been shown to control the secretion of specific subsets of effectors [START_REF] Cambronne | The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation[END_REF]. However, due to the low abundance of effectors and the small amounts that are secreted during infection, it is very difficult to determine secretion kinetics without artificially over-expressing the protein in question. Nonetheless, the prospect of a possible timed secretion of LphD and RomA, to first facilitate deacetylation of H3K14, followed by its methylation, represents a unique opportunity to assess secretion regulation in L. pneumophila.

To determine possible eukaryotic interaction partners, we transfected HEK293T cells with GFP or a GFP-LphD fusion construct, pulled down GFP proteins with GFP-trap beads, and analyzed those pulldowns by MS/MS for bound eukaryotic proteins. This led to the identification of KAT7, a histone acetyltransferase known to target H3K14, in a complex called HBO1 [START_REF] Kueh | HBO1 is required for H3K14 acetylation and normal transcriptional activity during embryonic development[END_REF]. KAT7 activity is connected to the promotor regions of highly transcribed genes and has recently been shown to have a wide variety of activities besides histone acetylation, for example histone propionylation and histone butyrylation [START_REF] Partridge | Occupancy maps of 208 chromatin-associated proteins in one human cell type[END_REF]Xiao et al., 2021). The interaction of LphD, as well as RomA, with KAT7 and other components of the HBO1 complex was confirmed in transfection followed by co-IP. In addition, the interaction of KAT7 with both bacterial effectors was also shown during infection.

Interestingly, in transfection experiments RomA seems not to bind every component of the complex, like LphD does. The reason for this is still unknown, but one possible explanation could be that the binding of RomA initiates structural changes in the HBO1 complex, either through its binding directly or through PTMs, leading to the loss of different components of the complex. This hypothesis could be tested by transfecting cells with RomA and performing co-IPs targeting the different components of the HBO1 complex and determining possible changes in its composition.

Moreover, a possible regulation of KAT7 activity by the two bacterial effectors cannot be excluded. Not much is known about the regulation of KAT7 activity, however, the activity of its homologue KAT8, was shown to be regulated by autoacetylation of a lysine residue, which is conserved in KAT7 [START_REF] Sun | Regulation of the histone acetyltransferase activity of hMOF via autoacetylation of Lys274[END_REF][START_REF] Yuan | MYST protein acetyltransferase activity requires active site lysine autoacetylation[END_REF]. As mentioned before, the investigation of non-histone targets of LphD could lead to very interesting results, and KAT7 could be one of those non-histone targets. It might be possible that LphD is not only hijacking the HBO1 complex to target the host chromatin, but in addition, deacetylates and thereby inactivates KAT7 to further impede histone acetylation. This multifunctional approach of LphD could ensure to minimize histone acetylation as efficiently as possible, thus promoting the subsequent methylation. In addition, other complexes connected to epigenetic regulation, like NuRD and Sin3A, have been identified during the GFP-pulldown. These complexes are known to contain eukaryotic HDACs, so they represent promising interaction partners of LphD (Adams et al., 2018;[START_REF] Torchy | Structure and function insights into the NuRD chromatin remodeling complex[END_REF]. Further experiments are necessary to investigate the possible interplay of LphD with these complexes.

The analysis of the RNA-seq experiments revealed drastic changes in the host's transcriptional response to L. pneumophila infection. However, if we compare up-and downregulated genes at these early stages of infection we can see that the up-regulation seems more robust, with around half of the genes being shared between the last three time points and a quarter shared between all time points. Whereas, in the group of down-regulated genes, genes unique for each time point represent the biggest groups. This indicates that the process of transcriptional up-regulation in response to an infection is highly organized and comprises a core set of genes involved in the immune response, a behavior that cannot be seen in the downregulated genes. The analysis of enriched GO terms (biological process) for the 100 most upand down-regulated genes only further supports this idea. For the up-regulation we see a strong enrichment in GO terms related to immune response with the biggest set of unique GO terms at 1 h post-infection. This set includes terms related to CD4+ T cell differentiation, a previously described phenomenon in L. pneumophila infection [START_REF] Trunk | Innate instruction of CD4+ T cell immunity in respiratory bacterial infection[END_REF]. Interestingly, another enriched GO term at early time points is "response to mechanical stimulus". However, this can be easily explained if we look at the genes attributed to this GO term. "Response to mechanical stimulus" includes genes such as CXCL10, NFKBIA (NF-kB inhibitor a) and IL1B, all of which can also be assigned to other, more expected GO terms. Later time points focus on overall immune response processes, such as interferon gamma production and regulation, as well as leukocyte migration. The results of this RNA-seq, concerning the reaction of the cell to an infection by L. pneumophila provide exciting insight into the early processes during bacterial invasion. Moreover, the cornucopia of results provided by these experiments can be used to further investigate the interplay between L. pneumophila and its host on a transcriptional level.

Although we show a clear synergy between LphD and RomA, this does not limit the possible interactions to these two proteins. L. pneumophila encodes several other putative nucleomodulins that might target histones. These putative secreted effectors are part of the PRMT (protein arginine methyl-transferase), DOT1 (histone lysine methyltranferase), HAT (histone acetyltransferases), and LSD1 (lysine-demethylase) families and could be part of a bigger interaction network of secreted effectors all targeting the host cell nucleus. The possible synergy of LphD with any of these effectors should be assessed to get a better understanding of LphD beyond its synergy with RomA.

Another aspect of this project that could be further investigated is the role of LphD and RomA during the infection of amoebae. We could clearly see a growth defect of the ∆lphD strain in A. castellanii, however no further experiments were possible due to the limited resources available for amoebae. Moreover, very little is known about the epigenetic landscape of A. castellanii, and even less is known about its regulation. A more thorough examination of the role of LphD and RomA in this context, could reveal new exciting results on how the bacteria manipulate different hosts with the same set of effectors.

Taken together, the results of my PhD work have, on one hand, brought new insight in how bacterial pathogens -in this case L. pneumophila -manipulate the chromatin landscape of the host cell, and, on the other hand, opened many new and exciting questions regarding the exact molecular mechanism. Answers to these questions might not only elucidate how pathogens manipulate host cell functions, but also contribute to discovering new host cell pathways. Moreover, the identification of new bacterial activities modifying host chromatin will help us to better understand the strategies employed by L. pneumophila, and possibly other bacteria, to modulate the host response for its advantage. Furthermore, inhibiting these chromatin modifying activities could represent a promising new target to treat bacterial infections, considering the ever-growing threat of antibiotic resistances. L. longbeachae is the second most common cause of Legionnaires' disease after L.

Annexes

pneumophila. Especially in Australia, New Zealand and Japan, L. longbeachae accounts for around 30% of all cases. Yet, L. longbeachae has not been studied to the same extent, leading to rather limited insight into its mode of infection and other aspects of its biology. While the research conducted on L. pneumophila can contribute to a better understanding of L.

longbeachae, however, this does not replace research performed specifically on L.

longbeachae, due to several key differences between the two species.

The aim of the project was the determination of the role of a secreted bacterial Rab-like small GTPase, RabL, during infection and assess its influence on bacterial virulence. I was involved in determining the intracellular localization of RabL during infection, by constructing an expression plasmid enabling the overexpression of V5-tagged RabL and following its translocation in the host cell by immunofluorescence microscopy.

In addition, I helped to exclude the involvement of specific phosphorylation sites of RabL in its intracellular localization when expressed ectopically in human cells, as well as its involvement in Golgi disruption during L. longbeachae infection. Last but not least, I was implicated in confirming the interaction of RabL with possible eukaryotic partners in the host cell.

This publication is in preparation and promises to grant an exciting new insight in hostpathogen interaction and manipulation of intracellular trafficking by pathogens.

2

Abstract

The zebrafish has become a powerful model organism to study host-pathogen interactions. Here, we developed a zebrafish model of Legionella pneumophila infection to dissect innate immune responses. We show that L. pneumophila cause zebrafish larvae death in a dose dependent manner, and that macrophages are the first line of defence, with neutrophils cooperating to clear the infection. When either macrophages or neutrophils are depleted, the larvae become lethally sensitive to L. pneumophila. As observed in human infections, the adaptor signalling molecule Myd88

is not required to control disease in the larvae. Furthermore, proinflammatory cytokines IL-1 and TNF were upregulated during infection, recapitulating key immune responses seen in human infection. We also uncovered a previously undescribed phenotype in zebrafish larvae, whereby bloodborne, wild type L. pneumophila invade and grow in the larval yolk region but not a T4SS mutant. Zebrafish larva represent an innovative L. pneumophila infection model closely mimicking important aspects of human infection.

.

INTRODUCTION

Legionella pneumophila, a gram negative, facultative intracellular bacterium inhabits natural, freshwater sources 1,2 . As an environmental, aquatic microbe L. pneumophila replicates intracellularly in aquatic protozoa 3 . Most interestingly, in contrast to other intracellular pathogens L. pneumophila is not adapted to a single host, but it exhibits a broad host range including Amoebozoa (amoebae), Percolozoa (excavates) and Ciliophora (ciliated protozoa) 3,4 . In the environment L. pneumophila can also be found within biofilms where it acquires nutrients from this mixed community, but it can also survive in a planktonic form for a certain time as well 5 . As fresh water and man-made systems are connected, L. pneumophila can also contaminate artificial water systems. Protected in its protozoan hosts L. pneumophila survives water disinfectants and may gain access to humans via aerosols produced by different man-made structures and devices. The inhalation of L. pneumophila contaminated aerosols can cause a severe pneumonia, the so-called 6 .

However, not every infection leads to disease. Disease outcome is determined by virulence of the bacterial strain, bacterial burden in the inhaled aerosols and most importantly by the host immune status. Host factors determining susceptibility include age above 50, smoking and/or having chronic lung disease, being immunocompromised and genetic factors that alter the immune response 2,7,8 .

Once the bacteria reach the lungs of susceptible individuals, they can infect alveolar macrophages and replicate therein. After being phagocytosed L. pneumophila avoids lysosomes and establishes an endoplasmic reticulum derived vacuole named the Legionella containing vacuole (LCV)

. The LCV, a safe haven for bacterial replication, is established by utilizing the Dot/Icm type IV secretion system (T4SS) that injects over 350 proteins into the host cell 9-11 . These effector proteins manipulate a myriad of host pathways to recruit vesicles derived from the endoplasmic reticulum to the LCV, to supply the bacteria with nutrients, restrain autophagy and supress apoptosis or to subvert the host cell immune response 9-11 . A surprising high number of these effectors mimic host proteins and encode eukaryotic functions helping L. pneumophila to subvert numerous host pathways in remarkable diverse ways 11-13 Intracellular bacterial replication and innate immune responses have been studied in vitro using both murine and human cell lines and in vivo using different animal models of L. pneumophila infection. However, results obtained with these models cannot be easily extrapolated to what is observed in human disease. Studies in invertebrate models, for example in Galleria mellonella and Caenorhabditis elegans, 14,15 require further validation in more developed models as their immune system greatly differs from that of vertebrates. More interestingly, mouse infection fails to recall the human disease phenotype, as most inbred mice strains are naturally resistant to L. pneumophila 16 .

Very early after the discovery of L. pneumophila the guinea was developed. Guinea pigs are highly susceptible to L. pneumophila when infected through injection into 4 the peritoneum 6 or when exposed to L. pneumophila containing aerosols 6 . Several studies thereafter have shown that the guinea pig infection model recalls human disease and allows to study the immune response to L. pneumophila infection 17,18 . However, the guinea pig model is now rarely used due to the limited availability of specific immunological reagents for these animals and the demanding laboratory and husbandry requirements to work with guinea pigs.

Since the above-mentioned models, including the widely used murine models, are limited for studying L. pneumophila infection in vivo and discrepancies exist between results obtained in mouse or human cells, the development of new, alternative models for Legionella infection is important. The zebrafish (Danio rerio) originally introduced as a model organism in developmental biology has emerged in recent years as a powerful non-mammalian model to study nearly every aspect of biology, including immune cell behaviour and host-pathogen interactions 19,20 .

Zebrafish are evolutionary closer to humans than fruit flies and nematodes, easier to manipulate than mice and their immune system is remarkably similar to the one of mammals, making them an attractive laboratory model for immunology and infection biology 19,20 . Its popularity is also due to its small size and the natural translucency of its embryos and larvae, which makes it possible to follow leukocyte behaviour and infection onset at the level of the whole organism in real-time and high resolution 21 . Additionally, although adult organisms display a fully developed immune system with both active innate and adaptive branches, studies can also be conducted at the early stages of life (embryonic or larvae) when the organism solely relies on innate immunity, allowing to dissect the mechanisms arising from different immune responses 21-23 . Thus, we sought to examine whether the zebrafish could be an alternative model for analysing host-pathogen interactions and the innate immune response to L. pneumophila infection.

We show that L. pneumophila infection of zebrafish larvae recapitulate human disease onset, as infected wild-type larvae are generally able to clear the infection, but immunocompromised fish fail to do so. Both macrophages and neutrophils quickly interact and engulf injected L. pneumophila.

Macrophage-depleted larvae show a dramatic increase of bacterial burden concomitant with host death, pointing to a crucial role of macrophages in controlling the infection. Interestingly, we discovered a new infection phenotype, as L. pneumophila replicates in the larvae yolk region, where it seems to be able to avoid the immune response of the host.

