Dr Florent Sebbane

Dr Ina Attree

Elena Sophia

Eric Jean

David

Christine Olivier

Mbia Chantal

Gaël

Baptiste, Cécile, Héloïse, Manon Justine Guillaume

Alain Charbit
email: alain.charbit@inserm.fr

Mathieu Coureuil
email: mathieu.coureuil@inserm.fr

Xin Tan

Elodie Ramond

Anne Jamet
email: anne.jamet@inserm.fr

Jean-Philippe Barnier

Baptiste Decaux-Tramoni

Marion Dupuis

Daniel Euphrasie

Fabiola Tros

Ivan Nemazanyy

Jason Ziveri

Xavier Nassif

Bâtiment Leriche

Maria 14 Rue

Vieira Helena

Da

Julie Meyer

Iva Nemazany

Cerina Chhuon

Ida Chiara Guerrera

Agnes Ferroni

Isabelle Sermet-Gaudelus

Transketolase of Staphylococcus aureus is involved in the control of master regulators of stress response during infection

Keywords: Staphylococcus aureus, transketolase, metabolic adaptation, Pentose Phosphate Pathway, Sigma B, RpiRc, cystic fibrosis, intracellular persistence, proteo-genomic Staphylococcus aureus, USA300, intracellular persistence, endothelial cells, SCV Transketolase, Staphylococcus aureus, metabolic adaptation, Pentose Phosphate Pathway, sigma B, RpiRc cystic ibrosis, Staphylococcus aureus, intracellular persistence, bioilm, proteogenomics

路漫漫其修远兮,吾将上下而求索。

屈原

The way ahead is long, I shall search high and low.

Yuan QU

Never give up on what you really want to do.

Part I Introduction bibliography

Chapter I: Intracellular bacteria: importance of metabolic adaptation Bacterial pathogens are a major danger for human health all over the world. In particular, with the rapid emergence of bacteria bearing multiple antibiotic resistances, developing new anti-bacterial approaches is becoming tremendously necessary. An important strategy used by several bacterial pathogens is the invasion of host cells in order to escape attack by the host immune system and evade antibiotic killing. These bacteria called "intracellular bacteria" include obligate intracellular bacteria and facultative intracellular bacteria.

Background

Intracellular bacterial pathogens fall into two major groups: obligate and facultative. Obligate intracellular bacteria, such as Chlamydia, Anaplasma, Ehrlichia, and Rickettsia, that have lost their capacity to replicate without their host cells, exclusively rely on host resources to enable development and ultimately transmission to a new host. In contrast, facultative intracellular bacteria, like Francisella tularensis, Listeria monocytogenes or Legionella pneumophila, have kept their capacity to replicate in different extracellular environment and this capacity to survive independently from the host cells sometimes is an advantage for their infectious life cycle [START_REF] Casadevall | Evolution of intracellular pathogens[END_REF]Fuchs, Eisenreich et al. 2012).

Growing evidence has demonstrated that several prototypical extracellular bacterial pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae or Streptococcus pyogenes also had an intracellular lifestyle in various host cell types, serving for transition or even as reservoir [START_REF] Osterlund | Intracellular reservoir of Streptococcus pyogenes in vivo: a possible explanation for recurrent pharyngotonsillitis[END_REF][START_REF] Hayes | Intracellular residency of Staphylococcus aureus within mast cells in nasal polyps: A novel observation[END_REF][START_REF] Shenoy | Streptococcus pneumoniae in the heart subvert the host response through biofilmmediated resident macrophage killing[END_REF].

Although intracellular pathogens possess various strategies to overcome host cell defenses, there is a common phenomenon shared by obligate and facultative intracellular bacterial pathogens: they generally display genome reduction. This genome reduction includes loss of genes and appearance of pseudogenes [START_REF] Andersson | Insights into the evolutionary process of genome degradation[END_REF][START_REF] Martinez-Cano | Evolution of small prokaryotic genomes[END_REF][START_REF] Wernegreen | Endosymbiont evolution: predictions from theory and surprises from genomes[END_REF]. Gene deletion event sometimes include genomic regions encoding entire metabolic pathways. In such cases, the bacterial pathogens have evolved to acquire the metabolites that they can no longer produce directly from the host. For example, Rickettsia spp. have lost large number of genes coding for sugar, purine, and amino acid metabolism [START_REF] Renesto | Some lessons from Rickettsia genomics[END_REF][START_REF] Merhej | Genotyping, evolution and epidemiological findings of Rickettsia species[END_REF]. Another example, genes loci of bacterial toxin-antitoxin (TA) are lost during the adaption of the intracellular lifestyle, which encode proteins for stress response for free-living prokaryotes [START_REF] Pandey | Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes[END_REF].

For some intracellular bacterial pathogens, genome reduction may be a selective advantage. This phenomenon, called "pathoadaptation", is a mechanism where the absence of specific genes could increase bacterial fitness in the host [START_REF] Pallen | Bacterial pathogenomics[END_REF]. For example, Shigella spp. have lost the gene coding for lysine decarboxylase, probably because the product of this enzyme could inhibit bacterial virulence factors [START_REF] Maurelli | Black holes" and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli[END_REF]. However, in some cases, gain of genes may also occur to favor the intracellular bacterial pathogens. For example, L. pneumophila is thought to produce numerous eukaryotic-like proteins, presumably due to a long co-evolution associated with environmental amoeba [START_REF] Cazalet | Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity[END_REF].

Although obligate intracellular bacterial pathogens always have an intimate relationship with their host, they have developed various strategies to escape from host immune responses. In contrast, facultative intracellular bacterial pathogens have more complex relationships with their host and outcome of the interaction of hosts with these bacterial pathogens usually depends on the immunological status of the host. For example, Mycobacterium tuberculosis can only cause diseases in a small population of infected hosts.

Different bacterial intracellular survival strategies

No matter which bacterial species, the intracellular lifestyle can be divided into three main phases: i) entry, ii) intracellular survival and/or replication and iii) exit. Of note, intracellular replication is not always needed.

The entry process, also called internalization, involves bacterial adherence and subsequent uptake. These two steps depend on both host and bacterial factors. Three main mechanisms of entry are currently described, namely zipper, trigger and looping [START_REF] Cossart | Manipulation of host membrane machinery by bacterial pathogens[END_REF][START_REF] Pizarro-Cerda | Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia[END_REF].

Zipper mechanism. Binding of bacterial proteins with host membrane proteins, leads to bacterial ingestion by a small-scale cytoskeletal rearrangement, which involves recruitment of various host factors. For example, L. monocytogenes induces its internalization into nonphagocytic cells via a prototypical zipper mechanism [START_REF] Pizarro-Cerda | Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia[END_REF].

Internalization of L. monocytogenes is mediated by two surface proteins, InlA and InlB, which respectively target E-cadherin and the hepatocyte growth factor receptor Met [START_REF] Mengaud | E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells[END_REF][START_REF] Shen | InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase[END_REF] (Figure 1A).

Trigger mechanism. Bacteria activate signaling pathways leading to large-scale cytoskeletal rearrangements by the formation of membrane protrusions named "ruffle" [START_REF] Cossart | Manipulation of host membrane machinery by bacterial pathogens[END_REF]. Then, the extending ruffles fold back and enclose the cell surface, thereby entrapping the nearby bacteria. For example, Salmonella induces its internalization into non-phagocytic cells via a trigger mechanism [START_REF] Pizarro-Cerda | Manipulation of host membranes by the bacterial pathogens Listeria, Francisella, Shigella and Yersinia[END_REF]. It injects directly into the host cell cytoplasm various bacterial effectors via one of its two Type III secretion system (T3SS) that trigger cellular responses [START_REF] Schlumberger | Salmonella type III secretion effectors: pulling the host cell's strings[END_REF][START_REF] Ribet | How bacterial pathogens colonize their hosts and invade deeper tissues[END_REF] (Figure 1B).

Looping mechanism. In the case of looping mechanism, bacterial internalization does not actively promote surface cytoskeletal rearrangements and instead rely on the internalization machineries of host cells. F. tularensis is the only intracellular pathogen described thus far to use this entry mechanism. Internalization into macrophages via this looping mechanism involves the initial formation of large loops or pseudopodia [START_REF] Clemens | Francisella tularensis enters macrophages via a novel process involving pseudopod loops[END_REF]) (Figure 1C).

Of note, in the course of infection, a given pathogen can enter in different cell types and express various sets of virulence genes. In these situations, each infected cell type may have a particular role in the progression of disease. Intracellular survival. After internalization, intracellular bacteria initially reside within a membrane-bound vacuole. Under normal conditions, the vacuole progressively acidifies, finally it develops into a mature degradative phagolysosome, which contains hydrolytic enzymes and microbicidal oxidative burst. Intracellular bacteria have evolved either to adapt to a vacuolar niche or to thrive freely in the cytoplasmic compartment.

A Zipper mechanism B Trigger mechanism C Looping mechanism

Intravacuolar niche. Some intracellular bacteria have developed various strategies to survive within vacuole, either by preventing phagolysosomal fusion or by modifying the environment within vacuole (e.g. Brucella, Legionella, Chlamydia, Salmonella, Mycobacteria, …).

For example, upon phagocytosis, Brucella resides within the Brucella-containing vacuole (BCV), which undergoes interactions with early endosomes (EE), late endosomes (LE) and partially fuse with lysosomes (Lys) to become the endosomal BCV (eBCV). Following a brief residence in this compartment, Brucella transfers eBCV to the endoplasmic reticulum (ER), which makes ER a niche for its replication [START_REF] Kumar | Leading a sheltered life: intracellular pathogens and maintenance of vacuolar compartments[END_REF][START_REF] Celli | The changing nature of the Brucella-containing vacuole[END_REF]) (Figure 2A).

After internalization into the host cell, Salmonella resides within the Salmonella-containing vacuole (SCV). The early SCV interacts with EE, LE and fuse with Lys to become the maturing SCV. Later, the maturing SCV progresses into the late SCV which interacts with EE and detoxified lysosomes (Detox. Lys), eventually resulting Salmonella-induced modified endosomal maturation. In parallel, lysosomal content is excluded from the maturing SCV, most probably via recycling endosomes (RE). The late SCV locates proximity to the Golgi, hijacks vesicles from this organelle. Meanwhile, the early SCV also interacts directly with the host ER, which might enable coat protein complex (COPII) vesicle association with the SCV. These processes are probably involved in vacuolar rupture and Salmonella hyper-replication in the host cytosol(Santos and Enninga 2016) (Figure 2B).

After internalization, Legionella pneumophila resides within the Legionella-containing vacuole (LCV). In phagocytes, LCV rapidly intercepts Rab1-positive ER-derived vesicles. The LCV then maturates into an ER-derived compartment that supports bacterial growth.

Additionally, the LCV interacts with EE but avoids fusion with Lys [START_REF] Santos | At the crossroads: communication of bacteria-containing vacuoles with host organelles[END_REF] (Figure 2C). Cytosolic niche. In contrast, other bacterial pathogens such as L. monocytogenes, Shigella.

flexneri and F. tularensis have developed strategies to escape from the phagosome in a very short time, to reside within the host cytosol [START_REF] Ray | Life on the inside: the intracellular lifestyle of cytosolic bacteria[END_REF]), see also Table 1.

Escaping from the vacuole is a bacterial driven process and each cytosolic pathogen has a unique strategy of entry into cytosol that depends on various sets of enzymes or lysins.

For L. monocytogenes, phagosomal escape is mediated by the pore-forming cholesteroldependent cytolysin listeriolysin O (LLO) and two type C phospholipases: phosphatidylinositol specific phospholipase C (PI-PLC) and the broad-range phosphatidylcholine-specific phospholipase C (PC-PLC). LLO transfers to the phagosomal membrane by binding cholesterol and forms pores, which leads to membrane disruption. Eventually this prevents Listeriacontaining vacuole fusion with lysosomes. PI-PLC is responsible for escape from the vacuole into the cytosol, whereas PC-PLC contributes to disruption of the double-membrane vacuole during cell-to-cell spread [START_REF] Ray | Life on the inside: the intracellular lifestyle of cytosolic bacteria[END_REF].

Phagosomal escape of S. flexneri is probably induced by the type III secretion system (T3SS) effectors/translocators IpaB and IpaC, due to their ability to transfer to the host cell membrane for the delivery of bacterial effectors. IpaB and IpaC can disrupt lipid vesicles (liposomes) in vitro and are required for contact-mediated hemolysis by Shigella as well as bacterial phagosomal escape in macrophages [START_REF] Mellouk | Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm[END_REF]. In addition, the T3SS effector IpaH7.8 may promote Shigella vacuolar escape both in macrophages and epithelial cells, although its function is not well understood [START_REF] Fernandez-Prada | Shigella flexneri IpaH(7.8) facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages[END_REF][START_REF] Paetzold | Shigella flexneri phagosomal escape is independent of invasion[END_REF].