RESULTS

L. pneumophila infection induces mortality in zebrafish larvae in a dose dependent manner

To analyse whether L. pneumophila can cause disease in zebrafish larvae we microinjected larvae 72 The copyright holder for this this version posted October 19, 2021. ; https://doi.org /10.1101/2021.10.18.464513 doi: bioRxiv preprint 5 secretion system (T4SS) deficient isogenic mutant expressing GFP ( dotA-GFP). The infected larvae were kept at 28°C and were monitored regularly until 72 hours post infection (hpi) to record survival or death using a stereomicroscope. Larvae infected with doses of up to 3x10 3 CFU of WT-GFP (defined as low dose, LD) all survived (100% survival). In contrast, larvae infected intravenously with doses of 10 4 CFU (defined as high dose, HD) resulted in approximately 30% of death within 72 hpi (Fig 1B). Importantly, all larvae injected with LD or HD of the dotA-GFP strain survived for the entire time of observation (Fig 1B) indicating that the T4SS is important for replication in zebrafish larvae as it is in other infection models and in humans.

We then set up a method to monitor the bacterial burden of the infected zebrafish larvae. The progression of the infection was followed by analysing the bacterial load at 0, 24, 48 and 72 hpi comparing three different methods. First, we quantified the pixel counts of GFP fluorescence of live larvae images (Fig. S1A), secondly, we analysed the number of GFP expressing bacteria present in lysed infected larvae by FACS (Fig. S1B) and thirdly we plated serial dilutions of homogenates of euthanized larvae on BCYE medium (Fig S1C). The results obtained with the three methods were comparable (Fig S1). We choose to routinely monitor the L. pneumophila load of zebrafish larvae by FACS. As shown in Fig. 1C, larvae injected with LD of WT-GFP progressively eliminate the bacteria, by 24 hpi. Similarly, with high doses dotA-GFP were progressively cleared by 24 hpi. In contrast, some zebrafish larvae injected with HD of WT-GFP were unable to eliminate the bacteria at 72hpi, and the bacterial burden even increased by . We also monitored infected larvae by fluorescent microscopy. Immediately upon injection (20 min to 2 hpi), bacteria were detectable as small foci, probably associated with professional phagocytes (Fig. 1D). By 24 hpi, in both, larvae injected with LD of WT-GFP as well as larvae injected with HD of the avirulent dotA-GFP strain, the GFP signal declined becoming undetectable by 48 hpi, suggesting that the bacteria were progressively cleared. Despite showing the same pattern 24 hpi, larvae injected with HD of WT-GFP displayed a radically different progression of infection at 48 hpi, as bacterial proliferation started in a fraction of the infected larvae as seen by an increase in GFP signal. Most interestingly, in these larvae, bacterial proliferation occurred mainly in the yolk region while the bacterial load in the body decreased simultaneously. These bacterial foci in the yolk increased dramatically over time, causing death of the infected larvae by 72 hpi (Fig 1D).

Collectively our results indicate that L. pneumophila WT, but not the T4SS mutant induces death of zebrafish larvae. Larvae that were unable to control infection by 72 hpi, showed a unique phenotype, an increase of the bacterial burden in the yolk region.

Bloodstream L. pneumophila establishes a proliferative niche in the yolk region causing a persistent infection

To characterise the L. pneumophila foci identified in the yolk region of zebrafish larvae, we used high resolution fluorescent microscopy of HD of WT-GFP bloodstream injected in 72hpf

Tg(mfap4::mCherryF) (herein referred as mfap4:mCherryF) (red macrophages) or Tg(Lyz::DsRed) nz50

(herein referred as lyz:DsRed )(red neutrophils) or Tg(kdrl::mCherry) is5 (red blood vessels) larvae.

Upon injection of HD of WT-GFP, bacteria were progressively eliminated by the rest of the body and appeared growing in the yolk region between 48 and 72hpi, with macrophages accumulating there (Fig. 2A). We observed that L. pneumophila foci in the yolk region are highly complex, aggregate-like structures of long, filamentous bacteria growing in the yolk cell region and not in the visceral organs of the zebrafish larva. Macrophages were recruited to the yolk region containing L. pneumophila, dotA mutant strain, did neither develop bacterial colonisation of the yolk nor larval death. This outcome was independent of the used dose, suggesting that zebrafish susceptibility to L. pneumophila infection and yolk penetration depends on a functional T4SS system.

(
Thus, blood-borne L. pneumophila is able to invade the yolk sac of zebrafish larvae, a previously undescribed phenotype of bacterial infection in this model. Once in the yolk, the bacteria replicate extensively, forming complex, organized, aggregate-like structures that cannot be removed by macrophages and neutrophils, thereby avoiding he host immune control and clearance, eventually leading to death of the larvae.

Infection of zebrafish larvae with high doses of L. pneumophila leads to macrophage and neutrophil death

In human infection, alveolar macrophages are the primary cell type infected by L. pneumophila supporting its intracellular replication. Following infection, neutrophils are recruited to the lung and are key players for controlling infection as they possess antimicrobial activity and kill L. pneumophila . To analyse whether zebrafish infection mirrors human infection we monitored the interaction of zebrafish macrophages or neutrophils with the bacteria in vivo. The transgenic zebrafish larvae mfap4:mCherryF and lyz:DsRed were injected with low or high doses of WT-GFP or with high doses of dotA-GFP. Infected larvae were monitored using widefield fluorescence microscopy and the number of leukocytes per larva was assessed by counting fluorescent macrophages and neutrophils over time until 72hpi. We observed that upon injection of high dose WT-GFP, the macrophage count decreased dramatically at 24hpi and then remained stable (Fig. 3A,B). Neutrophil counts gave similar results, as there was a dramatic decrease observed in neutrophil numbers starting at 24hpi, in particular after injection of high doses of WT bacteria Fig. 3C,D). Interestingly, upon infection with low doses of WT the neutrophil numbers decreased dramatically only at 24hpi but increased at 48hpi and 72hpi (Fig. 3D). In contrast macrophage and neutrophil counts remained unaffected upon dotA strain, suggesting that phagocyte death is linked to a functional T4SS system.

Taken together, these results show that high dose L. pneumophila infection leads to a decrease in the number of professional phagocytes dependent on the T4SS, similar to what is seen during human infection by L. pneumophila and Mycobacterium tuberculosis 24,25

Macrophages are the primary cells to phagocytise blood-borne L. pneumophila and neutrophils cooperate to decrease bacterial load

As macrophages and neutrophils are likely the phagocytes that interact with L. pneumophila we analysed phagocyte-L. pneumophila interactions in vivo by injecting mfap4:mCherryF or lyz:DsRed 72hpf larvae with WT-GFP dotA-GFP and recorded phagocyte-L. pneumophila interactions using high resolution confocal microscopy. This showed that upon injection of LD WT-GFP, macrophages immediately contacted and engulfed blood-borne bacteria, and the initial bacterial load was thereby unchanged for 8hpi. The GFP signal of the engulfed bacteria was present for a long time in macrophages, suggesting that live bacteria persist in macrophages in vivo over a certain period of time. However, macrophages were continuously recruited to the site of infection and by 16hpi the bacteria were mostly undetectable (Fig. 4A were not completely cleared but persisted, and at 72hpi L. pneumophila was found in macrophages, suggesting that the bacteria are also replicating in macrophages of zebrafish larvae. Indeed, high resolution confocal microscopy showed that at 72hpi, L. pneumophila can also be found inside of macrophages in replicative vacuoles (Fig. S2).

The analyses of L. pneumophila-neutrophil interactions showed that these engulfed the bacteria trapped in the mesenchyme around the site of injection, but they were less efficient at clearing blood-borne bacteria. This is similar to what has been previously observed for infection of zebrafish larvae with Escherichia coli or Shigella flexneri 22,28 . Indeed, upon infection with a high dose of WT-GFP, L. pneumophila persisted in neutrophils and massive death of infected neutrophils occurred Altogether this shows that upon bloodstream injection of L. pneumophila, macrophages and neutrophils efficiently cooperate to eliminate the majority of bacteria within 20-24 hpi, with macrophages playing the primary role. However, L. pneumophila is also able to persist and replicate in macrophages. In contrast, neutrophils interact with L. pneumophila by quickly engulfing bacteria trapped in the mesenchyme near the site of injection but are less efficient in clearing blood-borne bacteria.

Macrophages are the first line defence restricting L. pneumophila infection

In humans, innate immune responses, based essentially on the activities of professional phagocytes and pro-inflammatory cytokine induction, are the key players to control and restrict L. pneumophila proliferation. Thus, human disease develops primarily in immunocompromised individuals 10 . To investigate whether the phagocytes of the innate immune system, macrophages and neutrophils, are also responsible for controlling L. pneumophila infection in zebrafish larvae, we selectively and transiently depleted macrophages or neutrophils, respectively and infected these immunocompromised L. pneumophila. Depletion of macrophages was achieved by knocking down the expression of spi1b, a transcription factor involved in early myeloid progenitor formation. A low dose of spi1b morpholino was reported to impact macrophages without affecting neutrophils 29 . We monitored the effect of low doses spi1b morpholino injection on macrophage and neutrophil populations in double transgenic larvae with green neutrophils (mpx:GFP) and red Intravital imaging of infected spi1b knock down larvae also showed that both low and high doses of WT-GFP failed to be cleared and that the bacteria established a replicative niche in the yolk, where they proliferated extensively (Fig 5C). This highlights, that macrophages are critical to restrict the onset of infection and L. pneumophila proliferation in vivo. Furthermore, these results also suggest that neutrophils, which are not depleted in spi1b knockdown larvae, fail to control L. pneumophila infection in the absence of macrophages.

We next analysed the role of neutrophils in controlling the infection. Neutrophil development was disrupted by knocking down the G-CSF/GCSFR pathway using csf3R morpholino, previously reported to decrease up to 70% of the neutrophils present 30-32 . We then monitored the efficiency of the csf3R morpholino knockdown in double transgenic larvae confirming that 75% of the neutrophil population was depleted, while macrophage numbers were only slightly decreased (Fig S3B). When HD dotA-GFP was injected, neutrophil-depleted larvae survived, and the bacterial burden remained unchanged, similar to what we had observed in infections of macrophage-depleted larvae (Fig. 5D,E). However, when neutrophil-depleted larvae were injected with HD WT-GFP, larvae survival significantly decreased and bacterial burdens increased at 48hpi (Fig. 5D,E). Neutrophildepleted fish larvae showed an intermediate phenotype, displaying less survival and higher bacterial burden than in WT infected control larvae (Fig. 1A) but more survival and lower bacterial burden than in macrophage-depleted larvae (Fig. 5D,E). Intravital imaging showed that csf3R knockdown larvae that were unable to control L. pneumophila infection showed bacterial proliferation in the yolk comparable to WT control larvae (Fig 5F).

These results show that both neutrophils and macrophages are required for restricting and controlling L. pneumophila infection in the zebrafish model, but macrophages play the key role.

Although neutrophils contributed less to clear the bacteria upon bloodstream injection, neutrophils might impact the infection outcome through cytokine release that can modulate macrophage activity.

. CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

Key pro-inflammatory cytokines are induced upon L. pneumophila infection of zebrafish larvae

Proinflammatory cytokines produced by infected and bystander cells during L. pneumophila infection of humans and mice play crucial roles in orchestrating host defences to control infection 33,34 .

Infected cells produce IL---dependent translational bypass. In contrast, bystander cells produce IL-6, TNF-and IL-12 in an IL-1 receptor (IL-1R) dependant way 33,35 . To determine the pro-inflammatory responses of zebrafish larvae during L.

pneumophila infection, we analysed il1b, tnfa, and ifng1/2 (orthologues of mammalian Ifng) gene expression levels over time by qRT-PCR on RNA isolated from individual infected larvae. We found that infection of zebrafish larvae with LD or HD of WT-GFP induced a rapid (by 6hpi) and robust induction of il1b and tnfa gene expression. In larvae injected with low doses of WT-GFP the expression levels started to decrease by 24hpi, and gradually became undetectable at 72hpi. In contrast, larvae injected with HD of WT-GFP, expression of il1b and tnfa did not decrease over time (Fig. S3A andB) and a significant induction of ifng1 was observed at 48hpi (Fig. S3C) but not of ifng2 (Fig. S3D). In parallel, we scored the bacterial burden of the infected larvae before pro-inflammatory cytokine measurement at each time point under the microscope, which consistently showed that larvae with increased il1b and tnfa induction had also high bacterial burdens in the yolk and were not controlling the infection. These pro-inflammatory responses were T4SS dependent, as zebrafish larvae infected with HD of dotA-GFP did not show significant induction of transcription of tnfa, il1b

and ing1/2 (Fig. S3 A-D).

Collectively, these results reveal, that key pro-inflammatory cytokines known to orchestrate the host response during L. pneumophila infection in humans are also induced in zebrafish larvae, and that cytokine gene induction is sustained when uncontrolled L. pneumophila proliferation occurs.

The immune response of zebrafish larvae to L. pneumophila infection is independent of MyD88 signalling

In innate immunity, the myeloid differentiation factor 88 (MyD88) plays a pivotal role in immune cell activation through Toll-like receptors (TLRs). MyD88-deficient mice are highly susceptible to L. pneumophila infection 36-39 , however this is not the case when human macrophages are depleted of MyD88 40 . Therefore, we sought to analyse which role MyD88 plays in zebrafish larvae during L. pneumophila infection. We injected myd88-/and control larvae with LD or HD of WT-GFP, or with HD of dotA-GFP and monitored larvae survival and bacterial burden over time as described in Figure 1. Our results show that susceptibility to infection of myd88-/larvae injected with HD of WT-GFP, was comparable to that of WT larvae (Fig. 6A). Similarly, both control and myd88-/larvae injected with LD WT-GFP or with the avirulent dotA-GFP bacteria did not develop an infection, and the

Annexes

-160 -11 bacterial burden decreased over time indicating that bacteria were cleared (Fig. 6A,B). To determine if pro-inflammatory responses were affected in the absence of MyD88 signalling, we analysed il1b and tnfa gene expression levels over time in control and myd88-/larvae. Our results showed that il1b and tnfa gene expression levels were comparable in control and myd88-/infected larvae for all conditions tested (LD D).

Taken together, our results suggest that MyD88 signalling is not required for the innate immune response against L. pneumophila infection in the zebrafish larvae, which recapitulates human infection. However, MyD88 signalling may also be functionally compensated by other immune signalling pathways.

Legionella pneumophila replication in the yolk of zebrafish larvae is T4SS dependent

Interestingly, replication of L. pneumophila mainly took place in the yolk region of infected zebrafish larvae (Movie S1-4, Fig. 2), dependent on a functioning T4SS as dotA-GFP failed be detected in the yolk. To investigate whether the secretion mutant would be able to grow in the yolk cell when reaching it, we injected LD and HD of WT-GFP or dotA-GFP directly into the yolk cell cytoplasm of 72hpf lys:DsRed zebrafish larvae (Fig. S4A). WT-GFP replicated extensively in the yolk region with low and high dose infections leading to rapid bacterial proliferation followed by a marked increase of the bacterial burden and death of the larvae (Fig. 7A,B). Surprisingly, dotA-GFP did not replicate in the yolk even when injected directly but persisted over 72hpi. This result suggests that T4SS system is not only crucial for crossing the yolk sac syncytium but that its effectors are also necessary to obtain nutrients from the environment to allow replication. To further analyze this hypothesis, we selected a mutant in the gene encoding a sphingosine-1 phosphate lyase, (WT, spl) 41 as we reasoned that this enzyme might be implicated in degrading sphingolipids present in the yolk of zebrafish larvae and thereby might aid L. pneumophila to obtain nutrients. Injection of spl in the yolk sac region, and analyses of larvae death as compared to WT or dotA showed that survival of zebrafish larvae injected with the spl was slightly higher than with WT injected larvae (Fig. S4B), suggesting that the T4SS effector LpSpl might be implicated in nutrient acquisition in the yolk environment.

Interestingly, the first isolation of L. pneumophila was achieved by inoculating the yolk region of embryonated eggs probably due to the richness in nutrients provided by the yolk 6 . Later yolk sacs L. pneumophila 42 . Thus, we decided to analyse L. pneumophila WT and dotA phenotypes in the yolk sac of embryonated chicken eggs (ECE). We inoculated ECE directly in the yolk region with WT and with the dotA strain at a concentration of 9.2 log10 CFU/mL and 9.1 log10 CFU/mL, respectively and assessed mortality of the embryos daily. The total mortality during the 6-day observation period in WT-GFP infected eggs was significantly higher (88.9%) than in the dotA-infected eggs (14.3%; p=0.010) or PBS inoculated control eggs (28.6%; p=0.010 and p=0.021, respectively), which were not significantly different from each other (p=0.253) (Fig. S4C). The highest mortality was observed at 2 days post infection in WT inoculated eggs with 55.6% mortality versus 0% in dotA or 28.6% mortality in PBS inoculated eggs. Quantification of L. pneumophila in the yolk sac region at the day of mortality or at day 6 post infection revealed that the number of bacteria in the yolk sac of WTinfected ECE, was significantly higher than that dotA strain (7.8 log10 CFU/mL and 5.9 log10 CFU/mL, respectively, p=0.0127) (Fig. S4D). Controls inoculated with PBS (n=2) showed no L. pneumophila growth. Thus, like in zebrafish larvae only the WT strain is able to replicate in the yolk region and of inducing mortality in the embryos, while the T4SS mutant strain persists but is not able to replicate and does not induce high embryo mortality. This result further supports the finding that the T4SS system is crucial for obtaining nutrients when lipids are the major energy source available.

We next monitored neutrophil behaviour in the yolk-injected lyz:DsRed larvae in which neutrophils are labelled red. This showed that replication of WT-GFP in the yolk coincides with neutrophil death (Fig. 7C andD). The yolk cell is a single large cell where leukocytes were described to be unable to enter 43 , but interestingly, macrophages and neutrophils were highly recruited to the yolk of WT-GFP infected larvae (Figure 2B-E), suggesting that L. pneumophila is sensed by the immune system even when replicating in the yolk, and could It is likely neutrophils can partly counteract L. pneumophila growth in the yolk n in a zebrafish notochord infection model using non-pathogenic E. coli 32 .

Our results suggest that the L. pneumophila T4SS plays a crucial role for the bacteria to pass from the blood circulation into the yolk and that T4SS effectors play an important role to obtain nutrients for bacterial proliferation.

DISCUSSION

In this study, we developed a zebrafish larva infection model for L. pneumophila and have analysed host pathogen interactions and the innate immune response of the host. We have found that a successful infection of zebrafish larvae by L. pneumophila depends on the infection site, the infection dose, the T4SS Dot/Icm and the host innate immune response, in particular macrophages and neutrophils. Wild type zebrafish larvae are susceptible to infection in a dose dependent manner, as larvae infected with a highly concentrated bacterial inoculum displayed bacterial dissemination and replication, concomitant with host death. However, as only about 30% of the larvae displayed this phenotype, the innate host defence of the larvae against L. pneumophila infection is relatively efficient. Thus, similar to what is observed in L. pneumophila infection of immune competent but also by the capacity of the host immune system to quickly and efficiently respond to infection.

Only blood borne bacteria are able to proliferate and induce mortality in zebrafish larvae.

Once in the blood circulation, bacteria are actively engulfed and eliminated by both macrophages and neutrophils. However, some bacteria resist intracellular killing and replicate extensively inside macrophages (Fig. S2), get released into the blood flow and circulate in the zebrafish larvae. Some reach the yolk sac syncytium and T4SS competent L. pneumophila are able to cross this barrier and enter the yolk sac region. Once in the yolk, L. pneumophila gains a significant advantage in the pathogen-host arms race and establishes a replicative niche where it proliferates extensively. Indeed, in the yolk sac region L. pneumophila is protected from the host immune system as professional phagocytes are unable to enter in the yolk. Proliferation of the bacteria leads to host death, likely due to exhaustion of the nutrients present in the yolk, which are key in supporting the larvae development and due to the physical compression of the visceral developing organs, in particular the gastro-intestinal tract, exerted by the growing bacterial aggregate. Interestingly, we have also observed that in few cases the infected larvae were able to extrude the bacterial aggregates growing in the yolk and survived. This host defence mechanism has also been reported in a caudal fin model of Mycobacterium marinum infection, where infected zebrafish larvae extruded the bacteriacontaining granuloma 44 .

To our knowledge, the establishment of a replicative niche in the yolk upon injection in the bloodstream is unique to L. pneumophila. Most interestingly, direct yolk sac injection revealed that only the WT strain but not the T4SS knockout strain is able to replicate and establish a persistent infection, irrespective of the dose injected. This result points towards the involvement of the T4SS system and its secreted effectors in infection, replication and nutrient uptake in the yolk environment. Further analyses of this phenotype in embryonated chicken eggs, a commonly used model for antigen preparation, showed again, that only WT L. pneumophila are able to replicate in the yolk sac region, confirming the importance of the T4SS in nutrient uptake in addition to its known role in infection (Fig. S4A,B,C). L. pneumophila is known to mainly use amino acids as carbon and energy sources for growth 45 and secreted T4SS effectors have been shown to aid in amino acid uptake 46 , however, fatty acids, glucose and/or glycerol also serve as carbon sources during the later stages of the life cycle of L. pneumophila 47,48 , but no effectors connected to the uptake of these nutrients have been identified yet. The yolk cell is composed of a complex and dynamic mixture of different lipids on which the zebrafish larvae rely on for nutrition throughout development in the early larva phase. Cholesterol and phosphatidylcholine are the main constituents until 120hpf, with triacylglycerol, phosphatidylinositol, phosphatidylethanolamine, diacyl-glycerol, cholesteryl esters and sphingomyelins also present in significant concentrations 49 . L. pneumophila is known to secrete 14 several effectors with lipolytic activity through its T4SS which could be important for growth in a lipid rich environment like the yolk [START_REF] Hiller | Secreted phospholipases of the lung pathogen Legionella pneumophila[END_REF]. In a first attempt to identify one of these effectors we analysed the growth of a L. pneumophila mutant in a gene encoding a sphingosine-1 phosphate lyase (LpSpl) 41 compared to the WT strain after direct injection in the zebrafish larvae yolk sac.

Indeed, a small difference in larvae mortality was observed for the spl strain, suggesting that LpSpl is one of several effectors that might participate in nutrient acquisition from lipids (Fig. S4B).

However, further analyses are needed to identify all effectors implicated in this phenotype.

Studies of Legionella infection in humans, guinea pigs and mouse lungs have shown that L. pneumophila interacts closely with neutrophils and mononuclear phagocytes 50,51 . Professional phagocytes are the main replication niche for L. pneumophila with monocytes and macrophages, in particular alveolar macrophages, representing the main cells for replication in the lungs 52-55 . In vivo studies in mice have shown that upon lung infection with L. pneumophila neutrophils, cDCs, monocytes, and monocyte-like cells are rapidly recruited to the infection site, but although all these cells seem to engulf the bacteria, L. pneumophila appears to be able to translocate effectors only into neutrophils and alveolar macrophages. In zebrafish macrophages appear during the first days of development, followed by neutrophils a day later forming together an efficient immune system that protects the developing fish 23,56-58 . Therefore, the zebrafish larva offers a unique possibility to interrogate the role of innate immune responses to infection 21 . Indeed, macrophage depleted larvae showed a dramatically increased susceptibility to L. pneumophila infection as nearly 100% of larvae inoculated with HD of WT and 30% of larvae inoculated with LD of L. pneumophila died from the infection. Hence, macrophages are the first line of infection control against L. pneumophila and are essential for restricting and controlling blood-borne infections, similar to what was observed for Burkholderia cenocepacia or Staphylococcus aureus infection 59,60 . In contrast, when neutrophils were depleted, the innate immune response was impaired to a lesser extent, suggesting that neutrophils are required to ensure an effective innate immune response and, that macrophages alone are not able to contain high burdens of L. pneumophila infection (Fig. 5).

Human innate immune signalling relies strongly on activation of Toll-like receptors (TLRs) and respective adaptor molecules, all of which are highly conserved in the zebrafish 61,62 . One of these adaptors is MyD88, known as a central player in interleukin 1 receptor (IL-1R) and TLR signalling in humans and mammalian models 63 . MyD88 signalling is crucial for mice to combat L. pneumophila infection, as it triggers the early secretion of inflammatory cytokines, neutrophil recruitment, and the host immune response to the infection. Consequently, mice that lack MyD88 are highly susceptible to infection 35-38 . However, in MyD88 depleted human macrophages L. pneumophila replication is not different to replication in WT cells 40 Here we show, that L. pneumophila infected myd88-/zebrafish larvae have the same replication phenotype as WT larvae. Thus, Myd88 signalling does not play a key

Annexes

-164 -15 role or may be redundant in the control of the innate immune response to L. pneumophila in zebrafish larvae, indicating that zebrafish mirrors human infection better than the mouse model. In the mouse model infected macrophages are incapable of producing cytokines, such as tumor necrosis factor (TNF) and interleukin-12 (IL-12), which are necessary to control infection. In contrast, infection of zebrafish larvae with WT L. pneumophila induced a rapid (by 6hpi) and robust induction of il1b and tnfa gene expression. However, it is thought that IL-1 released initially by L. pneumophilainfected macrophages drives the production of critical cytokines by bystander cells 33 . Infection of zebrafish larvae with HD of WT L. pneumophila induced a rapid (by 6hpi) and robust induction of il1b and tnfa gene expression whereas WT LD infection leads only to a short induction of Il1b transcript levels at 6hpi before declining to CTRL levels at later time points, suggesting that a short boost of IL-L. pneumophila. However, for a high load of L. pneumophila even a high and long-term induction of IL-regulation of the immune response may be abrogated leading to a constant activation of ILexpression. Moreover, gene expression analyses also confirms that Myd88 has no influence on the control of the infection, as no difference in the transcript levels of il1b, tnfa, ifng1 or infg2 was observed further suggesting that activation of the IL1R and certain TLR pathways are not crucial for L. pneumophila clearance in zebrafish larvae. One may even hypothesise that ILbeneficial for L. pneumophila replication, as it was shown that ILthe metabolic state of the bystander cells as it was shown that ILmetabolically active cells and increased cellular glucose uptake 64 , which could aid L. pneumophila replication.