F. tularensis escapes from an acidified late-endosome-like vacuole before the fusion with lysosome. Lysis of the vacuole requires the recruitment of vacuolar ATPase pump on the membrane of vacuole, which allows import of protons and acidification [START_REF] Santic | Acquisition of the vacuolar ATPase proton pump and phagosome acidification are essential for escape of Francisella tularensis into the macrophage cytosol[END_REF]. The type VI secretion system (T6SS) also plays an essential role in F. tularensis phagosomal escape. The T6SS assembles a phage tail-like structure across the bacterial envelope and host cell membrane, enabling inject effector proteins (such as VgrG, IglI…) into the cytosol of the host cell [START_REF] Broms | The Role of the Francisella Tularensis Pathogenicity Island in Type VI Secretion, Intracellular Survival, and Modulation of Host Cell Signaling[END_REF][START_REF] Celli | Mechanisms of Francisella tularensis intracellular pathogenesis[END_REF][START_REF] Clemens | The Francisella Type VI Secretion System[END_REF][START_REF] Ledvina | A Phosphatidylinositol 3-Kinase Effector Alters Phagosomal Maturation to Promote Intracellular Growth of Francisella[END_REF]. Exit. Lastly, intracellular pathogens have also developed various strategies to exit the intracellular environment and disseminate to adjacent cells and tissues. Many cytosolic bacteria (L. monocytogenes, Ricketsia. spp, Shigella …) share a common mechanism to spread to neighboring cells. After escape from the phagosome into the cytosol, these bacteria polymerize actin at the bacterial pole, forming a comet-like tails on their surface, leads to the formation of bacteria-containing protrusions and cell-to-cell spread (Weddle and Agaisse 2018) (Figure 3). Cell-to-cell spread is particularly advantageous because it allows bacteria not to be exposed to the extracellular environment. Like this, pathogens are able to avoid potential detection or destruction by preexisting local inflammatory response [START_REF] Hybiske | Exit strategies of intracellular pathogens[END_REF].

However, some pathogens escape from host cells directly by inducing cell death through apoptosis or pyroptosis. The bacterial molecule that is responsible for apoptotic induction remains unknown. Intracellular pathogens can induce proinflammatory pyroptosis via inflammasome activation by sensing of bacterial molecules (flagellin of L. pneumophila and Salmonella spp. and chromosomal DNA in the case of F. tularensis) (Figure 4). For example, F. tularensis uses both apoptosis and pyroptosis to lyse infected cells and to infect adjacent cells. Apoptosis induced by F.tularensis is using the intrinsic pathway of activation: cytochrome c release from mitochondria and activation of the initiator caspase-9 and the effector capase-3 [START_REF] Lai | Francisella tularensis induces cytopathogenicity and apoptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication[END_REF][START_REF] Lai | Delineation of the molecular mechanisms of Francisella tularensis-induced apoptosis in murine macrophages[END_REF] (Figure 4).

Intracellular F. tularensis induce cell pyroptosis via absent in melanoma 2 (AIM2) inflammasome activation. AIM2 is a key sensor of pathogens that detects the presence of foreign DNA accumulating in the cytosol during the life cycle of intracellular pathogens (Figure 5). The molecular mechanisms of how pyroptosis is achieved following inflammasome activation are being unraveled. Very briefly, inflammatory caspases cleave the pore-forming protein gasdermin D that induces pyroptosis. Gasdermin D is able to oligomerize on the cell membrane. Its N-terminal fragment associates with the inner leaflet of the host cell membrane,

where it forms pores of 10-21 nm in diameter. These pores and/or the physical rupture of the cell allow passive release of interleukin (IL)-1β and IL-18 into the extracellular space which eventually causes cell death [START_REF] Franchi | Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages[END_REF][START_REF] Molofsky | Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection[END_REF][START_REF] Mariathasan | Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation[END_REF][START_REF] Craven | Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells[END_REF][START_REF] Soong | Staphylococcus aureus activation of caspase 1/calpain signaling mediates invasion through human keratinocytes[END_REF][START_REF] Lugrin | The AIM2 inflammasome: Sensor of pathogens and cellular perturbations[END_REF][START_REF] Mathur | Molecular mechanisms of inflammasome signaling[END_REF]) (Figure 5).

Importance of bacterial metabolism during the intracellular life cycle

All human bacterial pathogens are heterotrophs. They can subsist on a variety of carbon and nitrogen sources, including carbohydrates, lipids, glycolipids and amino acids. Carbon catabolism provides bacteria with energy in the form of reducing equivalents and ATP, as well as essential biosynthetic precursors including glucose-6-phosphate, fructose-6-phosphate, 3phosphoglycerate, phosphoenolpyruvate (PEP), acetyl-CoA, propionyl-CoA, oxaloacetate and α-ketoglutarate [START_REF] Munoz-Elias | Carbon metabolism of intracellular bacteria[END_REF].

The transport and use of nutrients, especially of the major carbon and nitrogen substrates, are affected by multiple specific uptake systems and pathways (Figure 6). The genes involved in these pathways are controlled by both global and specific regulators that may directly or indirectly sense key metabolites that are generated in the central carbon and nitrogen metabolic pathways [START_REF] Deutscher | How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria[END_REF][START_REF] Mao | Interplay between CRP-cAMP and PII-Ntr systems forms novel regulatory network between carbon metabolism and nitrogen assimilation in Escherichia coli[END_REF][START_REF] Sonenshein | Control of key metabolic intersections in Bacillus subtilis[END_REF][START_REF] Eisenreich | Carbon metabolism of intracellular bacterial pathogens and possible links to virulence[END_REF]. Some of these global regulators may also affect the expression of essential virulence factors according to the nutrient supply in these niches.

Intracellular nutrients

In order to survive within host cells, intracellular pathogens must adapt their metabolism to the available nutrients and physical conditions (pH, oxygen availability, osmotic pressure…) in these cells, and they must also coordinate their metabolism with their life cycle [START_REF] Eisenreich | Carbon metabolism of intracellular bacterial pathogens and possible links to virulence[END_REF].

The cytosol contains all of the nutrients that are provided to the host cells from the external medium and catabolic intermediates as well as all of the anabolic compounds that are produced by these cells. Thus, in principle, cytosolic bacteria have direct access to all of these host cell nutrients. However, the accurate constant concentrations of most of these compounds are very variable according the growth conditions of the cells, their genetic background and they also vary in different cell types. In some instances, host cells and bacteria may use similar nutrients, and so bacteria in the host cell cytosol may develop smart nutrient uptake systems to compete with that of host cell.

Replication in the apparently nutrient-rich cytosol would seem to require fewer metabolic adaptations compare to vacuolar environment. However, several studies suggest that life in the cytosol might be more complicated than formerly anticipated. Indeed, when bacterial pathogens are microinjected directly into the cytoplasm of mammalian cells, cytosolic intracellular pathogens such as L. monocytogenes and S. flexneri replicate freely, whereas intravacuolar intracellular pathogens such as S. typhimurium and L. pneumophila and extracellular pathogens such as Yersinia enterocolitica do not replicate at all [START_REF] Goetz | Microinjection and growth of bacteria in the cytosol of mammalian host cells[END_REF], suggesting that the host cell cytosol is not simply a nutrient-rich culture medium allowing efficient growth of heterotrophic bacteria. However, the factors responsible for growth restriction of intravacuolar and extracellular bacteria in the cytoplasm have not been identified yet.

Importance of carbon metabolism for intracellular bacteria

Here, I will briefly introduce the present understanding of intracellular carbon metabolism, using three extensively studied model intracellular pathogens, namely the cytosolicallyreplicating species L. monocytogenes and S. flexneri and the mainly phagosomally-replicating species S. typhimurium. Each of the three model pathogens seems to undergo specific intracellular metabolic adaptations, which are discussed below.

L. monocytogenes is a Gram-positive bacterium first described in 1926 during an outbreak that affected guinea pigs and rabbits. It was identified in the 1980s as a food-borne pathogen. It mainly affects immunocompromised individuals, pregnant women, and newborns. Severe infections are characterized by bacteremia, meningoencephalitis, abortion, or neonatal sepsis.

The most common vehicles of transmission of this saprophytic bacterium to humans are dairy products and other foods including eggs, seafood, and vegetables. Upon ingestion of contaminated food, after translocating across the gut epithelium, L. monocytogenes is capable to resist killing by professional phagocytes and can disseminate either freely or within infected circulating monocytes via the lymph and the blood to the liver and the spleen and even cross the blood-brain or the blood-placenta barrier. As recalled above, inside infected cells, L.

monocytogenes is capable to rapidly escape from the vacuole by disrupting the phagosomal membrane via the expression of listeriolysin O (LLO) and two phospholipases (PlcA and PlcB).

Within the cytosol of the host cell, L. monocytogenes multiplies with a generation time of approximately 1 h and spreads into neighboring host cells by co-opting actin-based motility (Fuchs, Eisenreich et al. 2012;[START_REF] Radoshevich | Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis[END_REF].

Transcriptional analyzes of L. monocytogenes in macrophages and epithelial cell lines showed a considerable upregulation of all genes involved in the facilitated uptake and catabolism of glycerol [START_REF] Chatterjee | Intracellular gene expression profile of Listeria monocytogenes[END_REF][START_REF] Joseph | Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening[END_REF]. A series of mutants unable to transport and catabolize glycerol due to a lack of glycerol uptake facilitator (GlpF), glycerol kinase (GlpK), glycerol-3-phosphate dehydrogenase (GlpD), and dihydroxyacetone kinase subunit K (DhaK) were also shown to have impaired intracellular growth [START_REF] Joseph | Glycerol metabolism and PrfA activity in Listeria monocytogenes[END_REF], suggesting that glycerol could be a major carbon source for carbon metabolism of intracellular bacteria.

Concomitantly, genes encoding glycolytic enzymes, as well as the pdhABC operon genes encoding pyruvate dehydrogenase, were downregulated in the intracellular environment in macrophages [START_REF] Chatterjee | Intracellular gene expression profile of Listeria monocytogenes[END_REF]. During intracellular growth in Caco-2 cells, strong repression of genes of the glycolytic pathway (from glucose to pyruvate) was observed, most likely reflecting the slower growth of L. monocytogenes in these cells (generation time about 40 min) compared to that in BHI (25 min) [START_REF] Joseph | Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening[END_REF]).

Expression of uhpT, which encodes a transporter involved in the exploitation of hexose phosphate from the host cell, was also upregulated in both cell types [START_REF] Chatterjee | Intracellular gene expression profile of Listeria monocytogenes[END_REF][START_REF] Joseph | Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening[END_REF]. Meanwhile, a ∆uhpT mutant was attenuated in vitro and in vivo whereas a mutant unable to uptake glucose does not show replication attenuation in macrophages or epithelial cells [START_REF] Stoll | The major PEP-phosphotransferase systems (PTSs) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extraand intracellular growth[END_REF]. Furthermore, the genes encoding the glucose-specific phosphoenolpyruvate (PEP)-dependent phosphotranferase system (PTS) permeases were not upregulated and expression of the PTS histidine-containing phosphocarrier protein (Hpr) was down regulated inside the host cell cytosol. Altogether these data supported the assumption that glucose-6-phosphate rather than glucose was used for bacterial metabolism within hosts.

Furthermore, Grubmuller et al. exploited the technique of 13 C -isotopologue profiling to study the carbon metabolism of L. monocytogenes in macrophages. They observed that glycerol was catabolized via the lower part of the glycolytic pathway, but not used for gluconeogenesis, and the intermediates generated from glucose 6-phosphate in the upper part of the glycolytic pathway and the pentose phosphate pathway probably serve primarily for anabolic purposes.

The intracellular L. monocytogenes metabolism which involves at least two major carbon substrates, also named "bipartite metabolism": glycerol mainly for energy supply and glucose 6-phosphate mainly for indispensable anabolic function [START_REF] Grubmuller | Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism[END_REF] (Figure 7). Of note, during intracellular survival, the expression of genes for enzymes involved in the synthesis of branched-chain amino acids (encoded by ilv and leu) was highly enhanced.

L. monocytogenes is auxotrophic for riboflavin, thiamine, biotin and lipoate and will totally depend on the host cell for the supply of these vitamins when replicating in the host cell cytosol.