In conclusion, we have set up a new infection model for L. pneumophila that mimics human infection better than the mouse model. The unique advantages of the zebrafish provide now exciting possibilities to further explore different aspects of the relationship between, L. pneumophila and its host: the dynamics of bacterial dissemination, the interactions of the bacteria with macrophages and neutrophils, as well as the host immune response by intravital imaging. Tg(mpx:GFP) i114 66 , Tg(kdrl::mCherry) is5 67 and myd88 hu3568 mutant line (obtained from the Hubrecht Laboratory and the Sanger Institute Zebrafish Mutation Resource) 68 , were raised in our facility. Eggs were obtained by natural spawning, bleached according to standard protocols, and kept in Petri dishes containing Volvic source water and, from 24 hours post fertilization (hpf) onwards 0.003% 1phenyl-2-thiourea (PTU) (Sigma-Aldrich) was added to prevent pigmentation. Embryos were reared at 28°C or 24°C according to the desired speed of development; infected larvae were kept at 28°C.

Timings in the text refer to the developmental stage at the reference temperature of 28.5°C. Larvae tricaine (Sigma-Aldrich) during the injection procedure as well as during in vivo imaging and processing for bacterial burden evaluation or cytokine expression studies.

Bacterial strains and growth conditions. Legionella pneumophila strain Paris carrying the pNT28 plasmid encoding for green fluorescent protein (constitutive GFP) 69 , wild-type (WT-dotA-GFP were plated from -80°C glycerol stocks on N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)buffered charcoal yeast-extract (BCYE) medium supplemented with 10 µg/ml of chloramphenicol and cultured for 3 days at 37°C. Suspensions were prepared by resuspending bacteria in sterile 1x

Phosphate Buffered Saline (PBS) and adjusting the OD 600 according to the desired bacterial concentrations for injection.

Morpholino injections. Morpholino antisense oligonucleotides (Gene Tools LLC, Philomath, OR, USA)

were injected at the one to two cell stage as described 70 A low dose (4ng) of spi1b (previously named pu1) translation blocking morpholino (GATATACTGATACTCCATTGGTGGT) 71 blocks macrophage development only, but can also block neutrophil development when it is injected at a higher dose (20ng in 2nl). The csf3r translation blocking morpholino (GAACTGGCGGATCTGTAAAGACAAA) (4ng) 30 was injected to block neutrophil development. Control morphants were injected with 4ng control morpholino, with no known target (GAAAGCATGGCATCTGGATCATCGA).

Zebrafish infections. The volume of injected suspension was deduced from the diameter of the drop obtained after mock microinjection, as described in 70 . Bacteria were recovered by centrifugation, washed, resuspended at the desired concentration in PBS. 72h post-fertilization (hpf) anesthetized zebrafish larvae were microinjected iv or in the yolk with 0.5-1nl of bacterial suspension at the desired dose (~10 3 bacteria/nl for Low Dose (LD) and ~10 4 bacteria/nl for High Dose (HD) as described 22,28 Evaluation of the bacterial burden in infected larvae. Infected zebrafish larvae were collected at 0, 24, 48 and 72hpi and lysed for analysing the bacterial burden by FACS. Each larva was placed in a 1.5 placed in 150 µl of sterile water. Larvae were then homogenized using a pestle motor mixer (Argos).

Each sample was transferred to an individual well of a 96 well plate, counted on a MACSQuant VYB FACS (Miltenyi Biotec) and data analysed using FlowJo version 7.6.5. For CFU enumeration, serial dilutions were plated on BCYE agar plates supplemented with Chloramphenicol and the Legionella Selective Supplement GVPN (Sigma). Plates were incubated for 4-5 days at 37°C and colonies with the appropriate morphology and colour were scored using the G-Box imaging system (Syngene) and colonies enumerated using the Gene Tools software (Syngene).

Dissociation of zebrafish larvae for FACS analysis of macrophages. Three to five

Tg(mfap4::mCherryF) larvae were pooled in single 1.5 ml Eppendorf tubes and anesthetized with tricaine. The supernatant was discarded, and the larvae were resuspended in 1ml of 1x trypsin-EDTA solution (SIGMA) and incubated in a dry heat block at 30 C for 10 -20 min. Every 2 minutes, the suspensions were homogenised by pipetting, until full homogenisation was reached. CaCl2 (final concentration of 2 M) and foetal bovine serum (final concentration of 10%) were added to each tube and samples were kept on ice. Lysates were filtered using 40 m strainers, washed with 20 ml ice cold 1X PBS and centrifuged 5 min at 1500 g, 4 C. Remaining pellets were resuspended in 250 l 1X PBS and analysed with a MACSQuant VYB FACS (Miltenyi Biotec).

Live imaging, image processing and analysis. Quantification of total neutrophils and/or macrophages on living transgenic reporter larvae was performed upon infection as previously described 28 . Briefly, bright field, DsRed and GFP images of whole living anesthetized larvae were taken using a Leica Macrofluo TM Z16 APOA (zoom 16:1) equipped with a Leica PlanApo 2.0X lens, and a Photometrics® CoolSNAP TM HQ2 camera. Images were captured using Metavue software 7.5.6.0 (MDS Analytical Technologies). Then larvae were washed and transferred in a new 24 wells plate filled with 1ml of fresh water per well, incubated at 28°C and imaged again under the same conditions the day after.

Pictures were analysed, and Tg(lyzC::DsRed) neutrophils or Tg(mfap4::mCherryF) macrophages manually counted using the ImageJ software (V 1.52a The bacterial burden was measured by counting the total number of pixels corresponding to the GFP channel (Metavue software 7.5.6.0). Briefly, images corresponding to the GFP channel were adjusted to a fixed threshold that allowed to abrogate the background of the autofluorescence of the yolk. The same threshold was used for all images of one experiment. Histogram in the Analyze menu was used to obtain the number of black and white pixels. As shown in figure S1A, number of white pixels corresponding to L. pneumophila are plotted using GraphPad Prism® software.

High resolution confocal live imaging of injected larvae was performed as previously described 72 . Briefly, injected larvae were positioned in lateral or ventral position in 35 mm glassbottom-Dishes (Ibidi Cat#: 81158). Larvae were immobilized using a 1% low-melting-point agarose (Promega; Cat#: V2111) solution and covered with Volvic water containing tricaine. A Leica SP8 confocal microscope equipped with two PMT and Hybrid detector, a 20X IMM objective (HC PL APO CS2 20X/0.75), a X Y motorized stage and with the LAS-X software was used to live image injected larvae. To generate images of the whole larvae, a mosaic of confocal z-stack of images was taken with the 20X objective using the Tile Scan tool of the LAS-X software and was stitched together using the Mosaic Merge tool of the LAS-X software. All samples were acquired using the same settings, allowing comparisons of independent experiments. After acquisition, larvae were washed and transferred in a new 24-per well, incubated at 28°C and imaged again under the same conditions over time. A Leica SPE inverted confocal microscope and a 40x oil immersion oil immersion objective (ACS APO 40 × 1.15 UV) was also used to live image larvae infected with L. pneumophila dotA-GFP (Figure 4).

The 4D files generated by the time-lapse acquisitions were processed, cropped, analysed, and annotated using the LAS-X and LAS-AF Leica software. Acquired Z-stacks were projected using maximum intensity projection and exported as AVI files. Frames were captured from the AVI files and handled with Photoshop software to mount figures. AVI files were also cropped and annotated with ImageJ software. Files generated with the LAS-X software were also processed and analysed with the Imaris software version9.5 (Bitplane, OXFORD Instruments) for 3D reconstruction, surfacing and volume rendering.

qRT-PCR to measure gene expression of cytokine encoding genes . RNA was extracted from individual larvae using the RNeasy ® Mini Kit (Qiagen). cDNA was obtained using M-MLV H-reversetranscriptase (Promega) with a dT 17 primer. Quantitative PCR was performed on an ABI7300 thermocycler (Applied Biosystems) using TakyonTM ROX SYBR ® 2X MasterMix (Eurogentec) in a final volume of 10 l. Primers used: ef1a (housekeeping gene used for normalization):

GCTGATCGTTGGAGTCAACA and ACAGACTTGACCTCAGTGGT; il1b: GAGACAGACGGTGCTGTTTA and

List of figures

RÉSUMÉ

Legionella pneumophila est une bactérie intracellulaire qui sécrète plus de 300 protéines dans la cellule hôte via un système de sécrétion spécialisé de type 4. L'un de ces effecteurs sécrétés, RomA, s'est avéré modifier directement la chromatine de l'hôte en méthylant la lysine 14 de l'Histone H3 (H3K14), un résidu généralement acétylé. Cela a conduit à la question de savoir comment la désacétylation de cette marque pourrait se produire pendant l'infection. Une recherche bioinformatique approfondie du genome de L. pneumophila a conduit à l'identification d'une protéine qui devrait coder pour une histone désacétylase (HDAC), nommée LphD. Au cours de ma thèse, j'ai montré que LphD est sécrétée dans la cellule hôte lors de l'infection et cible spécifiquement le noyau, où elle présente une activité désacétylase avec une efficacité élevée pour H3K14. En effet, j'ai montré que LphD désacétyle H3K14 également pendant l'infection, et que l'activité de LphD influence directement les niveaux de méthylation de H3K14 dans les cellules infectées, mettant en évidence une synergie entre LphD et RomA. J'ai également pu montrer que LphD et RomA ciblent un complexe de liaison à la chromatine endogène, nommé HBO1, qui contient l'histone acétyltransférase KAT7, contrôlant l'état d'acétylation de H3K14. Des RNAseq de cellules infectées soit par des bactéries de type sauvage, soit par le knockout LphD et RomA, ont mis en évidence l'influence de ces effecteurs bactériens sur le paysage transcriptionnel de l'hôte, en particulier sur les gènes liés à la réponse immunitaire. Le modèle que je propose est que les deux effecteurs sécrétés, LphD et RomA, travaillent ensemble pour détourner la machinerie épigénétique de l'hôte afin de faciliter la subversion de la réponse immunitaire et favoriser la réplication intracellulaire de L. pneumophila.

Mots clés: Legionella pneumophila, Histone désacétylases, nucleomodulines, épigénétique

Functional role of a histone deacetylase encoded by Legionella pneumophila

SUMMARY

Legionella pneumophila is an intracellular bacterium that secretes over 300 proteins in the host cell through a specialized type 4 secretion system. One of these secreted L. pneumophila effectors, RomA, was shown to directly modify the host chromatin by methylating lysine 14 of Histone H3 (H3K14), a usually acetylated residue. This led to the question how deacetylation of this mark might happen during infection. An in-depth bioinformatics search led to the identification of a protein predicted to code for a histone deacetylase (HDAC), named LphD. During my PhD, I showed that LphD is secreted into the host cell during infection and specifically targets the host cell nucleus, where it exhibits deacetylase activity with high efficiency for H3K14. Indeed, I showed that LphD deacetylates the H3K14 residue also during infection, and that the activity of LphD directly influences the levels of H3K14 methylation in infected cells, highlighting the synergy between LphD and RomA. I also could show that LphD and RomA target an endogenous chromatin binding complex, named HBO1, that contains the histone acetyltransferase KAT7, controlling the acetylation status of H3K14. RNAseq of cells infected with either wild type bacteria or the LphD and RomA knockout assessed the influence of these bacterial effectors on the host's transcriptional landscape, in particular on genes related to immune response. The model I propose is that the two secreted effectors, LphD and RomA, work together to hijack the host's epigenetic machinery in order to facilitate the subversion of the host immune response and promotes the intracellular replication of L. pneumophila. Key words: Legionella pneumophila, Histone deacetylases, nucleomodulins, epigenetics

Figure 1 :

 1 Figure 1: L. pneumophila in electron microscopy (A) and on a BCYE plate (B). (A) Electron microscopy picture of L. pneumophila growing in liquid culture with its polar flagellum. (B) L. pneumophila growing on BCYE agar. (A) Pictures taken by the group Biology of Intracellular Bacteria, Institut Pasteur, Paris. (B) Adapted from http://www.biomerieux-culturemedia.com/product/95legionella-agar-with-l-cysteine--bcye--and-without-l-cysteine--bcy-.

Figure 2 :

 2 Figure 2: Number of reported Legionnaires' disease cases in Europe and France. Graph comparing case numbers of Legionnaires' disease in Europe (left axis, green circles) and France (right axis, black squares). Data obtained from https://www.ecdc.europa.eu/en/legionnairesdisease/surveillance/atlas.

Figure 3 :

 3 Figure 3: Comparison of reported cases and fatal cases of Legionnaires' disease over different age groups in Europe in 2020. Graph represents the percentage of cases (reported and fatal) (Y axis) and the different age groups (X axis) in Europe in 2020. Data obtained from https://www.ecdc.europa.eu/en/legionnaires-disease/surveillance/atlas .

Figure 4 :

 4 Figure 4: Schematic representation of the intracellular life cycle of L. pneumophila in phagocytic cells and the cellular pathways targeted by the bacteria. After uptake of the bacteria, they establish the Legionella-containing vacuole (LCV). Through secretion of a wide variety of effectors, the bacteria promote the recruitment of mitochondria and ER-derived vesicles to the LCV. Within the LCV, the bacteria replicate and switch again to their more virulent, flagellated form, until they egress from the host cell. Adapted from Mondino et al. (2020).

Figure 5 :

 5 Figure 5: Electron microscopy of coiled phagocytosis of L. pneumophila by human monocytes. Human monocytes were incubated with L. pneumophila at 37˚C for 3.5 min to allow phagocytosis to begin. Then cells were fixed and analyzed by electron microscopy. Adapted from Horwitz (1984).

Figure 6 :

 6 Figure 6: Intracellular replication of L. pneumophila in macrophages. Confocal microscopy images of human monocyte-derived macrophages (hMDM), infected with L. pneumophila constitutively expressing GFP for 2 (left) and 20 (right) hours. DAPI (cyan), L. pneumophila (green), cytoplasm (red). (Pictures kindly provided by Pedro Escoll, Institut Pasteur)

Figure 7 :

 7 Figure 7: Dot/Icm secretion system of L. pneumophila. (A) Subtomogram of the Dot/Icm system of L. pneumophila depicting its connection between inner membrane (IM) and outer membrane (OM). Scale bar, 10 nm. (B) Schematic representation of the subtomogram, showing the known structures of the Dot/Icm system. Adapted from Ghosal et al. (2017).

Figure 8 :

 8 Figure 8: Schematic representation of selected Legionella effectors. Effectors are T4SS-dependently secreted into the host and target several different cellular pathways by either containing eukaryotic-domains or by mimicking eukaryotic proteins as a whole. Orange box, Legionella effector; pink oval, host target protein/molecule; orange oval, Legionella target protein; Me, methylation; U, ubiquitination; P, phosphorylation; G, glutamylation, CaM, calmodulin; IP6, inositol hexakisphosphate; ER, endoplasmic reticulum. Effectors highlighted in bold are discussed in this chapter. Adapted from Mondino et al. (2020).

Figure 9 :

 9 Figure 9: Schematic representation of chromatin organization in eukaryotic cells. DNA wraps around histone octamers, forming nucleosomes. Nucleosomes are connected by linker DNA and form chromatin fibers, which subsequently are organized into chromosomes. Adapted from https://www.abcam.com/epigenetics/chromatin-accessibility-and-architecture.

Figure 10 :

 10 Figure 10: Schematic representation of a nucleosome. DNA is wrapped around the core histone octamer, consisting of two copies of each H2A, H2B, H3 and H4. H1 acts as a clamp on the outside of the complex and stabilizes the protein/DNA interaction. Adapted from https://www.behance.net/gallery/83812381/How-DNA-is-Organised-Nucleosome-Structure?tracking_source=search_projects_recommended%7Cnucleosome.

Figure 11 :

 11 Figure 11: Schematic representation of the solenoid and zigzag model of nucleosome organization. (A) In the solenoid model interactions between the histone cores occur sequentially (1, 2, 3, 4, etc.). (B) In the zigzag model, the alternate octamers become interacting partners (1 and 3, 2 and 4, etc.), which is represented by two different colors of histone octamers. Adapted from https://www.mechanobio.info/genome-regulation/what-are-nucleosomes/.

Figure 12 :

 12 Figure 12: Selected nucleomodulins of S. flexneri, L. pneumophila, M. tuberculosis (MTB), L. monocytogenes and E. coli. Secreted nucleomodulins (in green) that enter the nucleus (represented by an orange oval; blue cylinders labeled "H" represent histones; host nuclear factors are in yellow). Post-translational modifications (PTMs): acetylation (Ac), methylation (Me), phosphorylation (P). Adapted from Bierne and Pourpre (2020).

122

  One of the most abundant histone modifications is the reversible acetylation of lysine 123 residues on histone tails. Histone acetylation is mostly connected to a transcriptional 124 activation(Barnes et al., 2019;[START_REF] Hebbes | A direct link between core histone 813 acetylation and transcriptionally active chromatin[END_REF]. The writers and 125 erasers controlling the acetylation status of histones are histone acetyltransferases (HAT) and 126 histone deacetylases (HDAC), respectively. In recent years, the role of HDACs in the immune 127 response and their manipulation by certain pathogens became evident. In addition, the 128 therapeutic use of HDAC inhibitors might open a new window to bypass the prominent 129 problem of antibiotic resistance. These topics will be the main focus of this review. 130 131 HDACs and their regulation 132 Five classes of HDAC proteins that are split up in two groups are known to date. The first 133 group, formed by Zn 2+ -dependent histone deacetylases, comprises class I (HDAC1, HDAC2, 134 HDAC3, and HDAC8), class IIa (HDAC4, HDAC5, HDAC7, and HDAC9), class IIb 135 (HDAC6 and HDAC10), and class IV (HDAC11). The second group, NAD + -dependent 136 histone deacetylases, contains class III, the so-called Sirtuins (SIRT1-7). This classification is 137 based on the homology of the enzymes to specific yeast proteins: Rpd3 for class I, Hda1 for 138 class II and Sir2 for class III. Class IV shares sequence similarity with both class I and class II 139 (H. P. Chen, Zhao, & Zhao, 2015). 140 However, despite their name, HDACs can also deacetylate non-histone proteins. The 141 tumor suppressor p53 and the adaptor protein MyD88 are two of the most prominent non-142 histone targets of HDAC enzymes (A

170

  activity through protein complex formation. HDAC1 and HDAC2 are crucial components of 171 at least five different protein complexes: Mi-2/NuRD (nuclear remodeling deacetylase), Sin3 172 (switch intensive 3), CoREST (corepressor of REST), MiDAC (mitotic deacetylase), and the 173 recently discovered BAHD1 (Bromo adjacent homology domain protein 1) (Kelly & Cowley,

  complex is an excellent example to illustrate such a combinatorial assembly. The core MTA 179 scaffolding proteins (MTA1, MTA2, MTA3) bridge HDAC1 and HDAC2 with subunits 180 involved in nucleosome remodeling (CHD3, CHD4), histone demethylation (LSD1), binding 181 to other subunits and histones (RBBP4, RBBP7, GATAD2A, GATAD2B), and binding to 182 methylated DNA (MBD2). The combinatorial assembly of these subunits determines the 183 function of NuRD in genomic targeting and association with specific transcription factors, in 184 the mediation of cell type-specific transcriptional regulations, such as the repression of tumor 185 suppressor genes[START_REF] Lai | Cancer biology and NuRD: a multifaceted chromatin remodelling 856 complex[END_REF].

402

  Subsequently, this ER-stress response induces apoptosis, a common consequence of T. gondii 403 infection. The phosphorylation of RTN1-C leads to a downregulation of HDAC3 activity, 404 causing the accumulation of acetylated GRP78, a chaperone present in the ER(An et al., 405 2018). GRP78 is one of the main regulators of the unfolded-protein response in the ER, which 406 can, under prolonged conditions, induce apoptosis(Madeo & Kroemer, 2009).407Interfering with histone deacetylase activity or recruiting these enzymes to specific 408 proteins or genetic regions is only one way, pathogens can manipulate host HDACs. Another 409 possibility to influence the activity of HDACs is to change their intracellular localization 410 (Figure 1). 411 412 Pathogens change the localization of HDACs 413 As mentioned before, the activity of many histone deacetylases is tightly linked to their 414 subcellular localization. Hence, this represents a major target for pathogens to manipulate 415 cellular functions.

428

  binding of InlB to c-Met activates the PI3-kinase, causing the translocation of SIRT2 from the 429 cytoplasm to the nucleus. Here, SIRT2 deacetylates lysine 18 of histone 3 (H3K18) at the 430 transcriptional start sites of SIRT2-regulated genes, altering the host gene expression to the 431 pathogen's advantage. Indeed, a lack of SIRT2, or its inhibition, impairs L. monocytogenes , the bacteria can evade the host response and facilitate their intracellular

  trichostatin A (TSA) decreases bacterial phagocytosis in cells challenged with either 565 S. typhimurium or Escherichia coli. In addition, co-treatment with these inhibitors on cells 566 already infected by the bacteria leads to an increase of bacterial clearance. This observation 567 could be explained by the enhanced production of reactive oxygen species from the 568 mitochondria caused by the HDACi treatment(Ariffin et al., 2015). Especially tubastatin A, a 569 specific HDAC6 inhibitor, seems to be very potent in reducing the intracellular survival of S.570 typhimurium and L. pneumophila(Ariffin et al., 2015;[START_REF] Yang | Mechanism of histone deacetylase inhibitor 1040 tubastatin A promoting autophagy of Legionella pneumophila infected RAW264.7 cells]. Xi bao 1041 yu fen zi mian yi[END_REF]. 571 Another example of the possible usage of HDACi during infections are chronic lung 572 infections, called non-resolving pneumonia. One of the most prevalent pathogens to cause this 573 type of infection is Klebsiella pneumoniae. During this infection cell death induces a release 574 of cardiolipin, the main lipid component of the inner mitochondrial membrane and a 575 mitochondrial damage-associated molecular pattern. Amid the lung infection, the released 576 cardiolipin causes the SUMOylation of nuclear receptor which consequently leads to 577 the recruitment of a protein complex containing HDAC3 to the promoter region of IL-10, 578 repressing its expression. This repression promotes a persistent inflammation in the lung. 579 Inhibiting HDAC3 restores the IL-10 production and increases the survival in an in vivo 580 mouse model (Chakraborty et al., 2017). 581 HDAC inhibition is not only a therapeutic option in fighting bacterial infections but 582 can also be used in viral infections. Lately, several studies assessed if HDACi could be used 583 to treat viral infections: e.g. SAHA drastically decreases human adenovirus replication by 584 interfering with viral gene expression, protein production, and DNA replication (Saha & 585 Parks, 2019).586 Also, Sirtuin-specific inhibitors show promising antiviral properties. One example is 587 Tenovin-1 that displays antiviral activity against different members of the Arbovirus 588 family[START_REF] Hackett | 797 Sirtuin Inhibitors Are Broadly Antiviral against Arboviruses[END_REF]. Arboviruses include major human pathogens such as West Nile 589 virus, Chikungunya virus, and Zika virus[START_REF] Barzon | Ongoing and emerging arbovirus threats in Europe[END_REF]. The treatment of infected cell 590 cultures with Tenovin-1 reduces the viral load of these three viruses. In addition, sirtinol, a SIRT2 inhibitor, interferes with the formation of West Nile virus replication foci, 592 a process where viral dsRNA accumulates in the host cell[START_REF] Hackett | 797 Sirtuin Inhibitors Are Broadly Antiviral against Arboviruses[END_REF]. These results593 support the potential of HDACi as therapeutic options for the treatment of viral infections in 594 the near future.595Pan HDAC inhibitors, such as vorinostat, not only show antiviral properties(Banerjee, 596 Moore, Broker, & Chow, 2018), but also can be used to treat protozoan infections. However,597the success of HDAC inhibition to alleviate protozoal infections is often not based on the 598 inhibition of the host HDAC, but rather on the inhibition of the parasite's HDACs. An 599 example is the use of HDACi as treatment of Cryptosporidium infections, a genus closely 600 related to Plasmodium and Toxoplasma. In a mouse model, treatment with low concentrations 601 of vorinostat led to a decrease of Cryptosporidium parvum oocysts. Oocysts are formed 602 during the life cycle of the parasite and are crucial for the spreading of the parasite from one 603 host to another. In addition, low concentrations of vorinostat killed the majority of C. parvum

607

  leishmaniasis is characterized by a dissemination of the parasites to internal organs such as 608 the liver, the spleen, and the bone marrow. The study demonstrates that the vorinostat 609 derivative shows a high efficacy against the parasite in vivo at very low doses. This 610 corroborates the potency of vorinostat, and its derivatives, as a possible treatment option for 611 parasitic infections(Corpas-López et al., 2019). Very recently, HDACi have also been 612 described to alleviate infections caused by multicellular pathogens such as nematodes and 613 fungi. Trichostatin A contributes to the alleviation of eosinophilic meningitis caused by the 614 nematode Angiostrongylus cantonensis in a mouse model. The main cause for this might be 615 the reduction of the inflammatory response in the animals[START_REF] Zhang | Trichostatin A, a 1048 Histone Deacetylase Inhibitor, Alleviates Eosinophilic Meningitis Induced by Angiostrongylus 1049 cantonensis Infection in Mice[END_REF].