Indeed, several studies show that the concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes to enable proliferation within the cytoplasm [START_REF] Schauer | Both thiamine uptake and biosynthesis of thiamine precursors are required for intracellular replication of Listeria monocytogenes[END_REF]. The importance of biosynthesis of branched-chain fatty acids for cytosolic growth of L. monocytogenes has also been demonstrated [START_REF] Keeney | Generation of branched-chain fatty acids through lipoate-dependent metabolism facilitates intracellular growth of Listeria monocytogenes[END_REF][START_REF] Sun | Branched-chain fatty acids promote Listeria monocytogenes intracellular infection and virulence[END_REF]. S. flexneri is a Gram-negative pathogen that is responsible for shigellosis. Shigella is closely related to E. coli, but has evolved specific traits of pathogenicity, physiology and serology.

Shigellosis is associated with fever and abdominal cramps; its diagnosis relies on the presence of erythrocytes, polymorphonuclear neutrophils (PMNs), and mucus in patient stools, which stand as diagnostic elements. S. flexneri specifically invades the human colon, but not the small intestine. The molecular basis of this specificity remains unknown. Before reaching the epithelial layer, S. flexneri has to survive in the gastrointestinal environment, to outcompete the colonic luminal commensal bacteria and to disrupt a thick protective mucus layer. In addition, the inflammatory response induced by S. flexneri invasion might result in a perturbation of the composition and the function of these barriers, although these aspects are not well understood.

Following ingestion, S. flexneri transits to the large intestine, and it uses its T3SS to induce uptake by colonic epithelial cells. S. flexneri firstly resides within a vacuole, it quickly lyses the vacuole membrane and replicates within the host cell cytoplasm and spreads from cell-to-cell [START_REF] Waligora | Role of intracellular carbon metabolism pathways in Shigella flexneri virulence[END_REF][START_REF] Anderson | Shigella Diversity and Changing Landscape: Insights for the Twenty-First Century[END_REF].

Comparing transcriptional profiles from bacteria growing in the cytosol of macrophages with those from bacteria growing in LB (Luria-Bertani) culture show an upregulation of the genes encoding glucose uptake proteins (the glucose-specific PTS component gene ptsG and the mannose-specific PTS component genes manX, manY and manZ) and ugp gene encoding the glycerol-3P transporter, as well as uhpT gene encoding the hexose phosphate transporter (Runyen-Janecky and Payne 2002; [START_REF] Lucchini | Transcriptional adaptation of Shigella flexneri during infection of macrophages and epithelial cells: insights into the strategies of a cytosolic bacterial pathogen[END_REF]. The ∆uhpT mutant showed reduced growth on glucose 6-phosphate as sole carbon source in vitro; however, no reduction in growth was observed in epithelial cells compared with the wild-type strain for Shigella (Runyen-Janecky and Payne 2002), suggesting either that glucose 6-phosphate is not the only available intracellular carbon source or that it is transported by other sugar transporters during bacterial intracellular life cycle.

Global proteome analysis of Shigella inside host cells identified that mixed-acid fermentation, and metabolism of pyruvate in particular, is required for optimal intracellular growth of S. flexneri [START_REF] Pieper | Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth[END_REF]. In contrast to fermentation pathway enzymes, components of the tricarboxylic acid (TCA) cycle were less expressed in the intracellular bacteria.

S. flexneri uses three glycolytic pathways to produce pyruvate from glucose: the Embden-Meyerhof-Parnas (EMP) pathway, the pentose-phosphate pathway (PPP) and the Entner-Doudoroff (ED) pathway. Analysis in cell culture shows that the EMP pathway is necessary, while the PPP and ED pathways were dispensable for S. flexneri intracellular growth [START_REF] Waligora | Role of intracellular carbon metabolism pathways in Shigella flexneri virulence[END_REF]. Importantly, growth defects caused by glycolysis mutants could be overcome by supplementation with pyruvate, showing that this molecule is an essential carbon source for S. flexneri intracellular growth [START_REF] Killackey | Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes[END_REF]. Furthermore, Kentner et al. observed that host glycolytic pathways were not significantly affected by the intracellular pathogen, but infected cells excreted acetate instead of pyruvate and lactate, suggesting that the latter molecules had been captured and used by Shigella for its metabolism [START_REF] Kentner | Shigella reroutes host cell central metabolism to obtain highflux nutrient supply for vigorous intracellular growth[END_REF]. This study supported the importance of pyruvate for Shigella intracellular growth. See also Figure 8. replicate [START_REF] Finlay | Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication[END_REF][START_REF] Fabrega | Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation[END_REF][START_REF] Dandekar | Salmonellahow a metabolic generalist adopts an intracellular lifestyle during infection[END_REF].

Large-scale transcriptome and proteome data are available for S. Typhimurium in host cell intracellular environment and in infected animals [START_REF] Eriksson | Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica[END_REF][START_REF] Becker | Robust Salmonella metabolism limits possibilities for new antimicrobials[END_REF]. Comparing transcriptional profiles from bacteria growing in Hela cells with those from bacteria growing in LB culture show up regulation of ptsG and uhpT [START_REF] Hautefort | During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems[END_REF], suggesting that both glucose and glucose phosphates might be available to intraepithelial Salmonella.

13

C-isotopologue profiling data showed that S. Typhimurium replicating in the SCV of Caco-2 cells prior to use glucose (but not glucose-6-phosphate) as carbon substrate. However, a mutant, which is impaired in glucose uptake, was still able to replicate in Caco-2 cells, indicating that S. Typhimurium can switch to other nutrients as carbon and energy sources in the absence of glucose, most likely to C3-substrates, such as glycerol, glycerol 3-phosphate… [START_REF] Gotz | Carbon metabolism of enterobacterial human pathogens growing in epithelial colorectal adenocarcinoma (Caco-2) cells[END_REF].

Genes encoding enzymes for glycolysis and the ED pathway are upregulated in these bacteria, meanwhile most genes encoding enzymes in the TCA cycle are downregulated.

Expression of these genes is more pronounced in Hela cells than in J774 macrophage cell lines [START_REF] Hautefort | During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems[END_REF]. The ED pathway facilitates growth on sugar acids such as galacturonate that are found in the gastrointestinal tract [START_REF] Hautefort | During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems[END_REF].

Together these data suggest that S. Typhimurium may use host-sugar acids as intracellular carbon sources, in both epithelial and macrophages cells, in addition to host-derived glucose in epithelial cells.

Upon systemic infections in mice, strong attenuation is observed in S. Typhimurium mutants which are impaired in the biosynthesis of aromatic amino acids (including histidine) and purines (especially adenine), meanwhile moderate attenuation is found for pyrimidine and methionine mutants, which demonstrated the dependency of in vivo growing S. Typhimurium on the biosynthesis of these anabolic monomers [START_REF] Gotz | Carbon metabolism of enterobacterial human pathogens growing in epithelial colorectal adenocarcinoma (Caco-2) cells[END_REF][START_REF] Eisenreich | Metabolic Adaptations of Intracellullar Bacterial Pathogens and their Mammalian Host Cells during Infection ("Pathometabolism")[END_REF]. The in vitro data also supported this assumption: the transcriptome studies showed enhanced expression of the genes encoding enzymes for these anabolic pathways. In addition, the 13 C-isotopologue profiling results also showed significant de novo synthesis of amino acids in S. Typhimurium replicating in Caco-2 cells [START_REF] Gotz | Carbon metabolism of enterobacterial human pathogens growing in epithelial colorectal adenocarcinoma (Caco-2) cells[END_REF].

In systemically infected mice, S. Typhimurium replicates mainly in macrophages, bacterial growth depends on a complete TCA cycle, and mutants unable to convert succinate to oxaloacetate are avirulent [START_REF] Tchawa Yimga | Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice[END_REF][START_REF] Mercado-Lubo | Salmonella enterica serovar Typhimurium mutants unable to convert malate to pyruvate and oxaloacetate are avirulent and immunogenic in BALB/c mice[END_REF]. Removal of malate from the TCA cycle to produce pyruvate requires that TCA cycle intermediates be replenished to keep the TCA cycle operative. However, since it appeared that the glyoxylate shunt was not needed for full virulence, replenishment of TCA cycle intermediates did not seem to occur by this pathway in a systemic S. Typhimurium infection model [START_REF] Tchawa Yimga | Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice[END_REF]). However, this pathway might have a role in persistent infections of animals [START_REF] Fang | Isocitrate lyase (AceA) is required for Salmonella persistence but not for acute lethal infection in mice[END_REF]. Mutants blocked in gluconeogenesis are still fully virulent in systemically infected mice [START_REF] Tchawa Yimga | Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice[END_REF]. From these data, it can be suggested that S. Typhimurium grows in mouse macrophages mainly depend on glucose or other C6 carbohydrates rather than glycolytic carbon sources.

Host cell response

On the other hand, host cells develop various defensive strategies against bacterial infections.

Here, I will highlight two important defense mechanisms: phagosomal elimination and autophagy.

Phagosomal eliminition

Following phagocytosis by host cells, intracellular bacterial pathogens initially reside in phagosome. Hence, the phagosome is the first defend strategy from the infected host cell and phagosome maturation is critical for macrophages to eliminate the ingested pathogens. There are three major mechanisms of pathogen eradication during this process: i) the oxidative burst, inducing by the production of reactive oxygen species (ROS); ii) phagosomal acidification; and

iii) the release of antimicrobial proteins and peptides (AMPs) [START_REF] Flannagan | Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus[END_REF][START_REF] Weiss | Macrophage defense mechanisms against intracellular bacteria[END_REF].

The process of phagosome maturation comprises a series of strictly coordinated membrane fusion/fission events between the phagosome and compartments of the endo/lysosomal network and eventually with the formation of the phagolysosome, a degradative organelle [START_REF] Desjardins | Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus[END_REF]). It contains three major stages (Figure 10). The fusion of the early phagosome and early endosome is the first stage of phagosome maturation. This process, tightly coordinated by a family of proteins, Rab GTPases [START_REF] Stenmark | Rab GTPases as coordinators of vesicle traffic[END_REF], is characterized with the acquisition of several specific membrane marker: the small GTPase Rab5, early endosomal antigen 1 (EEA1), the phosphatidylinositol 3 kinase (PI3K)

Vps34, and phosphatidylinositol 3-phosphate (PI3P) [START_REF] Weiss | Macrophage defense mechanisms against intracellular bacteria[END_REF]. Other molecules are also involved in this process, such as the family of soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins, vesicle associated membrane proteins (VAMPs), and syntaxins. The early phagosome lacks the proton pumping vesicular ATPase (vATPase) and is therefore characterized by a mildly acidic pH of 6-6.5 (Sturgill-Koszycki, Schlesinger et al. 1994). It is also at this stage, the ROS is produced by NADPH oxidase (Nox2), an enzyme complex. When the macrophage is not activated, this complex remains inactive and becomes active when the macrophage is activated. The Nox2 NADPH oxidase complex is comprised of the membranal proteins gp22 phox and gp91 phox (also known collectively as the cytochrome b558) and four regulatory cytosolic subunits: p40 phox , p47 phox , p67 phox and Rac2 [START_REF] Assari | Chronic Granulomatous Disease; fundamental stages in our understanding of CGD[END_REF][START_REF] Lam | The many roles of NOX2 NADPH oxidasederived ROS in immunity[END_REF]) (Figure 11). This complex can produce a large quantity of ROS which are derived from initial production of superoxide (O2-. ). O2-. is extremely unstable and weakly bactericidal. It is subsequently transformed into hydrogen peroxide (H2O2) and then transforms into hydroxyl radical (OH-. ). The high ROS concentration generated within the phagosome are thought to kill pathogens directly due to their oxidizing capacity. This procedure of ROS production is called the respiratory or oxidative burst [START_REF] Assari | Chronic Granulomatous Disease; fundamental stages in our understanding of CGD[END_REF][START_REF] Lam | The many roles of NOX2 NADPH oxidasederived ROS in immunity[END_REF]. During the second stage of phagosome maturation, the early phagosome transforms into late phagosome by the fusion with late endosomes. A hallmark of this stage is the progressive acidification of the phagosomal lumen, pH is ~5.5. This drastic drop in pH is catalyzed by the V-ATPase, a proton pump that drives H + uptake into the phagosome lumen from the cytosol via using the energy from ATP hydrolysis. The late phagosome is characterized by the acquisition of the Rab7 and Rab9 lysosome-associated membrane proteins (LAMP1 and LAMP2) and the loss of Rab5 and PI3P [START_REF] Rink | Rab conversion as a mechanism of progression from early to late endosomes[END_REF][START_REF] Poteryaev | Identification of the switch in early-to-late endosome transition[END_REF].