629

  required to elucidate the mechanism of action of these inhibitors. Moreover, clinical studies in 623 this field are scarce and many questions are yet to be answered. Additionally, the 624 development of new HDACi derivatives might help to minimize negative side effects as well their antimicrobial properties, so that these molecules might become novel 626 options to treat infectious diseases. The examples discussed in this review show the importance of histone deacetylases in the 630 immune response, as targets of pathogens and as possible treatment options. However, there 631 are still many unanswered questions surrounding HDACs and their function. For example, 632 how bacterial, viral or protozoan pathogens exploit or mimic eukaryotic HDACs is a wide-633 open field of investigation and only little is known to date. Excitingly, as shown here, 634 Legionella and probably also other bacterial pathogens encode HDAC mimics in their 635 genome and might secrete them into the host cell to manipulate it to their advantage. Further 636 analyses will elucidate in which ways these HDAC mimics function. This knowledge might 637 lead to the discovery of new targets to fight bacterial infections. However, as already shown 638 in cellular and animal models, inhibition of HDACs might also be a promising alterative to 639 fight infections caused by antibiotic resistant bacteria. Future research will proof if this 640 approach is applicable. We are convinced that the research on the function of eukaryotic and 641 bacterial HDACs, as well as on the mechanisms employed by bacterial, viral or protozoan 642 pathogens to subvert HDAC function will lead to a wealth of new knowledge. This will 643 ultimately lead to a better understanding of many fundamental processes but may also lead to 644 the development of new therapeutics. 645 646 ACKNOWLEDGEMENTS 647 Work in the CB laboratory is financed by the Institut Pasteur and funding has been received 648 from the Agence National de recherche grants ANR-10-LABX-62-IBEID and ANR-15-649 CE17-0014-03 to CB and ANR-18-CE15-0005-01 to MR, and the "Fondation de la 650 Recherche Médicale" grant EQU201903007847 to CB. DS was supported by the École 651 Doctorale "ED515: Complexité du vivant".

  chromatin. Furthermore, to determine if LphD, together with RomA, might play a concerted role to modulate the host chromatin landscape to promote bacterial replication. To elucidate the function of LphD and its role during infection this project focused on three main objectives: I. Decipher the functional role of the LphD in the eukaryotic cell II. Solve the structure and the biochemical function of LphD III. Understand the mechanisms of function of LphD in a context of multi-effector synergy as well as its implication in transcriptional changes during infection Beside my main project on the characterization of LphD nuclear functions, I was also involved in two other projects during the time I spent in the lab. In particular, I was working on the characterization of a L. longbeachae effector (Annex 1) and in setting up a new animal model, zebrafish (Danio rerio), for L. pneumophila infection (Annex 2).

Figure 13 :

 13 Figure 13: Crystallization of LphD under two different experimental conditions. Purified LphD -together with its inhibitor TSA (molar ratio 1:4) -was screened for crystallization under different conditions using the sitting-drop vapour diffusion method and a Mosquito nanoliter-dispensing crystallization robot. The two conditions are as follows: I) 0.2 M MgCl2, 0.1 M MES pH 6.5, 25% PEG 4K; II) 22% (w/v) PEG 8k, 250 mM MgCl2, 0.1 M MES pH 6.5.

Figure 14 :

 14 Figure 14: AlphaFold2 prediction of LphD structure.The structure of LphD shows the presence of a possible substrate binding pocket, also seen in eukaryotic HDACs. In addition, conserved residues (Y392 red; H177/178 pink; D216/330 green; N218 blue; Y248 yellow) know to be implicated in substrate binding are positioned within this probable binding pocket. The core of the protein -including the HDAC domain -is predicted with high confidence.

Figure 15 : 1 TITLETwo

 151 Figure 15: Superposition of the HDAC domains of LphD and HDAC10. The HDAC domain of a catalytic inactive HDAC10 (Y307F) from Danio rerio is superposed with the HDAC domain of LphD. In both proteins a clear overlap in the position of the binding pocket can be seen. In addition, when comparing the position of the catalytic residue of each HDAC domain (Y307F, orange for HDAC10; Y392 red for LphD) they closely colocalize, further highlighting the similarity between the two domains.

b-lactamase translocation assays Around 1 ´

 1 10 5 THP-1 cells are seeded in a 96-well plate and differentiated for 72 hours using 10 nM phorbol 12-myristate 13-acetate (PMA). One day before infection Legionella pneumophila wild type carrying responding plasmids for the expression of either b-lactamase alone or a b-lactamase LphD fusion are cultured in BYE broth containing chloramphenicol and IPTG to induce protein production. After differentiation the cells are washed and fresh RPMI medium with IPTG (1 mM) is added. Cells are infected with the b-lactamase fusion protein expressing bacteria at an MOI of 50. Spin the plates for 5 minutes at 300 g and then incubate the plate for 2 hours at 37˚C in a humidified 5% CO2 atmosphere. After this incubation, LiveBLAzer CCF4-AM solution (Thermo Fisher Scientific) is added to all the wells and the plate is incubated in the dark at room temperature for 2 hours. The cells are washed and cell dissociation solution (SIGMA) is added. After another incubation of 30 minutes at 37˚C with 5% CO2 the samples are analyzed by flow cytometry (MACS Quant, Miltenyi Biotec). Noninfected cells are used as negative control.

  Figure 1

  Figure 2

Figure 1 (

 1 Figure 1 (A) Fluor de Lys ® HDAC activity assay of LphD. Increasing amounts of purified LphD and its predicted catalytic inactive mutant (LphD Y392F) were used to assess the lysine deacetylase activity in vitro. Trichostatin A (TSA), a known HDAC inhibitor, was added to LphD as a control (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.01, **** p < 0.0001). (B) Top: Steady-state kinetics of purified LphD on fluorogenic H3 peptide substrates acetylated at different lysine residues (H3K4ac, H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac). Bottom: Michaelis-Menten constants (KM and kcat) were individually calculated for each peptide from non-linear regression fit using OriginPro and are embedded below the graph. Data represents mean value ± SD (n = 3). (C) Representative western blot of LphD activity on H3K14ac levels on histone octamers. Wild type protein (LphD) is compared to the catalytic inactive mutant (LphD Y392F). H3K14ac levels are assessed every minute for 5 minutes. Ponceau S staining of H3 is used as loading control.

Figure 2 (

 2 Figure 2 (A) b-lactamase secretion assay for LphD. Percentage of blue cells after 2 hours of infection with L. pneumophila wild type or ∆dotA overexpressing expressing either b-lactamase alone (b-lac), b-lactamase fused to LphD (b-lac+LphD) or b-lactamase fused to RomA (b-lac+RomA). Cells were incubated with LiveBLAzer CCF4-AM solution (Thermo Fisher Scientific) and analyzed by flow cytometry (n = 3, ** p < 0.01). (B) Immunofluorescence analysis of subcellular localization of EGFP-LphD. HeLa cells were transfected either with EGFP (top) or EGFP-LphD (bottom). Nuclei are stained by DAPI (cyan) and actin cytoskeleton with phalloidin (gray). Scale bars 10 µm. (C) Immunofluorescence analysis of subcellular localization of LphD during infection. Differentiated THP-1 cells were infected with L. pneumophila wildtype expressing LphD and DsRed. DAPI (cyan), LphD (green), L. pneumophila (red), and phalloidin (gray). Scale bars 10 µm. (D) Immunofluorescence analysis of H3K14ac in LphD transfected cells. HeLa cells were either transfected with EGFP-LphD (top) or EGFP-LphD Y392F (bottom) and then stained for H3K14ac using a specific antibody. DAPI (cyan), EGFP-LphD/LphD Y392F (green), H3K14ac (red), and phalloidin (gray). Scale bars 10 µm. (E) Quantification of western blot signal for H3K14 acetylation. THP-1 cells were infected with L. pneumophila wild type (green) or ∆lphD (white) strain expressing EGFP. Cells

Figure 3 (

 3 Figure 3 (A) Representative western blot signal for H3K14 methylation (top) and quantification (n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001) (bottom). THP-1 cells were infected with L. pneumophila wild type (green) or ∆lphD (white) strain expressing EGFP. Cells were sorted at different times post-infection by FACS, histones were isolated and analyzed by western blot. Histone H1 was used as loading control and signal is compared to non-infected cells. Data are represented as mean ± SD. (B) Volcano plot of EGFP-LphD interacting proteins. The log2 fold change of EGFP-LphD to control (GFP) is plotted against the -log10 of the false discovery rate (FDR). Protein selected for further tests (KAT7) is highlighted in red. Thresholds were set at log2 fold change > 2 and false discovery rate < 0.1 (dashed red lines). (C) Schematic representation of the HBO1 histone acetyltransferase complex in the configuration with BRPF1-3 targeting H3K14. (D) Immunoblots showing the interaction of LphD with KAT7 and histone H3. Co-immunoprecipitation using GFP-trap beads (GFP-trap) in HEK293T cells transfected with EGFP, EGFP-LphD, or EGFP-LphD Y392F. Input shows the expression level of endogenous KAT7 and Histone H3 in total lysates. (E) Immunoblots showing the interaction of LphD and RomA with the HBO1 complex and histone H3. Co-immunoprecipitation using GFP-trap beads in HEK293T cells transfected with EGFP, EGFP-LphD, or EGFP-RomA. Samples were analyzed for the presence of the different components of the HBO1 complex (BRPF1, KAT7, MEAF6, ING5) as well as its target, histone H3. Input shows the expression level of endogenous HBO1 components and histone H3 in total lysates (F) Immunoblots showing the interaction of LphD and RomA with KAT7 and Histone H3 during infection. Coimmunoprecipitation using GFP-trap beads in HEK293T cells transfected with either EGFP or

Figure 4 ( 42 (

 442 Figure 4 (A) Principal component analysis (PCA) plot of RNA-seq results of L. pneumophila wild type (WT) infected THP-1 cells over a time course, as indicated. Each dot represents a sample and each color represents a sample group. (B) Upset plot of intersection of sets of genes at multiple time points comparing the 100 most up-and down-regulated genes in L. pneumophila wild type infected cells. Each column represents the number differently expressed genes (DEGs) corresponding to a single time point (last four columns) or set of time points (dots connected by lines below the X axis). The number of genes in each set appears above the column, while the time points shared are indicated in the graphic below the column. (C) Dot plot of enriched biological processes of the 100 most up-regulated genes of THP-1 cells infected with L. pneumophila wild type at different time points compared to non-infected cells. Dot size represents the gene count per GO-term and color represents adjusted p-value. (D) Bar graph of the number of DEGs (up-regulated = green, down-regulated = red) compared to non-infected cells. Comparison of each time point (1 h, 3 h, 5 h, and 7 h post-infection) for each tested infection condition (wild type (WT), ∆lphD, ∆romA, and ∆lphD∆romA). (E) Dot plot of enriched biological processes of the 100 most up-regulated genes of THP-1 cells infected seven hours with L. pneumophila knockout mutants (∆lphD, ∆romA, and ∆lphD∆romA) compared to wild type infected cells. Dot size represents the gene count per GO-term and color represents adjusted p-value (p.adjust). (F) Relative mRNA expression for selected genes after seven hours infection with wild type (WT), ∆lphD, ∆romA, and ∆lphD∆romA strains, compared to noninfected cells (set to 0). Transcript levels were assessed by RT-qPCR and normalized to those of ActB, GAPDH and 36B4. Data are represented as mean ± SD (n = 9, * p < 0.05, ** p < 0.01, *** p < 0.01, **** p < 0.0001).

  the lab for my Master thesis in January 2018, I was working in collaboration with Sonia Mondino, Postdoc in the lab, on the characterization of a secreted effector of L. longbeachae.

  hours post fertilisation (hpf) intravenously in the caudal vessels near the cloaca (UGO) (Fig 1A), with 10 3 or 10 4 CFU of wild type (WT) L. pneumophila strain Paris expressing GFP (WT-GFP) or the type IV . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

  Fig. 2B, D Movie S1). Similarly, upon injection of HD of WT-GFP in lyz:DsRed larvae (red neutrophils), neutrophils were recruited to and accumulated around the growing bacterial aggregates, but seem unable to engulf them (Fig 2E, Movie S2 ).Moreover, confocal microscopy revealed that L.pneumophila exhibits grow in aggregates, and that these growing complex bacterial structures localize in the yolk and or in the yolk tube (Fig.2F, Movie S3 ). Upon injection HD of WT-GFP in Tg(kdrl::mCherry) is5 (red blood vessels) larvae, we also showed that, the fast growing bacterial aggregates interact with the blood vessels (Fig 2G, Movie S4). It should be noted that the yolk is the only food source of the larvae during this developmental stage. The fast proliferation of the bacteria in the yolk region probably depletes its nutritional content, leading to larvae death (Fig 2, Movie S1).

  top panel, Movie S5). Macrophages that had engulfed a large amount of L. pneumophila stopped moving and rounded-up, suggesting cell death. Similarly, the inhibition of the migration of phagocytes by L. pneumophila has been observed previously during infection of RAW 264.7 macrophages and the amoeba Dictyostelium discoideum and Acanthamoeba castellanii,26,27 . In contrast, zebrafish infected with HD of WT-GFP were not able to restrict the bacterial growth by 16hpi. HD of L pneumophila formed big aggregates, that were not easily engulfed and cleared by macrophages (Fig 4A, bottom panel, Movie S5). Remarkably, macrophages were very efficient in engulfing and rapidly clearing high doses of blood-dotA-GFP bacteria. By 10hpi 8 most of the bacteria had been engulfed and cleared as suggested by the diffuse GFP staining in phagocytes (Fig. 4A, bottom panel, Movie S5). However, upon infection with a HD WT-GFP, bacteria

(

  Fig. 4B, second panel, Movies S6). In sharp contrast, neutrophils very efficiently engulfed and cleared large amounts dotA-GFP aggregated and trapped in the mesenchyme (Fig. 4B, lower panel, Movie S6) as well as low doses of WT-GFP (Fig 4B upper panel, Movie S6).

  mCherryF). The specific depletion of the two cell types was confirmed by counting macrophages and neutrophils 72hpf (Fig S3A). We then infected macrophage depleted larvae (spi1b knockdown) by intravenous injection of LD or HD of WT-GFP. Independently of the infection dose, a dramatic decrease in survival occurred, as even injection of low doses of WT-GFP resulted in the death of 30% of the larvae (Fig 5A). When injecting high doses of WT-GFP nearly all of the infected larvae died by 72hpi, with the earliest deaths starting 48hpi (Fig 5A). In contrast, spi1b knockdown larvae injected with high doses of dotA-GFP did not show impaired survival (Fig 5A). The increased mortality correlated with an increased bacterial burden in spi1b knockdown larvae compared to control larvae as judged from counting bacteria growing on BCYE agar from homogenates of individual larvae by FACS analyses (Fig 5B).