A large number of AMPs has been found within late phagosomes, such as defensins, cathelicidins, and ubiquitin-derived AMPs, which are responsible for the degradation of bacterial pathogens. The anti-bacterial activity of cathelicidins is specifically effective for Shigella. spp, S. aureus, Group A and B Streptococci (Guani-Guerra, [START_REF] Guani-Guerra | Antimicrobial peptides: general overview and clinical implications in human health and disease[END_REF]. The AMPs also play a role of pro-inflammatory and chemotactic activities on immune cells, which may modulate the immune response, serving as a bridge between the innate and adaptive immune responses [START_REF] Ganz | Defensins: antimicrobial peptides of innate immunity[END_REF]. AMPs probably synergize with other degradative enzymes, including cysteine, serine, and aspartate-proteases (such as the cathepsins B, L, and D), peptidases, glycosidases (such as N-acetylglycosaminidase, β-glucuronidase, βgalactosidase) as well as (phospho-) lipases.

Other anti-bacterial activity mechanisms also exist, like the sequestration of essential nutrients for bacterial pathogens. For example, lactoferrin sequesters irons in infected macrophages to limit iron availability to intracellular pathogens (Cassat and Skaar 2013). Natural resistance-associated macrophage protein 1 (NRAMP1) is expressed in the late phagolysosome and has been characterized as a transporter for divalent metals (Fe 2+ , Zn 2+ , and Mn 2+ ) and protons. For example, Mn 2+ and Zn 2+ are needed by microbes as part of the antioxidant defense protein (Mn 2+ / Zn 2+ -superoxide dismutase) and as a catalytic component of several central proteins [START_REF] Weiss | Macrophage defense mechanisms against intracellular bacteria[END_REF]. In addition, theses ions are the cofactors for some important enzymes to exert their anti-microbial activity [START_REF] Hood | Nutritional immunity: transition metals at the pathogenhost interface[END_REF].

Late phagosomes eventually transform into phagolysosome via the fusion with lysosome. This is the last stage for phagosome maturation. This compartment can be defined by its enrichment with an abundance of degradative enzymes including proteases, lipases, nucleases, phosphatases, glycosidases and LAMPs. For example, cathepsin D, a protease implicates with the degradation of protein, is used as a specific marker for immunofluorescence. A drastic drop of pH (~4.5) also occurs in this compartment due to the presence of V-ATPase. In addition, the accumulation of H + in the maturing phagosome is enhanced by the decreased passive permeability or "proton leak" of the limiting phagosomal membrane, which assists in the maintenance of an acidic pH [START_REF] Canton | Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages[END_REF]. The phagosome acidification is essential to the function of this organelle. Luminal acidification is not only a consequence of phagosome maturation but is also a prerequisite as indicated by the fact that the presence of weak bases (e.g., NH4Cl) or agents that specifically inhibit the function of the v-ATPase also interrupt phagosome maturation [START_REF] Canton | Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages[END_REF]. Furthermore, some lysosomal proteases, such as cathepsins, catalyze optimally at this low pH while the metal transporter NRAMP1 also requires phagosomal H + for its activity at low pH [START_REF] Jabado | Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane[END_REF][START_REF] Flannagan | Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus[END_REF].

Autophagy

Autophagy occurs constitutively at basal rate under normal cellular conditions to maintain homeostasis for intracellular recycling and metabolic regulation [START_REF] Ravanan | Autophagy: The spotlight for cellular stress responses[END_REF]. Autophagy is stimulated during various pathological and physiological conditions such as starvation, growth factor deprivation, ER stress, and intracellular pathogen infection [START_REF] He | Regulation mechanisms and signaling pathways of autophagy[END_REF]. The cells adapt to these conditions by converting these signals into several anabolic and catabolic responses. Autophagy degrades the damaged organelles, longlived proteins and even invasive bacterial pathogens. Indeed, autophagy has been extensively demonstrated to confer resistance to numerous bacterial pathogens, most notably by the direct destruction of internalized bacteria residing in the cytosol or damaged vesicles, a mechanism also named xenophage [START_REF] Bauckman | Selective autophagy: xenophagy[END_REF].

Autophagy is initiated by the formation of double-membrane-bound vesicles, called autophagosomes, which sequester cytoplasmic material in a non-degradative compartment [START_REF] Rubinsztein | Autophagy modulation as a potential therapeutic target for diverse diseases[END_REF]. The outer-membrane of the autophagosome fuses with the lysosomal membrane, and the inner vesicle, together with its cargo, is degraded [START_REF] Randow | Autophagy in the regulation of pathogen replication and adaptive immunity[END_REF]. During autophagosome formation in eukaryotic cells, a membrane is marked with phosphatidylinositol-3-phosphate (PI3P) by a complex of the type III PI3 kinase Vps34 which induces activation of protein Atg5, Atg12, and Atg16L1. At the PI3P marked site membrane elongation results in the formation of an isolation membrane (also known as a phagophore), which eventually closes around the substrate into a completely sealed autophagosome. Then, the ATG5-ATG12-ATG16L1 complex mediates the covalent attachment of ATG8/LC3B to phosphatidylethanolamine (PE) in the isolation membrane [START_REF] Saitoh | Regulation of innate immune responses by autophagy-related proteins[END_REF][START_REF] Randow | Autophagy in the regulation of pathogen replication and adaptive immunity[END_REF]) (Figure 10 and12). Once the autophagosome is completed, it rapidly fuses with lysosomes in a Rab7-dependent way, following subsequent degradation.

However, some intracellular pathogens develop various strategies to avoid autophagy. For example, when L. monocytogenes escapes into the cytosol, it expresses a surface protein ActA to polymerize actin at the bacterial pole, which mediates bacterial motility. ActA also prevents the coating of L. monocytogenes with ubiquitin and thereby interferes with autophagy, possibly because of ActA-recruited host protein (Arp2/3 complex and Ena/VASP) which disguise the bacterial surface [START_REF] Yoshikawa | Listeria monocytogenes ActA-mediated escape from autophagic recognition[END_REF]. The phospholipases PlcA and PlcB, which are critical for Listeria escape from the double membrane surrounding the bacteria following cell-to-cell spread, also reduce autophagic flux and PI3P levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria [START_REF] Tattoli | Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures[END_REF].

Furthermore, some intracellular bacterial pathogens involve various mechanisms to take advantage of autophagy, converting autophagosomes into a niche that sustains bacterial replication and allows unorthodox trafficking. For example, S. aureus can proliferate in doubleor multilamellar LC3 + vesicles, which most probably due to canonical autophagosomes failing to acidify and mature [START_REF] Schnaith | Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death[END_REF][START_REF] Mestre | Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells[END_REF]. Schnaith et al.

observe that S. aureus fails to grow in atg5 -/-mouse embryonic fibroblasts, while autophagy induction by rapamycin enhances the proliferation. However, how these bacteria co-opt autophagy and how autophagy contributes to bacterial growth remains unknown. Chapter II: Staphylococcus aureus [START_REF] Gould | New insights into meticillin-resistant Staphylococcus aureus (MRSA) pathogenesis, treatment and resistance[END_REF].

The emergence of community associated MRSA (CA-MRSA) infections has dramatically increased. In contrast to HA-MRSA infections, for which there is a predisposing risk factor or condition, CA-MRSA infections can also occur in healthy individuals [START_REF] Herold | Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk[END_REF], suggesting that CA-MRSA strains have enhanced virulence compared with traditional HA-MRSA strains. In particular, S. aureus USA300 has now emerged as the predominant cause of CA-MRSA infections in the United States and is continuously spreading around the world [START_REF] Nimmo | USA300 abroad: global spread of a virulent strain of communityassociated methicillin-resistant Staphylococcus aureus[END_REF].

With the increasing incidence of both community and hospital acquired staphylococcal infections and the prevalence of multidrug resistance strains, S. aureus infections remain a huge challenge to clinicians and scientists [START_REF] Vandenesch | Community-acquired methicillinresistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence[END_REF][START_REF] Tristan | Global distribution of Panton-Valentine leukocidin--positive methicillin-resistant Staphylococcus aureus, 2006[END_REF]).

Virulence factors

S. aureus virulence is related to the production of a wide variety of virulence factors that are responsible for adhesion to surface /tissues, lysis of host cells, tissue invasion and destruction [START_REF] Foster | Surface protein adhesins of Staphylococcus aureus[END_REF][START_REF] Lowy | Staphylococcus aureus infections[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF]. Importantly, many specific virulence factors produced by S. aureus are dedicated to specifically manipulate both innate and adaptive immune responses [START_REF] Rooijakkers | Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases[END_REF][START_REF] Haupt | The Staphylococcus aureus protein Sbi acts as a complement inhibitor and forms a tripartite complex with host complement Factor H and C3b[END_REF][START_REF] Thammavongsa | Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death[END_REF][START_REF] Stapels | Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors[END_REF]).

We will briefly recall below the main and best studied virulence attributes of S. aureus (Table 2).

Adhesins. The pathophysiology of S. aureus infections usually begins with the colonization in host skin and/or mucosa by its adhesion factors. These factors allow the bacterium to bind to the components of the extracellular matrix of the host, but also to surgical materials (catheter, prosthesis, mechanical valve ...). These factors, also called microbial surface components recognizing adhesive matrix molecules (Foster, Geoghegan et al. 2014), include staphylococcal protein A (SpA), fibronectin-binding proteins A and B (FnbpA and FnbpB), clumping factor proteins (ClfA and ClfB) and collagen-binding protein (Cna).

Toxins. S. aureus is also able to produce three types of super-antigenic toxins: staphylococcal enterotoxins, the staphylococcal enterotoxin-like proteins, and toxic shock syndrome toxin-1 [START_REF] Xu | Staphylococcal superantigens in colonization and disease[END_REF]. So far, more than 20 staphylococcal superantigens (SAgs) have been characterized and at least 80% of clinical strains harbor at least one SAg gene, although most strains encode many [START_REF] Xu | Staphylococcal superantigens in colonization and disease[END_REF].

Panton-Valentine leukocidins (PVL) of S. aureus are protein toxins composed of two subunits, named LukF-PV and LukS-PV (32 kDa and 38 kDa,respectively). PVLs can induce cell lysis on target cells. Of note, PVL genes are present in only a small percentage (~5%) of overall S. aureus clinical strains, whereas they are strongly associated with CA-MRSA strains (~85%), in particular those causing pneumonia, and skin and soft tissue infections [START_REF] Naimi | Comparison of community-and health care-associated methicillin-resistant Staphylococcus aureus infection[END_REF][START_REF] Yoong | The effects of Staphylococcus aureus leukotoxins on the host: cell lysis and beyond[END_REF].

Phenol-Soluble Modulins (PSMs) are a family of amphipathic and α-helices peptides [START_REF] Peschel | Phenol-soluble modulins and staphylococcal infection[END_REF][START_REF] Cheung | Phenol-soluble modulins-critical determinants of staphylococcal virulence[END_REF]. In S. aureus, there are two major families of PSMs: PSMβ (43-45 amino acids) and PSMα (20-25 amino acids) [START_REF] Wang | Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA[END_REF]). δ-hemolysin (Hld), encoded by the agr locus (see below), also belongs to the family of PSMα.

α-hemolysin (Hla) is a secreted 33 kDa protein toxin that is able to lyse cells by forming pores on the target cells via interaction with its receptor ADAM10 [START_REF] Powers | ADAM10 mediates vascular injury induced by Staphylococcus aureus alpha-hemolysin[END_REF][START_REF] Berube | Staphylococcus aureus alpha-toxin: nearly a century of intrigue[END_REF]. This toxin, produced massively by the strains of USA 300, contributes to the pathogenicity of these strains by favoring the bacterial invasion by destruction of the epithelial and endothelial barriers. On the other hand, β-hemolysin (Hlb) is a sphingomyelinase, which does not involve pore-forming in target cells, but causes destabilization of the lipid bilayer and an alteration of its fluidity leading to cell death [START_REF] Katayama | Beta-hemolysin promotes skin colonization by Staphylococcus aureus[END_REF]. The majority of S. aureus strains possess the hlb gene encoding Hlb. However, since this gene constitutes a bacteriophage insertion site, more than 80% of human clinical isolates do not express Hlb [START_REF] Roetzer | Genotypic and phenotypic analysis of clinical isolates of Staphylococcus aureus revealed production patterns and hemolytic potentials unlinked to gene profiles and source[END_REF].

Virulence regulators

Production of S. aureus virulence determinants is controlled by several global regulatory loci, which include agr, sar, sigB, rot, arlR/S, svrA, saeR/S and the metabolite-responsive regulators [START_REF] Goerke | Role of Staphylococcus aureus global regulators sae and sigmaB in virulence gene expression during device-related infection[END_REF]. The virulence gene regulators could affect the expression of target genes directly, by binding to their promoters, or indirectly, via other regulators. Here, we will describe two important global virulence regulators agr and sigB, and briefly mention some other metabolite-responsive virulence regulators.

The agr operon

The accessory gene regulator system (agr) operon was discovered by [START_REF] Recsei | Regulation of exoprotein gene expression in Staphylococcus aureus by agar[END_REF][START_REF] Recsei | Regulation of exoprotein gene expression in Staphylococcus aureus by agar[END_REF]). The agr operon constitutes a global regulatory system that controls cell density-dependent virulence factor expression. In acute mice infection models, including skin and soft tissue infections (SSTIs), intracranial abscesses and necrotizing pneumonia, agr activity is necessary [START_REF] Bubeck Wardenburg | Surface proteins and exotoxins are required for the pathogenesis of Staphylococcus aureus pneumonia[END_REF][START_REF] Novick | Quorum sensing in staphylococci[END_REF][START_REF] Cheung | Role of the accessory gene regulator agr in community-associated methicillin-resistant Staphylococcus aureus pathogenesis[END_REF]. On the other side, there is a link between agr dysfunction and persistent colonization in nosocomial infections with S. aureus (Fowler, Phenol-Soluble Modulins (PSMs) Induce Activates plasminogen; inactivates antimicrobial peptides [START_REF] Fowler | Persistent bacteremia due to methicillin-resistant Staphylococcus aureus infection is associated with agr dysfunction and low-level in vitro resistance to thrombininduced platelet microbicidal protein[END_REF][START_REF] Shopsin | Prevalence of agr dysfunction among colonizing Staphylococcus aureus strains[END_REF][START_REF] Shopsin | Mutations in agr do not persist in natural populations of methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Kang | agr dysfunction affects staphylococcal cassette chromosome mec type-dependent clinical outcomes in methicillin-resistant Staphylococcus aureus bacteremia[END_REF].

The agr operon consists of two divergent promoters, P2 and P3. Expression from P2 produces the components of a quorum-sensing system (AgrB, D, C, A) and expression from P3 produces the agr effector molecule RNAIII (Figure 13). RNAIII is responsible for the regulation of various virulence factors, leading to a switch from expression of cell-surface associated proteins, such as staphylococcal protein A (SpA) and fibronectin-binding proteins A and B (FnBPA and FnBPB), to secreted toxins such as α-haemolysin (Hla) and -haemolysin (Hld, encoded within the RNAIII transcript), phenol-soluble modulins (PSMs), and Panton-Valentine leucocidin (PVL) [START_REF] Novick | Quorum sensing in staphylococci[END_REF][START_REF] Queck | RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus[END_REF][START_REF] Kane | Virulence Factor Targeting of the Bacterial Pathogen Staphylococcus aureus for Vaccine and Therapeutics[END_REF]. At low cell density, the agr genes are continuously expressed at basal levels [START_REF] Recsei | Regulation of exoprotein gene expression in Staphylococcus aureus by agar[END_REF]. A signal molecule, the autoinducing peptide (AIP), produced and secreted by the bacteria, accumulates outside of the cells. When the cell density increases and the AIP concentration reaches a threshold, it activates the agr response by increasing RNAIII expression. AgrB and AgrD are essential for the production of AIP that functions as a signal for quorum sensing. AgrB is a transmembrane protein, involved in the proteolytic processing of AgrD and facilitating the export of the processed AgrD peptide.

There are four allelic variants (types I-IV) of agr, each encoding a distinct AIP molecule, which functions as a specific ligand for the corresponding sensor histidine kinase AgrC, but as an inhibitor of other AgrC variants. AgrC possesses an extracellular N-terminal domain and seven trans-membrane domains. Binding of AIP induces a conformational change within the cytoplasmic helix that links the sensor and kinase domains of AgrC, activating its autophosphorylation [START_REF] Novick | Quorum sensing in staphylococci[END_REF][START_REF] Wang | New insight into transmembrane topology of Staphylococcus aureus histidine kinase AgrC[END_REF]. However, binding with heterologous AIP (from S. aureus expressing one of the three other allelic variants of Agr)

causes the helical linker to twist in the opposite direction, preventing auto-kinase activity [START_REF] Wang | Activation and inhibition of the receptor histidine kinase AgrC occurs through opposite helical transduction motions[END_REF].

AgrC and AgrA act as a classical two-component system (TCS), where AgrC transfers the phosphoryl group, carried on a Histidine residue to an Aspartate residue of the conserved response regulator AgrA. Phosphorylated AgrA dimers bind to the agr intergenic region (agr-IR) between P2 and P3 and up-regulate expression of these promoters [START_REF] Novick | Quorum sensing in staphylococci[END_REF][START_REF] Painter | What role does the quorum-sensing accessory gene regulator system play during Staphylococcus aureus bacteremia?[END_REF].

Recently Sully et al. identified a small molecule, designated Savirin (for S. aureus virulence inhibitor), which inhibits agr-associated virulence gene expression by preventing AgrA binding to the agr-IR [START_REF] Sully | Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance[END_REF]. ω-Hydroxyemodin (OHM), a small compound isolated from Penicillium restrictum is also able to inhibit the quorum sensing function of the agr operon, by binding to the agrA promoter and preventing interaction with DNA [START_REF] Daly | omega-Hydroxyemodin limits staphylococcus aureus quorum sensing-mediated pathogenesis and inflammation[END_REF]. Another inhibitor of the Agr activity has been developed, named Solonamide B, which prevents AgrC activation in all allelic variants of agr, hence inhibits quorum-sensing and subsequent virulence gene expression via agr locus [START_REF] Nielsen | Solonamide B inhibits quorum sensing and reduces Staphylococcus aureus mediated killing of human neutrophils[END_REF]. are essential for the production of AIP that functions as a signal for quorum sensing. AgrB is a transmembrane protein, involved in the proteolytic processing of AgrD and facilitating the export of the processed AgrD peptide.

Savirin, which inhibits agr-associated virulence gene expression by preventing AgrA binding to the agr-IR. ω-Hydroxyemodin, a small compound isolated from Penicillium restrictum is also able to inhibit the quorum sensing function of the agr operon, by binding to the agrA promoter and preventing interaction with DNA. Another inhibitor of the Agr activity has been developed, named Solonamide B, which prevents AgrC activation in all allelic variants of agr, hence inhibits quorum-sensing and subsequent virulence gene expression via agr locus.

ω-Hydroxyemodin

SigB

The alternative transcription factor σ B (SigB) is known to affect the expression of numerous genes (including virulence factors and regulatory elements), upon changes in the environment (starvation, heat shock, osmotic shock, etc…). Wu et al. first described and sequenced a chromosomal cluster of four S. aureus genes comprising sigB (rsbU, rsbV, rsbW, sigB) [START_REF] Wu | Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing[END_REF] (Figure 14B). The regulation of the σ B activity is modulated by the rsbU, rsbV, and rsbW gene products. RsbW acts as an anti-σ B factor. RsbV is an anti-antiσ B factor, which competes with σ B for RsbW. RsbV is modulated by both the phosphatase RsbU and the phosphorylase RsbW [START_REF] Senn | Molecular analysis and organization of the sigmaB operon in Staphylococcus aureus[END_REF][START_REF] Pane-Farre | Role of RsbU in controlling SigB activity in Staphylococcus aureus following alkaline stress[END_REF]). In S.

aureus, σ B is activated via the RsbU-dependent pathway (Pane-Farre, Jonas et al. 2009). RsbW inactivates σ B through the complex RsbW-σ B , and thus suppresses the aggregation of the σ B subunit with the RNA polymerase (RNAP) core enzyme. During the exponential growth phase, RsbV is phosphorylated and inactivated by RsbW. Consequently, the σ B factor is sequestered in a RsbW-σ B complex. In response to environmental stress, RsbV is dephosphorylated by RsbU, leading to the formation of the highly specific RsbV-RsbW complex and the release of activated σ B factor, which is able to bind to the RNAP core enzyme to form an active σ B holoenzyme [START_REF] Bronner | Regulation of virulence determinants in Staphylococcus aureus: complexity and applications[END_REF]). Nevertheless, the rsbU natural mutants (e.g. RN6390) can still produce a functional σ B protein, suggesting that the activity of σ B is also dependent on factors other than RsbU. Thus, it has been suggested that a second σ B activation pathway that is independent of RsbU but dependent on RsbV exists [START_REF] Palma | sigma(B) activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth[END_REF]) (Figure 14A).

Various S. aureus genes, most of which are involved in stress response, have been shown to be controlled by σ B [START_REF] Gertz | Characterization of the sigma(B) regulon in Staphylococcus aureus[END_REF][START_REF] Pane-Farre | Role of RsbU in controlling SigB activity in Staphylococcus aureus following alkaline stress[END_REF]. Notably, SigB is involved in bacterial aggregation by modulating the expression of the genes encoding clumping factor or other adhesins. Of interest, SigB was also described to down-regulate the agr system [START_REF] Bischoff | Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus[END_REF], which is most likely responsible for the enhanced inflammatory activity. The down-regulated agr system helps the bacteria to form biofilm and silences bacterial aggressiveness for persistence within host cell [START_REF] Boles | Agr-mediated dispersal of Staphylococcus aureus biofilms[END_REF][START_REF] Grundmeier | Staphylococcal strains vary greatly in their ability to induce an inflammatory response in endothelial cells[END_REF]. Remarkably, Tuchscherr showed that SigB also played a crucial role during bacterial adaptation during long-term persistence [START_REF] Tuchscherr | Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections[END_REF].

Initially description of persistence phenomenon is almost 60 years ago, Bigger et al. observed a subpopulation of staphylococci is able to survive with treatment of penicillin without acquiring resistance-conferring genetic changes, these cells regrow a new population that is sensitive to the antibiotic [START_REF] Bigger | TREATMENT OF STAPHYLOCOCCAL INFECTIONS WITH PENICILLIN BY INTERMITTENT STERILISATION[END_REF]). It has since been observed in many bacterial species.

These microorganisms, called persisters, are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics [START_REF] Lewis | Persister cells[END_REF]. Persisters are associated with chronic

Metabolite-responsive regulators

Modification of central metabolic pathways, such as the tricarboxylic acid (TCA) cycle, can affect virulence determinant biosynthesis by feedback and feedforward alteration of enzymatic activities or by activating metabolite-responsive regulators via the alteration of intracellular concentrations of specific metabolites [START_REF] Chatterjee | Enhanced poststationary-phase survival of a clinical thymidine-dependent small-colony variant of Staphylococcus aureus results from lack of a functional tricarboxylic acid cycle[END_REF][START_REF] Somerville | At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci[END_REF]. Several metabolite-responsive regulators have been identified in S. aureus (e.g., CcpA, CcpE, CodY, and Rex) that are involved in the regulation of both metabolism and virulence. For examples, the carbon catabolite protein A (CcpA) responds to glucose-associated metabolic signals [START_REF] Deutscher | Protein kinasedependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria[END_REF] and inactivation of ccpA results in a lower level of RNAIII. The carbon catabolite protein E (CcpE) responds to citrate and inactivation of ccpE results in increased staphyloxanthin production and enhanced bacterial virulence in a mouse model of systemic infection [START_REF] Ding | Metabolic sensor governing bacterial virulence in Staphylococcus aureus[END_REF]. CodY responds to GTP and branched-chain amino acids and inactivation of codY results in derepressed synthesis of the agr locus enhanced bacterial virulence of USA300 strain in the mouse model [START_REF] Majerczyk | Staphylococcus aureus CodY negatively regulates virulence gene expression[END_REF][START_REF] Pohl | CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression[END_REF][START_REF] Montgomery | CodY deletion enhances in vivo virulence of community-associated methicillinresistant Staphylococcus aureus clone USA300[END_REF][START_REF] Gaupp | RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus[END_REF]) (Figure 15).

Finally, the PPP-responsive regulator RpiR, which catalyzes the reversible isomerization of ribulose-5-phosphate and ribose-5-phosphate, was also suggested to down-regulate expression of RNAIII [START_REF] Gaupp | RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus[END_REF]. Members of the RpiR family have been identified in both Gram-negative and Gram-positive bacteria, including E. coli and Bacillus subtilis [START_REF] Yamamoto | Regulation of the glv operon in Bacillus subtilis: YfiA (GlvR) is a positive regulator of the operon that is repressed through CcpA and cre[END_REF][START_REF] Sonenshein | Control of key metabolic intersections in Bacillus subtilis[END_REF]. As sugar-responsive regulators, members of the RpiR family of proteins have N-terminal helix-turn-helix DNA binding motifs and C-terminal sugar isomerase binding domains [START_REF] Zhu | RpiR homologues may link Staphylococcus aureus RNAIII synthesis and pentose phosphate pathway regulation[END_REF]. Two of the three homologues (RpiRB and RpiRC) of S. aureus positively influence the transcription of the PPP genes rpiA and zwf [START_REF] Zhu | RpiR homologues may link Staphylococcus aureus RNAIII synthesis and pentose phosphate pathway regulation[END_REF]. The significance of RpiRc was demonstrated in an acute pneumonia model and a systemic abscess formation model, where inactivation of rpiRc markedly increased mortality and the bacterial burden in mice [START_REF] Gaupp | RpiRc Is a Pleiotropic Effector of Virulence Determinant Synthesis and Attenuates Pathogenicity in Staphylococcus aureus[END_REF].