12

 12

17 0.

 17 003% PTU in 24-well culture plates), incubated at 28°C and regularly observed under a stereomicroscope.

18

 18

19 GTAAGACGGCACTGAATCCA;Figure 1 .

 191 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. Bloodstream L. pneumophila establish a proliferative niche in the yolk causing a persistent local infection. Characterization of the L. pneumophila foci growing in the yolk region of zebrafish larvae. Maximum intensity projection of confocal acquisition using high resolution fluorescent microscope. A) 72hpf mfap4: mCherry larva (red macrophages) injected in the bloodstream with HD of WT-GFP and followed over time with confocal fluorescent microscopy. B) Imaris 3D reconstruction and volume rendering of the L. pneumophila growth in the yolk of the same infected larva at 72hpi, shown laterally. Inset shows the maximum intensity projection of the L. pneumophila foci in the same larva mounted ventrally. C) Scheme of 72hpf larva indicating with green dots the yolk sustaining L. pneumophila growing. D) Imaris 3D reconstruction and volume rendering of the L. pneumophila growth (GFP labelling) in the yolk of the same infected larva at 72hpi, showed ventrally. E) Imaris 3D reconstruction and volume rendering of the L. pneumophila growth in the yolk of lyz:DsRed (red neutrophils) infected larva at 72hpi, showed laterally. F) Imaris 3D reconstruction and volume rendering of the L. pneumophila growth (GFP labelling) in the yolk of wild type AB infected larva at 72hpi, showed laterally. Overlay of GFP and mCherry, or DsRed fluorescence is shown (2B, E, G), and BF is shown to help to visualize the yolk region and host anatomy (2A, D, F). See also related Movies S1-S4.

Figure 3 .

 3 Figure 3. L. pneumophila high dose infection results in (systemic) macrophage and neutrophil death. A) Representative images of L. pneumophila dissemination, determined by live imaging using a fluorescence stereomicroscope of zebrafish Tg(mfap4::mCherryF) larvae infected with a Low Dose or a HD of WT-GFP, or a HD of dotA-GFP. The same infected larvae were live imaged 4h, 24h, 48h, and 72h post L. pneumophila injection. Overlay of GFP and mCherry fluorescence is shown. B) Macrophage counts in uninfected larvae (black symbols) or upon Low Dose (blue symbols) or High Dose of WT-GFP (red symbols), or High Dose (green symbols) of dotA -GFP injections. Macrophages were counted manually from images taken on live infected larvae, using ImageJ software, and results were plotted using GraphPad Prism® software. Mean±SEM are also shown (horizontal bars). Data plotted are from two pooled independent experiments (n=12 larvae scored for each condition). C) Representative images of L. pneumophila dissemination, determined by live imaging using a fluorescence stereomicroscope, of zebrafish Tg(LysC::DsRed) nz50 larvae infected with a Low Dose or a High Dose of WT-GFP or a High Dose of dotA-GFP. The same infected larvae were live imaged 4h, 24h, 48h, and 72h post L. pneumophila injection. Overlay of GFP and DsRed fluorescence is shown. D) Neutrophil counts in uninfected (CTRL, black symbols) or upon Low Dose or High Dose of WT-GFP (blue or red symbols), or High Dose of dotA-GFP (green symbols) injections. Data plotted in the same way as for macrophage counts, are from two pooled independent experiments (n=10 larvae scored for each condition).

Figure 4 .

 4 Figure 4. Live imaging of macrophage and neutrophil interaction with L. pneumophila Frames extracted from maximum intensity projection of in vivo time-lapse confocal fluorescent microscopy of 72hpf Tg(mfap4::mCherryF) larvae injected in the bloodstream (iv) with a LD, HD (of WT-GFP or a HD of dotA-GFP (upper panel) or Tg(LysC::DsRed) nz50 in the bloodstream (iv) with a LD, HD of WT-GFP or a HD of dotA-GFP (lower panel) to follow macrophage and neutrophil interaction with L. pneumophila respectively. Images were taken from time lapse at different time points (0hpi, 2hpi, 4hpi, 8hpi and 16hpi). Overlay of green (L. pneumophila) and red (leucocytes) fluorescence of the caudal area of the larvae (region boxed in the scheme on the right of the panel) is shown. Scale bar: 50 m. See also related Movies S5, S6.

Figure 5 .

 5 Figure 5. Macrophages are crucial to restrict Legionella pneumophila dissemination A) Survival curves of CTRL morphant zebrafish larvae injected with a Low Dose (LD) (blue dashed curve, n=34 larvae) or a High Dose (HD) (red dashed curve, n=34) of WT-GFP, or with a HD (green dashed curve, n=24) of dotA -GFP, and spi1b morphant zebrafish larvae injected with a LD (blue curve, n=48) or a HD (red curve, n=48) of WT-GFP, or with a High Dose (HD) (green curve, n=48) of dotA -GFP. Non-injected CTRL morphant fish (black dashed curve, n=48), and spi1b morphant fish

27 (

 27 black curves, n=48) were used as control. Infected and control larvae were incubated at 28°C. Data plotted are from two pooled independent experiments. B) and E) Bacterial burden quantification by enumerating live bacteria in homogenates from individual larvae infected with LD of WT-GFP (blue symbols) or HD (red symbols), or with LD of dotA-GFP (magneta symbols) or HD (green symbols), measured by plating onto BCYE agar plates supplemented with Chloramphenicol and the Legionella Selective Supplement GVPN immediately after L. pneumophila injection and 24h, 48h and 48h post L. pneumophila injection. n=10 larvae for each condition. D) Survival curves of CTRL morphant zebrafish larvae injected with a LD (blue dashed curve, n=36) or a HD (red dashed curve, n=36) of WT-GFP, or with a HD (green dashed curve, n=24) of dotA -GFP, and csf3r morphant zebrafish larvae injected with a LD (blue curve, n=24) or a HD (red curve, n=36) of WT-GFP, or with a HD (green curve, n=36) of dotA -GFP. Non-injected CTRL morphant fish (black dashed curve, n=48), and csf3r morphant fish (black curve, n=36) were used as control. Data plotted are from two pooled independent experiments. C) and F) Representative images of L. pneumophila dissemination, determined by live imaging using a fluorescence stereomicroscope, of Tg(mfap4::mCherryF) spe1b morphant larvae (C) and of Tg(LysC::DsRed) nz50 (F) csf3r morphant larvae non infected, or infected with a LD or a HD of WT-GFP, or a HD of dotA-GFP. The same infected larvae were live imaged 4h, 24h, 48h, and 72h post L. pneumophila injection. Overlay of GFP and mCherry fluorescence is shown.

Figure 6 .

 6 Figure 6. Zebrafish larva Immunity to L. pneumophila is independent from signalling through MyD88 or compensated by other signalling pathways. A) Survival curves of CTRL zebrafish larvae injected with WT-GFP Low Dose (LD) (blue dashed curve) or High Dose (HD) (red dashed curve), or with dotA -GFP HD (green dashed curve), and myd88 hu3568 mutant zebrafish larvae injected with WT-GFP LD (blue curve) or HD (red curve), or with dotA -GFP HD (green curve). Non-injected CTRL larvae (black dashed curves), and myd88 hu3568 mutant larvae (black curves) were used as control. Infected and control larvae (n= 72 fish for myd88 hu3568 mutant conditions and n= 57 fish for CTRL conditions) were incubated at 28°C. Data plotted are from 3 pooled independent experiments. B) Bacterial Burden of myd88 hu3568 mutant zebrafish larvae are the same as what is observed for control larvae. Bacterial burden quantification by enumerating live bacteria in homogenates from individual larvae infected with WT-GFP LD (blue symbols) or HD (red symbols), or with dotA -GFP HD (green symbols) were measured by plating onto BCYE agar plates supplemented with Chloramphenicol and the L. pneumophila Selective Supplement GVPN immediately after Legionella injection and 24h, 48h and 48h post Legionella injection. n=15 larvae for each condition. C-D) Cytokine (il1b, tnfa) induction was measured from individual myd88 hu3568 mutant larvae injected with a HD (red curves) of WT-GFP and non-injected fish as control (CTRL, black curves). The same colours are used in individual CTRL zebrafish with dashed curves. Data plotted are from one experiment (n=5 larvae for each condition);

28 individual

 28 values are shown, and curves correspond to the medians. There is no statistically significant difference between CTRL and myd88 hu3568 mutant curves over time for all the conditions analysed.

Figure 7 .

 7 Figure 7. L. pneumophila replication in the yolk of zebrafish larvae is T4SS dependent. A) Survival curves of zebrafish larvae injected with WT-GFP Low Dose (LD) (blue curve) or High Dose (HD) (red curve), or with dotA-GFP LD (magenta curve) or HD (green curve). Non-injected larvae (black curves) were used as control. n= 48 larvae per conditions. All larvae were incubated at 28°C. Data plotted are from two pooled independent experiments. B) Bacterial burden quantification of zebrafish larvae injected with L. pneumophila in the yolk cell, by enumerating live bacteria in homogenates from individual larvae infected with WT-GFP LD (blue symbols) or HD (red symbols), or with dotA-GFP Low Dose (LD) (magenta symbols) or HD (green symbols). They were measured by plating onto BCYE agar plates supplemented with Chloramphenicol and the Legionella Selective Supplement GVPN immediately after L. pneumophila injection and 24h, 48h and 48h post Legionella injection. n=10 larvae for each condition. C-D) Representative images of L. pneumophila dissemination, determined by live imaging using a fluorescence stereomicroscope, of Tg(LysC::DsRed) nz50 not infected zebrafish larvae, or infected with a Low Dose of WT-GFP or dotA -GFP (C), or infected with a High Dose of WT-GFP or dotA -GFP (D). The same infected larvae were live imaged 4h, 24h, 48h, and 72h post L. pneumophila injection. Overlay of GFP and mCherry fluorescence is shown.

Figure 2 .Figure 3 .

 23 Figure 2

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :

 123456789101112131415 Figure 1: L. pneumophila in electron microscopy (A) and on a BCYE plate (B)…….………-2-

  283 amino acids for group 2. Interestingly, although the second group contains 533 smaller proteins with smaller HDAC domains, the domain covers almost the whole protein 534 length (92%), in contrast, in the first group the HDAC domain represents on average 79% of 535 the amino acid sequence of the protein.

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  therapeutic targets during infection.