Intracellular life cycle of Staphylococcus aureus

S. aureus is generally considered as an extracellular pathogen. However, several studies have demonstrated that S. aureus was able to survive in vivo within host cells [START_REF] Zautner | Intracellular persisting Staphylococcus aureus is the major pathogen in recurrent tonsillitis[END_REF][START_REF] Hayes | Intracellular residency of Staphylococcus aureus within mast cells in nasal polyps: A novel observation[END_REF][START_REF] Hanssen | Localization of Staphylococcus aureus in tissue from the nasal vestibule in healthy carriers[END_REF]. Indeed, S. aureus is able to invade a variety of non-professional phagocytic cells (NPPCs) such as endothelial and epithelial CcpE CcpE Citrate cells, osteoblasts, fibroblasts, keratinocytes ... [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF][START_REF] Moldovan | In or out: Phagosomal escape of Staphylococcus aureus[END_REF]. The intracellular environment could protect staphylococci from host's immune system and antibiotics. Infected cells can thus constitute reservoirs likely to play an important role for bacterial persistence in the host and relapses of infection.

Like other intracellular bacteria, the S. aureus intracellular lifestyle can be divided into three main phases: entry, survival or replication and exit. See also (Figure 16).

Entry

Invasion of NPPCs by S. aureus is mediated by a zipper-type mechanism and involves several bacterial adhesins. The most dominant staphylococcal adhesins participating to bacterial entry into NPCCs are FnBPs [START_REF] Dziewanowska | Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells[END_REF][START_REF] Jevon | Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts[END_REF][START_REF] Edwards | Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA[END_REF], which can recruit α5β1 integrins by a fibronectin bridge. This interaction is sufficient to induce zipper-type uptake of staphylococci [START_REF] Sinha | Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells[END_REF]. However, FnBP-independent invasion was also observed in S. aureus. For example, it has been shown that S. aureus Newman strain invaded epithelial cells and fibroblasts by using extracellular adherence protein [START_REF] Harraghy | The adhesive and immunomodulating properties of the multifunctional Staphylococcus aureus protein Eap[END_REF]. Zapotoczna et al. found that the protein IsdB directly bound to β3-containing integrins and that this interaction could promote bacterial adhesions to, and internalization by, host cells [START_REF] Zapotoczna | Iron-regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non-phagocytic human cells[END_REF]). In addition, the wall teichoic acid (WTA) has been shown to play an important role for nasal colonization, by promoting binding to a type-F scavenger receptor [START_REF] Weidenmaier | Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections[END_REF][START_REF] Baur | A nasal epithelial receptor for Staphylococcus aureus WTA governs adhesion to epithelial cells and modulates nasal colonization[END_REF]. The staphylococcal autolysin was also identified as an adhesion mediating binding to its host cell receptor, the heat shock cognate protein Hsc70 [START_REF] Hirschhausen | A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor[END_REF]. Finally, ClfB has been shown to bind to cytokeratins as well as to the α-chain of fibrinogen [START_REF] Ni Eidhin | Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus[END_REF][START_REF] Ganesh | Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions[END_REF], and Cna to the collagen triple helix [START_REF] Zong | A 'Collagen Hug' model for Staphylococcus aureus CNA binding to collagen[END_REF]. The internalization process of the pathogen mediated by WTA, ClfB, Cna and other molecules remains unknown so far.

Survival

After internalization within host cells, S. aureus either resides in a vacuolar compartment or in host cytosol, subsequently replicates intracellularly and eventually causes host cell death, leading to bacterial release and dissemination.

Within vacuole

S. aureus has evolved various mechanisms to survive phagocytosis by host cells. Following phagocytosis, S. aureus initially resides in a phagosome. Few minutes after uptake, the S. aureus containing phagosome (SaCP) acquires early endosomal markers such as the small GTPase Rab5 [START_REF] Flannagan | Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination[END_REF]. Then, SaCP quicky acquires the late phagosomal marker LAMP-1. Other lysosomal markers such as LAMP-2 have been identified in SaCP.

However, a fully functional phagolysosomal microenvironment is absent for the internalized S.

aureus. The reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D, β-glucuronidase, and lysosomal integral membrane protein (LIMP-II), were discovered in S. aureus-infected macrophages (Jubrail, Morris et al. 2016;[START_REF] Tranchemontagne | Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages[END_REF]. Jubrail et al. suggested that macrophages kill the ingested bacteria at early time post infection. However, these macrophages eventually lose their ability to eliminate S. aureus which can then replicate within such "exhausted" macrophages. Other studies have supported this observation [START_REF] Tranchemontagne | Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages[END_REF][START_REF] Lacoma | Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line[END_REF].

Of note, S. aureus is able to tolerate in acidic environments, which may contribute to its survival in phagolysosomes [START_REF] Weinrick | Effect of mild acid on gene expression in Staphylococcus aureus[END_REF]. For this, S. aureus has evolved various mechanisms that lead to alkalinization of its environment. For example, S. aureus can generate ammonia through arginine deiminase, urease, nitrite and nitrate reductase. Inside host phagocytes, the bacteria also suffer the oxidative burst provoked by the rapid release of reactive oxygen species (ROS) and reactive nitrogen species (RNS). S. aureus has developed strategies to resist ROS and RNS by producing the carotenoid staphyloxanthin and by expressing protective enzymes such as catalase, superoxide dismutase and peroxiredoxins [START_REF] Karavolos | Role and regulation of the superoxide dismutases of Staphylococcus aureus[END_REF][START_REF] Clauditz | Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress[END_REF][START_REF] Cosgrove | Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus[END_REF][START_REF] Richardson | A nitric oxide-inducible lactate dehydrogenase enables Staphylococcus aureus to resist innate immunity[END_REF]. S. aureus has also adapted to increase its resistance against antimicrobial peptides (AMPs) like defensins and lysosome by modifying its cell surface wall via upregulation expression D-alanyl-lipoteichoic acid (DltA, responsible for D-alanine esterification of teichoic acids) and O-acetyltransferase A (OatA, responsible for O-acetylation of the peptidoglycan) [START_REF] Peschel | How do bacteria resist human antimicrobial peptides?[END_REF][START_REF] Bera | Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus[END_REF].

Interestingly, a low phagosomal pH is required for survival and replication of the CA-MRSA S. aureus strain USA300 in macrophages [START_REF] Tranchemontagne | Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages[END_REF]) and a functional glycopeptide resistance-associated (GraRS) two-component system has been shown to be crucial for USA300 adaptation and replication with in acidified phagolysosomes [START_REF] Flannagan | Staphylococcus aureus Uses the GraXRS Regulatory System To Sense and Adapt to the Acidified Phagolysosome in Macrophages[END_REF] as well as for a stronger resistance to cationic AMPs via upregulation with the multiple peptide resistance factor (MprF) [START_REF] Joo | Mechanisms of resistance to antimicrobial peptides in staphylococci[END_REF][START_REF] Flannagan | Staphylococcus aureus Uses the GraXRS Regulatory System To Sense and Adapt to the Acidified Phagolysosome in Macrophages[END_REF].

S. aureus survival in motile phagocytes may be responsible for dissemination of the infection into deeper tissues and infected phagocytes have been considered as possible "Trojan horse" for S. aureus dissemination [START_REF] Thwaites | Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus?[END_REF]. During bloodstream infections, S. aureus is efficiently sequestered in the liver where bacteria are captured by liver macrophages, Kuffer cells. In these cells, S. aureus replicate within acid vesicles, decorated with LAMP-1, for several days, without being detected by innate immune system [START_REF] Surewaard | Identification and treatment of the Staphylococcus aureus reservoir in vivo[END_REF]. Furthermore, infected neutrophils from the liver can re-enter the circulation and disseminate the pathogen to secondary infection sites [START_REF] Pollitt | Staphylococcus aureus infection dynamics[END_REF]).

Within cytosol

S. aureus phagosomal escape has been observed in different epithelial cell lines: A549 (human alveolar basal epithelial cells), Hela (human cervix epithelial cell), HEK923 (human embryonic kidney epithelial cell), S9 (upper airway epithelial cell), CFBE (Cystic fibrosis bronchial epithelial cell) as well as in endothelial cell-like line (Eahy.926), NIH/3T3 (fibroblast cell) or keratinocytes lines (such as HaCaT or RHEK-1), suggesting that these cell types are probably a comfortable niche for subsequent cytoplasmic bacterial replication [START_REF] Jarry | Staphylococcus aureus escapes more efficiently from the phagosome of a cystic fibrosis bronchial epithelial cell line than from its normal counterpart[END_REF][START_REF] Giese | Expression of delta-toxin by Staphylococcus aureus mediates escape from phagoendosomes of human epithelial and endothelial cells in the presence of beta-toxin[END_REF][START_REF] Chi | Panton-Valentine leukocidin facilitates the escape of Staphylococcus aureus from human keratinocyte endosomes and induces apoptosis[END_REF]Grosz, Kolter et al. 2014;[START_REF] Neumann | Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase[END_REF][START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. Of note, phagosomal escape seems to be a strain-dependent process. Generally, the highly cytotoxic strains can translocate to cytoplasm environment whereas non-cytotoxic remain stuck in the phagosome. However, the non-cytotoxic strain Cowan I is also able to induce phagosomal escape in A549 cell line [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF].

S. aureus phagosomal escape has been linked with agr activity [START_REF] Jarry | Staphylococcus aureus escapes more efficiently from the phagosome of a cystic fibrosis bronchial epithelial cell line than from its normal counterpart[END_REF][START_REF] Kubica | A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages[END_REF]. Jarry et al. suggested that the agr regulated α-toxin was involved in S. aureus phagosomal escape because α-toxin is a pore forming toxin which induces phagosomal membrane disruption [START_REF] Jarry | Staphylococcus aureus escapes more efficiently from the phagosome of a cystic fibrosis bronchial epithelial cell line than from its normal counterpart[END_REF]. The role of α-toxin in phagosomal escape has been reported in epithelial cells with cystic fibrosis phenotype as well as in marcophages [START_REF] Jarry | Staphylococcus aureus escapes more efficiently from the phagosome of a cystic fibrosis bronchial epithelial cell line than from its normal counterpart[END_REF][START_REF] Jarry | The expression of alpha-haemolysin is required for Staphylococcus aureus phagosomal escape after internalization in CFT-1 cells[END_REF][START_REF] Kubica | A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages[END_REF].

However, in the LCSFN cell line complemented with the wild-type CF transmembrane conductance regulator (CFTR), α-toxin had no effect [START_REF] Jarry | Staphylococcus aureus escapes more efficiently from the phagosome of a cystic fibrosis bronchial epithelial cell line than from its normal counterpart[END_REF]. (2) late phagosomes. These are lysosome-associated membrane protein-1 (LAMP-I) and Rab7-positive, and pH is continuously decreasing by v-ATPase activity. ( 3) The SaCP may fuse with lysosomes forming a phagolysosome.

The phagolysosome maturation may be incomplete (no cathepsin D or beta-glucuronidase activity) or delayed.

Following phagocytosis, S. aureus faces a different fate, depending on the host cell type: (4) in macrophages, S. aureus stays confined to the SaCP and replicates in the acidic compartment. The exhausted macrophage dies by a mode of death, which has both apoptotic and necrotic hallmarks. Intracellular bacteria are released and are phagocytosed again. Functional agr, graRS and sigB operons as well as α-toxin (Hla) and the protease aureolysin (Aur) are necessary for survival and persistence in macrophages. (5) In non-professional phagocytes, bacteria do not replicate in the SaCP. Cytolytic strains can escape from the phagosome and replicate in the cytosol. The host cell is killed from within. A functional agr system is essential for phagosomal escape. Several bacterial factors such as phenol soluble modulin PSMα and the nonribosomal peptide synthase AusAB or Panton-Valentine leukocidin (in keratinocytes only) appear to be required.

Another agr-regulated virulence factor, PSMα, was identified as a key mediator of S. aureus phagosomal escape (Grosz, Kolter et al. 2014). As mentioned earlier, PSM is a family of amphipathic and α-helices peptides [START_REF] Peschel | Phenol-soluble modulins and staphylococcal infection[END_REF][START_REF] Cheung | Phenol-soluble modulins-critical determinants of staphylococcal virulence[END_REF] and δtoxin, encoded by the agr locus also belongs to the family of PSMα. Inactivation of psmα dramatically decreased the translation of S. aureus clinical strain to the cytosol in epithelial cells (Grosz, Kolter et al. 2014). Blattner at al. also showed that inactivation of PSM-specific ABC-transporter component pmtC [START_REF] Chatterjee | Essential Staphylococcus aureus toxin export system[END_REF]) drastically reduced phagasomal escape of S. aureus [START_REF] Blattner | Staphylococcus aureus Exploits a Nonribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes[END_REF]. Proteomic analysis showed that all phagosomal escape strains were PSM producers [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. Of note, whereas overexpressing δ-toxin or PSMβ in S. aureus RN4220 (non-cytotoxic laboratory strain) led staphylococci phagosomal escape, overexpressing PSMα did not translocate S. aureus RN4220 to the cytosol [START_REF] Giese | Expression of delta-toxin by Staphylococcus aureus mediates escape from phagoendosomes of human epithelial and endothelial cells in the presence of beta-toxin[END_REF]. In addition, membrane disruption by δ-toxin and PSMβ depend on the presence of the staphylococcal β-toxin (a sphingomyelinase). However, S. aureus strain USA300, an expert for phagosomal escape, does not code for a functional βtoxin, due to the integration of prophage ϕSA3usa within the β-haemolysin gene [START_REF] Diep | Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus[END_REF]. Alternatively, prophage might be lost (for example, during exposure to phagosomal ROS) and then the strain can produce β-toxin, eventually contributing to phagosomal escape.

These results support the importance of PSMs for S. aureus phagosomal escape in NPPCs.

However, the mode of action of PSM during this process remains currently unknown.

Recently Blattner et al. identified two genes, ausA and ausB, that were required for efficient phagosomal escape [START_REF] Blattner | Staphylococcus aureus Exploits a Nonribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes[END_REF]. These two genes encode subunits of the nonribosomal peptide synthase (NRPS) AusA and a phosphopantetheinyl transferase AusB, which is involved in AusA activation [START_REF] Zimmermann | A family of pyrazinone natural products from a conserved nonribosomal peptide synthetase in Staphylococcus aureus[END_REF]. The NRPS AusAB produces three cyclic dipeptides: phevalin, tyrvalin, and leuvalin. During infection in HeLa cells, both ausA and ausB transposon-insertion mutants showed significant decreased phagosomal escape rate compared with the parent S. aureus. The lower escape rates correlated with lower cytotoxicity of the mutants. The mode of action of AusAB or its dipeptide products are still unresolved.

Most current studies suggest that cytosolic replication after phagosomal escape is the predominant fate of S. aureus in NPCCs. However, in cystic fibrosis-derived pulmonary epithelial cells, S. aureus start replicating 9 hours after their internalization and 24 hours after infection, S. aureus were found in vacuoles which had not fused with lysosomes [START_REF] Kahl | Staphylococcus aureus RN6390 replicates and induces apoptosis in a pulmonary epithelial cell line[END_REF]. Thus, another compartment may exist in host cells for bacterial replication.

Role of autophagy in S. aureus intracellular survival. It has been demonstrated that SaCP

is targeted by autophagy [START_REF] Schnaith | Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death[END_REF][START_REF] Mestre | Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells[END_REF][START_REF] Neumann | Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase[END_REF]. However, the molecular mechanisms for autophagosome formation, autophagosomal escape and intracellular survival of S. aureus are still controversial. Notably, Schnaith et al. report that S. aureus use autophagosomes as a replicative niche, whereas Mestre et al. observe replication of S. aureus in the cytosol [START_REF] Schnaith | Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death[END_REF][START_REF] Mestre | Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells[END_REF]. Mestre et al. notably showed that an α-toxin-deficient mutant failed to recruit the autophagic marker microtubule-associated protein 1 light chain 3 (LC3B) to the SaCP [START_REF] Mestre | Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells[END_REF], suggesting that α-toxin plays an important role for induction of autophagy. -toxin may induce SaCP membrane disruption in NPPCs. Autophagy would be then induced to remove these leaky vesicles [START_REF] Maurer | Autophagy mediates tolerance to Staphylococcus aureus alpha-toxin[END_REF]. Neumann et al. observed that more S. aureus survived inside atg5 (encoding ATG5 protein which involves in autophagy flux) knockout (KO) than ATG5-reconstitued mouse embryonic fibroblast (MEFs), indicating that autophagy is not essential for intracellular survival [START_REF] Neumann | Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase[END_REF]). The higher replication of S. aureus in atg5 KO MEFs suggested that bacterial replication inside the host cells can occur without autophagosomes. In addition, transmission electron microscopy of ultrathin sections revealed infected NIH/3T3 fibroblast cells containing dividing bacteria residing in the cytoplasm without a surrounding membrane.

Neumann et al. suggested that replication of S. aureus inside non-professional phagocytes was independent from autophagy induction. These authors employed the strain SH1000, which is devoid of prophages and has a repaired rsbU gene resulting in normal SigB activity [START_REF] Neumann | Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase[END_REF]. The community acquired methicillin-resistant S. aureus (MRSA) strain MW2 and USA300, which were used by Schnaith et al., contain a mecA cassette conferring methicillin resistance and often also the Panton-Valentine leukocidin (PVL) poreforming toxin [START_REF] Neumann | Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase[END_REF]. In conclusion, the different strains used in the study are likely to express virulence factors and pore-forming toxins at different levels which probably affects the interplay between S. aureus and the host cell.

Exit

Following S. aureus intracellular replication, host cells die by programmed cell death (including apoptosis, pyroptosis and necroptosis) and necrosis, thereby releasing S. aureus to extracellular environment (Munoz-Planillo, [START_REF] Munoz-Planillo | A critical role for hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of the Nlrp3 inflammasome[END_REF][START_REF] Flannagan | Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus[END_REF][START_REF] Flannagan | Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination[END_REF][START_REF] Man | Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases[END_REF].

Apoptosis is a programmed cell death procedure that relies on an active cascade of cysteine endopeptidases called caspases [START_REF] Taylor | Apoptosis: controlled demolition at the cellular level[END_REF]. Various virulence factors of S. aureus are responsible to host cell death. For example, Haslinger-Loffler et al. showed that when overexpress α-toxin in strain Cowan I, which is barely hemolytic, it marked more potent for induction of apoptosis in HUVEC than the parental strain [START_REF] Haslinger-Loffler | Multiple virulence factors are required for Staphylococcus aureus-induced apoptosis in endothelial cells[END_REF]. α-toxin causes leukocytes cell death either by apoptosis or necrosis [START_REF] Essmann | Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation[END_REF][START_REF] Haslinger | Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway[END_REF] and low doses of α-toxin induce apoptosis accompanied by a breakdown of the mitochondrial membrane potential [START_REF] Haslinger | Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway[END_REF]. In contrast, high doses of α-toxin induce cell necrotic death [START_REF] Essmann | Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation[END_REF][START_REF] Haslinger | Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway[END_REF]. Meanwhile, PVL induces a rapid caspase-9/caspase-3-dependent cell death in PMN [START_REF] Genestier | Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils[END_REF]. Besides from apoptosis S. aureus is also able to induce pyroptosis. Pyroptosis is induced by the sensing of bacterial molecules through the host inflammasome. The inflammasome activates caspase-1, which leads to interleukin (IL)-1β and IL-18 secretion and causes cell death [START_REF] Hybiske | Exit strategies of intracellular pathogens[END_REF]. S. aureus can function as a stimulus for NOD-like receptor protein 3 (NLRP3) inflammasome, however, the molecular mechanism of the stimulating signal is yet known so far.

Since releasing lysosomal protease cathepsin B into the cytoplasm contributes to NLRP3 activation [START_REF] Bergsbaken | Pyroptosis: host cell death and inflammation[END_REF]), thus, it seems that lysosomal rupture or permeabilization by S. aureus toxins releases cathepsin which then activates the inflammasome. Indeed S. aureus α, β, and γ-toxins have been shown to be important for activation of the NLRP3 inflammasome (Munoz-Planillo, [START_REF] Munoz-Planillo | A critical role for hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of the Nlrp3 inflammasome[END_REF].

Importance of staphylococcal metabolism during infection

S. aureus is a typical heterotrophic and essentially prototrophic microorganism (Fuchs, Eisenreich et al. 2012) which can survive in diverse host environments. This versatility depends on its ability to acquire and utilize nutrients from different sources, but glucose seems to be the preferred one. Glucose uptake is achieved by the glucose-specific phosphotransferase system (PtsG). S. aureus is also able to import glucose-6-phosphate by a hexose phosphate transport protein (UhpT) [START_REF] Park | Characterization of a novel two-component regulatory system, HptRS, the regulator for the hexose phosphate transport system in Staphylococcus aureus[END_REF]. The central carbon metabolism of S. aureus contains the complete glycolytic pathway, TCA cycle and PPP.

S. aureus possesses a quinone-dependent malate oxidoreductase encoded by mqo2 that may replace the missing TCA cycle enzyme under certain growth conditions (e.g. upon growth on pyruvate or lactate as carbon source). Among the anaplerotic pathways, pyruvate kinase (PYC) and PEP-carboxykinase (PckA) are present. The glyoxylate shunt (GS) is absent, whereas the malic enzyme converting malate to pyruvate is present (Fuchs, Eisenreich et al. 2012). In the exponential growth phase, where is a nutrient-rich environment (especially in presence of glucose), the TCA cycle is repressed, and acetyl-CoA is converted predominantly via acetylphosphate (acetyl-P) to acetate with ATP generation by substrate-level phosphorylation.

Together with carbon substrates of lower energy (e.g. pyruvate or TCA cycle intermediates), acetyl-CoA enters the active TCA cycle. This is accompanied by an increased oxidative phosphorylation activity through the electron transfer chain to remove NADH generated in the TCA cycle.

At the end of the exponential phase, the concentration of an essential nutrient (e.g., glucose) decreases to a level where it can no longer sustain rapid growth. If conditions are favorable, S. aureus can enter into post-exponential growth, where it can catabolize of non-preferred carbon sources such as acetate [START_REF] Somerville | Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival[END_REF]. The post-exponential catabolism of non-preferred carbon sources requires TCA cycle activity and coincides with a large increase in TCA cycle enzymatic activity [START_REF] Somerville | Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions[END_REF]. Because the TCA cycle provides biosynthetic intermediates, ATP, and reducing potential, the transition to TCA cycle-driven metabolism dramatically alters the staphylococcal metabolome [START_REF] Somerville | At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci[END_REF].

Growth of S. aureus can occur under aerobic and anaerobic conditions, and oxygen, nitrite or nitrate can be used as the final electron acceptors in respiration. During aerobic growth, electrons enter into the electron transport chain from NADH and are transferred to menaquinone by the NADH dehydrogenase complex [START_REF] Bayer | Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein 1[END_REF]. Menaquinone transfers the electrons to oxidized cytochrome c, generating the reduced form of cytochrome c. The electrons are then transferred to oxygen by cytochrome c oxidase (this process can also generate ROS), then ATP is produced via ATP synthase complex. The inactivation of the electron transfer chain, either by genetic inactivation [START_REF] Kohler | Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach[END_REF] or by extreme iron restriction [START_REF] Friedman | Staphylococcus aureus redirects central metabolism to increase iron availability[END_REF], results in a growth phenotype similar to that of anaerobic/fermentative growth. The inactivation of the electron transfer chain produces a clinically relevant phenotype in staphylococci known as the small-colony variant (SCV) phenotype [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF].

During acute infection, S. aureus grows predominantly extracellularly and the above described metabolic capabilities allow a perfect adaption to the varying nutrient conditions that this pathogen may encounter. With the failure of antibiotic treatment or inefficient host response, the staphylococci acute infections will develop to a chronic infection. The intracellular life cycle of S. aureus may be involved in persistence and dissemination of the bacteria in the infected host [START_REF] Garzoni | A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells[END_REF][START_REF] Kubica | A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages[END_REF]. During some persistent and recurrent S. aureus infections, such as osteomyelitis, implant infections and airway infections in cystic fibrosis patients, a SCV phenotype was observed. SCV was also observed from intracellular persistence assay within non-phagocytic host cells such as epithelial cells, endothelial cells and bone cells [START_REF] Von Eiff | A sitedirected Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly[END_REF][START_REF] Atalla | Antibody and cell-mediated immune responses to Staphylococcus aureus small colony variants and their parental strains associated with bovine mastitis[END_REF][START_REF] Tuchscherr | Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections[END_REF]). SCV appears to express high level of sigB and low level of agr, and seems to represent a general adaptation response [START_REF] Mitchell | A role for sigma factor B in the emergence of Staphylococcus aureus small-colony variants and elevated biofilm production resulting from an exposure to aminoglycosides[END_REF].