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Table 1 :

 1 Pathogens known to target different eukaryotic histone deacetylases

	manuscriptcentral.com/microlife

acetylation and methylation changes. BMC Microbiology, 10, 172. https://doi.org/10.1186/1471-664 2180-10-172

665 Arechederra, M.,

Daian, F., Yim, A., Bazai, S. K., Richelme, S., Dono, R., … Maina, F. (2018)

. 666 Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in 667 liver cancer. Nature Communications, 9(1), 3164. https://doi.org/10.1038/s41467-018-05550-5 668 Ariffin, J. K., das Gupta, K., Kapetanovic, R., Iyer, A., Reid, R. C., Fairlie, D. P., & Sweet, M. J. 669 (2015). Histone Deacetylase Inhibitors Promote Mitochondrial Reactive Oxygen Species 670 Production and Bacterial Clearance by Human Macrophages. Antimicrobial Agents and 671 Chemotherapy, 60(3), 1521-1529. https://doi.org/10.1128/AAC.01876-15 672 Atif, F. A. (2015). Anaplasma marginale and Anaplasma phagocytophilum: Rickettsiales pathogens of 673 veterinary and public health significance. Parasitology Research, 114(11), 3941-3957. 674 https://doi.org/10.1007/s00436-015-4698-2 675 Aung, H. T., Schroder, K., Himes, S. R., Brion, K., van Zuylen, W., Trieu, A., … Ravasi, T. (2006). 676 LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase 677 expression. The FASEB Journal, 20(9), 1315-1327. https://doi.org/10.1096/fj.05-5360com 678 Baek, Y.-S., Haas, S., Hackstein, H., Bein, G., Santana, M. H., Lehrach, H., … Seitz, H. (2009). 679 Identification of novel transcriptional regulators involved in macrophage differentiation and 680 activation in U937 cells. BMC Immunology, 10(1), 18. https://doi.org/10.1186/1471-2172-10-18 681 Banerjee, N. S., Moore, D. W., Broker, T. R., & Chow, L. T. (2018). Vorinostat, a pan-HDAC 682 inhibitor, abrogates productive HPV-18 DNA amplification. Proceedings of the National 683 Academy of Sciences of the United States of America, 115(47), E11138-E11147. 684 https://doi.org/10.1073/pnas.1801156115 685 Bao, N., Lye, K.-W., & Barton, M. K. (2004). MicroRNA binding sites in Arabidopsis class III HD-C 887 Lurie-Weinberger, M. N., Gomez-Valero, L., Merault, N., Glöckner, G., Buchrieser, C., & Gophna, U. 888 (2010). The origins of eukaryotic-like proteins in Legionella pneumophila. International Journal 889 of Medical Microbiology : IJMM, 300(7), 470-481. https://doi.org/10.1016/j.ijmm.2010.04.016 890 Madeo, F., & Kroemer, G. (2009). Intricate Links between ER Stress and Apoptosis. Molecular Cell,

Table 2 :

 2 HDAC inhibitors shown to affect different pathogens

				Page 36 of 38
	Inhibitor	Target HDAC	Pathogen	Reference
	Suberanilohydroxamic acid Class I, II and IV Salmonella typhimurium	(Ariffin et al., 2015)
	(SAHA/Vorinostat)		Escherichia coli	(Ariffin et al., 2015)
			Adenovirus	(Saha & Parks, 2019)
			Human papillomavirus	(Banerjee et al., 2018)
			Cryptosporidium parvum	(Guo et al., 2018)
			Leishmania
				(Corpas-López et al., 2019)
			infantum/donovani
			Fusarium solani	(X. Li et al., 2019)
	Trichostatin A	Class I and II	Salmonella typhimurium	(Ariffin et al., 2015)
	(TSA)		Escherichia coli	(Ariffin et al., 2015)
			Angiostrongylus
				(Zhang et al., 2019)
			cantonensis
	Tubastatin A	HDAC6	Salmonella typhimurium	(Ariffin et al., 2015)
			Legionella pneumophila	(X. Yang et al., 2021)
	Sodium butyrate	Class I	Klebsiella pneumoniae	(Chakraborty et al., 2017)
	Tenovin-1	SIRT1 and SIRT 2	Arboviruses	(Hackett et al., 2019)
	Sirtinol	SIRT1 and SIRT2	West Nile virus	(Hackett et al., 2019)

Pathogens interfering with HDAC localization often promote HDAC degradation. Distribution of histone deacetylase proteins on the phylogeny of the genus Legionella. Page 38 of 38 https://mc.manuscriptcentral.com/microlife Manuscripts submitted to microLife

Table S1 :

 S1 LphD interacting proteins identified through GFP-trap analysis directly connected to epigenetic regulation.

	Protein	Epigenetic complex	FDR	Fold change
	KAT7	HBO1	0.06	75.70
	HDAC2	NuRD/Sin3	0.08	8.44
	MTA2	NuRD	0.03	5.41
	SAP18	Sin3	0.005	9.46
	PRKDC	DNA-PK	0.005	4.71
	XRCC5	DNA-PK	0.02	14.47
	SSRP1	FACT	0.02	3.08
	SUPT16H	FACT	0.001	3.39
	EED	PRC2/EED-EZH2	0.03	32.19
	SUZ12	PRC2/EED-EZH2	0.002	15.45
	EZH2	PRC2/EED-EZH2	0.09	9.17
				36

Table S2 :

 S2 Primers used in this study

	Primer	Sequence (5'-3')	Purpose	Reference
	52H	GATGAAGGCACGAACCCAGTTGACA	Deletion of lphD gene	This study
	52B	CGGCTTGAACGAATTGTTAGGTGGC	Deletion of lphD gene	This study
	195H	GACCCTCGACTTAATTGGATAACGG	Deletion of lphD gene	This study
	195B	GGAGGGTAAACGGAAAACAAACTG	Deletion of lphD gene	This study
	196H	GCCACCTAACAATTCGTTCAAGCCGGGTGATC TAAGCTACTTCTATGAATCCATATTC	Deletion of lphD gene	This study
	196B	TGTCAACTGGGTTCGTGCCTTCATCGTTTCCTG AGTTGTAATAATTGGTAAATTCC	Deletion of lphD gene	This study
	11H	TCCAATATACAAGCATTCATGTGCTATCTG	Deletion of romA gene	(Rolando et al., 2013)
	11B	GAAGTTTCTCGAATTCTTTGGACAAGC	Deletion of romA gene	(Rolando et al., 2013)
	60H	CATCGATGAATTGTGTCTCAAAA	Deletion of romA gene	(Rolando et al., 2013)
	60B	GTCCCGTCAAGTCAGCGTA	Deletion of romA gene	(Rolando et al., 2013)
	66H	TTTTGAGACACAATTCATCGATG GCTCTATTT TGCATGTGATTTTCATT	Deletion of romA gene	(Rolando et al., 2013)
	66B	TACGCTGACTTGACGGGAC GCAAGTTTTTTTG ATTTGATATTTCCTG	Deletion of romA gene	(Rolando et al., 2013)
	217H	ATCTATTGGGGCTTGGAAGGTGGATTTGACAG GACCATGTATGAA	Base pair substitution (LphD Y392F)	This study
	217B	TTCATACATGGTCCTGTCAAATCCACCTTCCAA GCCCCAATAGAT	Base pair substitution (LphD Y392F)	This study
	222H	GGATCCGAAAGTAGCGCCTCTTGTCTTGATA	Complementation of lphD	This study
	222B	GGTACCTTAACAGGACATATTATGCGAATTGG Complementation of lphD	This study
	CCL2_2_fwd	ACCTTCATTCCCCAAGGGC	qPCR analysis of CCL2 mRNA	This study
	CCL2_2_rev	CTCCTTGGCCACAATGGTCT	qPCR analysis of CCL2 mRNA	This study
	CCR2_1_fwd	TTCCAGGAATTCTTCGGCCT	qPCR analysis of CCR2 mRNA	This study
	CCR2_1_rev	GCAATCCTACAGCCAAGAGC	qPCR analysis of CCR2 mRNA	This study
	TXNIP_2_fwd	GCCACACTTACCTTGCCA AT	qPCR analysis of TXNIP mRNA	(Cui et al., 2019)
	TXNIP_2_rev	TTGGATCCAGGAACGCTA AC	qPCR analysis of TXNIP mRNA	(Cui et al., 2019)
	AJUBA_2_fwd	GCTTTGCTTCGCCTGACG	qPCR analysis of AJUBA mRNA	This study
	AJUBA_2_rev	CCATAGATGCCTTTGTTGCACT	qPCR analysis of AJUBA mRNA	This study
	ADAM19_2_fwd	GGGACTGGGCTCTTCAGTTT	qPCR analysis of ADAM19 mRNA This study
	ADAM19_2_rev	GTCCTGGTCTCGTCGATTCT	qPCR analysis of ADAM19 mRNA This study
	CLEC2D_1_fwd	TCTGAATTGCCTGCAAACCC	qPCR analysis of CLEC2D mRNA This study
	CLEC2D_1_rev	GGCATGCAGCTTGAAGACAT	qPCR analysis of CLEC2D mRNA This study
	ACTB_2_fwd	GTTGTCGACGACGAGCG	qPCR analysis of ACTB mRNA	(Rolando et al., 2013)
	ACTB_2_rev	GCACAGAGCCTCGCCTT	qPCR analysis of ACTB mRNA	(Rolando et al., 2013)
	36B4_1_fwd	TGGCAGCATCTACAACCCTG	qPCR analysis of 36B4 mRNA	(Rolando et al., 2013)
	36B4_1_rev	AAGGTGTAATCCGTCTCCACAGA	qPCR analysis of 36B4 mRNA	(Rolando et al., 2013)
	GAPDH_fwd	GAGTCAACGGATTTGGTCGT	qPCR analysis of GAPDH mRNA	(Neves-Costa and Moita, 2013)
	GAPDH_rev	TTGATTTTGGAGGGATCTCG	qPCR analysis of GAPDH mRNA	(Neves-Costa and Moita, 2013)

Table S3 :

 S3 Antibodies and dyes used in this study.

	Target	Manufacturer	Product code
	Western blot:
	b-Actin	Sigma	A5316
	BRPF1	Active Motif	61541
	EGFP	Thermo Fisher	A11122
	H1	Active Motif	61201
	H3	Active Motif	39163
	H3K14ac	Milipore	07-353
	H3K14me2	Euromedex	H3-2B10
	H3K18ac	Active Motif	39755
	H3K23ac	Active Motif	39131
	HA	Sigma	H6908
	ING5	Active Motif	91329
	KAT7	Santa Cruz Biotechnology	sc-39846
	MEAF6	Thermo Fisher	PA5-40704
	V5	Thermo Fisher	46-0705
	anti-Mouse HRP-conjugated	Cell signaling	7076S
	anti-Rabbit HRP-conjugated	Cell signaling	7074S
	Immunofluorescence:
	LphD	Thermo Fisher	Custom (see Supp Methods)
	Alexa488 goat anti-Rabbit	Thermo Fisher	A32731
	Alexa546 goat anti-Rabbit	Thermo Fisher	A11010
	DAPI	Thermo Fisher	D21490
	Alexa633 phalloidin	Thermo Fisher	A22284

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	-168 -

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ACKNOWLEDGMENTS

Work in the CB laboratory is financed by the Institut Pasteur, the Fondation pour la Recherche Médicale (FRM) grant N° EQU201903007847 and the Agence Nationale de la Recherche grant n°ANR-10-LABX-62-IBEID to CB and grant n° ANR-18-CE15-0005-. DS was funded by a Sorbonne University doctoral contract. We thank Jacques Monod Institute, the UMR 7592 Paris University/CNRS and the region Île-de-France for support. We also thank the group of Craig Roy for providing the HEK293-FcgRII cells.

ACKNOWLEDGEMENTS

We thank Pedro Escoll and Jean-Pierre Levraud for help and ideas in the initial set up of the project and Tobias Sahr for critical reading and helpful discussions and Yohann Rolin and Noël Aimar for their excellent care of the fish. Work in the CB laboratory is financed by the Institut Pasteur, the Fondation pour la Recherche Médicale (FRM) grant N° EQU201903007847 and the grant n°ANR-10-LABX-62-IBEID. Work in ECG group is financed by Institut Pasteur, CNRS, and ANR grant N° 17-CE15-0026.

Valerio Laghi is funded by ANR grant TIFAsomes. We wish to thank the fish facility (Yohann Rolin and Noël Aimar) for their excellent care of the fish. The funders, other than the authors, did not play any role in the study or in the preparation of the article or decision to publish.

2

ABSTRACT

Legionella pneumophila -a facultative intracellular bacterium -is the causative agent of a severe pneumonia called Legionnaires' disease. It has been shown that L. pneumophila directly modifies host chromatin by secreting RomA, a eukaryotic-like SET-domain methyltransferase.

RomA specifically methylates a usually acetylated lysine residue on histone H3 (H3K14), a usually acetylated residue, leading to a transcriptional repression of the host's immune response. Here we show that L. pneumophila is directly involved in the process of H3K14 deacetylation, by secreting a histone deacetylase, which we named LphD. LphD targets the host cell nucleus and modifies host chromatin in synergy with RomA. Both bacterial effectors target the host chromatin by interacting with the HBO1 histone acetyltransferase complex, a complex known to target H3K14. RNA-seq analyses of wild-type L. pneumophila and mutants deleted of the two bacterial effectors revealed that RomA and LphD influence the expression of genes implicated in the host immune response in a cumulative way. Thus, we provide unique insight into how a bacterial pathogen manipulates their host to ensure survival in an otherwise hostile environment.

GRAPHICAL ABSTRACT

LphD

RomA

Input

GFP-trap

Legionella longbeachae secretes a Rab GTPase protein to hijack NLRP3 during infection

INTRODUCTION AND AIM OF THE PROJECT

In the lab, we aimed at establishing a new animal model for L. pneumophila infection.

At this time, very few animal models are available for this purpose, and all of them have significant drawbacks. We wanted to assess if a common model organism, like the zebrafish (Danio rerio) could be used as a new tool to better understand how L. pneumophila infects eukaryotic organisms and how the innate immune system reacts to this bacterial invasion. In this project, led by Flávia Viana, I was involved in preparing the bacteria for the infection of the zebrafish larvae, as well as the following analyses of bacterial burden in the larvae after several days of infection. The publication associated with this project was recently submitted at PLOS biology and illustrates that zebrafish larvae represent an innovative L. pneumophila infection model that closely mimics important aspects of human infection.

Annexes

-169 -20 due to bad inoculation. Dead embryos were stored at 4°C overnight prior to harvesting the yolk sacs.

Remaining live embryos at 6-days post injection were euthanized by refrigeration overnight and the Octo Dissociator (Miltenyi Biotec, Germany) and 100 µL of serial dilutions at 10 -2 , 10 -4 and 10 -6 were automatically plated using easySpiral® automatic plater (Interscience, France) in triplicates on BCYE agar. L. pneumophila were quantified after 5 days-incubation using Scan® 1200 Automatic HD colony counter (Interscience, France).

Author contributions

Competing Interest

The authors declare there are no competing interests.

List of abbreviations