The presence of SCV may be an important mechanism for chronic infections. Thus, S. aureus SCVs have been particularly used as a model to study persistence [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF]Garzoni and Kelley 2009;Sendi and Proctor 2009;[START_REF] Mitchell | Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells[END_REF][START_REF] Tuchscherr | Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection[END_REF][START_REF] Kalinka | Staphylococcus aureus isolates from chronic osteomyelitis are characterized by high host cell invasion and intracellular adaptation, but still induce inflammation[END_REF][START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. As recalled earlier, the SCV phenotype is generally associated with the formation of tiny colonies on plates, increased biofilm capacity, antibiotic resistance and culture supernatants of SCVs generally show reduced cytotoxicity on cell cultures. Furthermore, live cell imaging shows that SCVs persist intracellularly mainly in late endosomal compartments without significant replication [START_REF] Schroder | Live cell imaging of phagosome maturation in Staphylococcus aureus infected human endothelial cells: small colony variants are able to survive in lysosomes[END_REF]. A number of "Omics" approaches have been carried out, which show that S. aureus SCVs have a reduced metabolic activity [START_REF] Seggewiss | Reporter metabolite analysis of transcriptional profiles of a Staphylococcus aureus strain with normal phenotype and its isogenic hemB mutant displaying the small-colony-variant phenotype[END_REF][START_REF] Von Eiff | Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype[END_REF][START_REF] Kriegeskorte | Small colony variants of Staphylococcus aureus reveal distinct protein profiles[END_REF]. Several advances studies have provided further support for the close linkage between metabolism and virulence synthesis [START_REF] Somerville | At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci[END_REF]. The reduced metabolic activity of SCV could explain the persistence.

Besides, recent works shed light on lipid pathways of S. aureus with its persistent infection.

The potential of further deciphering lipid pathways is broad since in addition of being major energy sources and important component of membranes, lipid related molecules released by bacteria are signal molecules sensed by other bacteria (as in quorum sensing) or sensed by host immune cells. Indeed, bacterial lipoproteins and lipopeptides are pathogen-associated molecular pattern (PAMP), which are major agonists for Toll-like receptor (TLR) signaling during innate immune response. In S. aureus, lipoproteins are triacylated and bind to TLR1 and 2. Grayczyk et al. found that a mutation in the lipoic acid synthetase gene (lipA) elicits a hyperinflammatory response of macrophages [START_REF] Grayczyk | A Lipoylated Metabolic Protein Released by Staphylococcus aureus Suppresses Macrophage Activation[END_REF]. Lipoic acid (LA) is a fatty acid related cofactor covalently linked to proteins such as the pyruvate dehydrogenase (PDH) complex yielding lipoyl-PDH [START_REF] Grayczyk | A Lipoylated Metabolic Protein Released by Staphylococcus aureus Suppresses Macrophage Activation[END_REF]. Wild type S. aureus, but not lipA mutant, releases nanomolar quantities of lipoyl-PDH in the culture supernatant, which is able to reduce TLR2-dependent activation of macrophages. It is likely that lipoyl-proteins antagonize lipoprotein activation of macrophage by a competition for TLR binding, inducing immune evasion, which ultimately promote persistence.

More recently, Chen et al. demonstrated that, in addition to its known nutrient acquisition function, the Geh secreted lipase is also a potent immunomodulator [START_REF] Chen | Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses[END_REF]. Indeed, Geh hydrolyzes environmental long chain FAs including S. aureus own lipoproteins released in the milieu. Thus, by hydrolyzing self-released lipoproteins, S. aureus Geh prevents their recognition by host immune cells and fulfills an elegant immune evasion function, which ultimately promotes persistence [START_REF] Chen | Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses[END_REF].

Chapter III: Adaptation of bacterial pathogens in the cystic fibrosis model

Patients with cystic fibrosis are highly susceptible to chronic colonization by various pathogens including S. aureus, which can cause recurrent and persistent infections. By now, S. aureus adaptation in the long-term cystic fibrosis infection is still poorly understood.

General aspects of bacterial adaptation

When a bacterium is moved from one environment to another, such as from soil to water, or from an external environment to a mammalian host, adaptation is an absolute necessity. The adaptation to a new environment is guided by the selective pressures found in the environment.

Host adaptation allows bacteria to switch from an intermittent colonization to a chronic colonization or infection [START_REF] Sokurenko | Pathoadaptive mutations: gene loss and variation in bacterial pathogens[END_REF]. Identification of the selective forces shaping this evolution can inspire new therapeutic for combating bacterial infections.

Furthermore, an understanding of how bacteria diversify as they adapt can illuminate the molecular mechanisms of pathogenesis and is crucial for tracking transmission networks.

In general, adaptation includes two aspects: phenotypic acclimation and genetic adaptation.

Phenotypic acclimation is based on alterations in gene regulatory mechanisms caused by environmental factors, such as toxins or nutrients, resulting in a phenotypic change without genotypic alterations. This initial adaptive mechanism is quickly brought into play. On the other hand, genetic adaptation is mediated by either acquisition of exogenous genetic material acquired from the environment, a phenomenon known as horizontal gene transfer (HGT), or changes to the pre-existing genetic content by mutation [START_REF] Nielsen | Detecting rare gene transfer events in bacterial populations[END_REF]. Phenotypic acclimation and genetic adaptation have been found in laboratory experiments with E. coli and in CF model for P. aeruginosa [START_REF] Cooper | The population genetics of ecological specialization in evolving Escherichia coli populations[END_REF][START_REF] Yang | Evolutionary dynamics of bacteria in a human host environment[END_REF].

By now, the study of genomic evolution holds enormous potential for research. By tracking genomic changes that facilitate a pathogen to persist within our bodies, we can identify its weaknesses and design therapeutic strategies against these infections. Additionally, even if within-patient bacterial evolution generates diversity, related sequential isolates recovered from the same patient are separated by relatively few mutations, enabling straightforward analysis for identifying the genomic basis of phenotypic changes [START_REF] Rolain | Realtime sequencing to decipher the molecular mechanism of resistance of a clinical pan-drugresistant Acinetobacter baumannii isolate from Marseille, France[END_REF].

Furthermore, we can use these genomic mutations to track the progression and transmission of pathogens throughout a population [START_REF] Lieberman | Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes[END_REF][START_REF] Yang | Evolutionary dynamics of bacteria in a human host environment[END_REF].

With the speed and cost of genomic sequencing becoming more and more available and affordable (a few days and less than $100 per genome, including sample preparation), the potential for rapid insight is enormous.

Evolution of S. aureus during human infection

The primary niche of S. aureus is the human anterior nares but the skin, and mucosa have also been identified as secondary niches [START_REF] Wertheim | The role of nasal carriage in Staphylococcus aureus infections[END_REF][START_REF] Sivaraman | Staphylococcus aureus nasal carriage and its contributing factors[END_REF]. It is reasonable to assume that S. aureus is highly adapted towards asymptomatic carriage in the human nasopharynx, which leads to its long term co-evolution within the human host (van Belkum, [START_REF] Van Belkum | Co-evolutionary aspects of human colonisation and infection by Staphylococcus aureus[END_REF]). In addition to carriage, S. aureus can cause opportunistic acute infections at different anatomical sites, or persistent chronic infections such as osteomyelitis, endocarditis or respiratory infections (especially of cystic fibrosis patients). In particular, CF patients are often persistently colonized with S. aureus and the majority of CF S. aureus infections are caused by single clones as determined by genotyping analysis of sequential isolates [START_REF] Branger | Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time[END_REF][START_REF] Kahl | Impact of Staphylococcus aureus on the pathogenesis of chronic cystic fibrosis lung disease[END_REF]. However, polyclonal infections may occur and periods of strain replacement have been observed over time [START_REF] Branger | Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time[END_REF][START_REF] Kahl | Impact of Staphylococcus aureus on the pathogenesis of chronic cystic fibrosis lung disease[END_REF]. Several evidences for genetic diversification during infection have been reported such as variation of the polymorphic region of the gene encoding staphylococcal protein A (SpA) [START_REF] Kahl | Variation of the polymorphic region X of the protein A gene during persistent airway infection of cystic fibrosis patients reflects two independent mechanisms of genetic change in Staphylococcus aureus[END_REF] and loss or acquisition of mobile genetic elements (MGE) [START_REF] Goerke | Increased frequency of genomic alterations in Staphylococcus aureus during chronic infection is in part due to phage mobilization[END_REF]).

Since the emergence of penicillin-resistant strains of S. aureus over a half century ago [START_REF] Kirby | Extraction of a Highly Potent Penicillin Inactivator from Penicillin Resistant Staphylococci[END_REF], the ability of S. aureus to readily acquire resistance to antibiotics has made it complicated to control infection. In particular, methicillin-resistant strains which are resistant to all β-lactam antibiotics are causing a global health problem (DeLeo and Chambers 2009).

β-lactam resistance is mediated by the mecA gene which encodes the protein PBP2A. mecA is found on a family of MGE known as staphylococcal cassette chromosome mec (SCCmec) [START_REF] Witte | Emergence and spread of antibiotic-resistant Gram-positive bacterial pathogens[END_REF]. In fact, acquisition of methicillin resistance during infection has not been observed frequently, probably due to the fact that most S. aureus infections are monoclonal. Since different clonal lineages have distinct restriction modification systems, this may eventually limit horizontal gene transfer (HGT) [START_REF] Waldron | Sau1: a novel lineage-specific type I restrictionmodification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages[END_REF]. However, several studies have reported the HGT of SCCmec during infection, which eventually leads to the emergence of new methicillin-resistant S. aureus (MRSA) strains [START_REF] Berglund | The origin of a methicillin-resistant Staphylococcus aureus isolate at a neonatal ward in Sweden-possible horizontal transfer of a staphylococcal cassette chromosome mec between methicillin-resistant Staphylococcus haemolyticus and Staphylococcus aureus[END_REF][START_REF] Bloemendaal | Methicillin resistance transfer from Staphylocccus epidermidis to methicillin-susceptible Staphylococcus aureus in a patient during antibiotic therapy[END_REF]. The first case report of a vancomcyin-resistant S. aureus (VRSA) which is also resistant to methicillin, was from a diabetic patient with a coinfection by S. aureus and Enterococcus faecalis [START_REF] Chang | Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene[END_REF]. MRSA strains have been shown to acquire vancomycin resistance via transfer of the Tn1546 transposon encoding the vancomcycin resistance gene (vanA) from a 57.9 Kb multi-resistance conjugative plasmid from the E. faecalis strain [START_REF] Weigel | Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus[END_REF].

Besides the acquisition of MGE encoding antibiotic resistance, several studies have confirmed, using comparative genomic approaches, that the acquisition of specific mutations were also responsible for the emergence of antibiotic resistance during infection. For example, Mwangi et al. investigated the development of multidrug resistance in S. aureus during a persistent infective endocarditis infection [START_REF] Mwangi | Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing[END_REF]). The whole genome sequencing (WGS) of isolates obtained during infection, revealed the acquisition of 35 mutations in 31 loci in a time-dependent manner. These events correlated with the emergence of resistance to antibiotics which were used to treat the infection. Mutations associated with the development of resistance to rifampicin, β-lactam antibiotics, and vancomycin have been identified [START_REF] Mwangi | Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing[END_REF]. Overall, this work demonstrated the tremendous power of using WGS of sequential isolates to explore the evolution of bacteria during infections. An understanding of the molecular mechanisms of the emergence of antibiotic resistance during infection, may help therapeutic decisions. Besides the gain of antibiotic resistance, S. aureus adapt infections via other mechanisms, such as alteration of agr activity and switch to SCV phenotype (see also chapter II above).

Agr. The accessory gene regulator system (agr) operon in S. aureus was discovered by [START_REF] Recsei | Regulation of exoprotein gene expression in Staphylococcus aureus by agar[END_REF][START_REF] Recsei | Regulation of exoprotein gene expression in Staphylococcus aureus by agar[END_REF], and agr has been shown to play a critical role in virulence in several animal models of infection [START_REF] Abdelnour | The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model[END_REF][START_REF] Cheung | Diminished virulence of a sar-/agr-mutant of Staphylococcus aureus in the rabbit model of endocarditis[END_REF]. Interestingly, some clinical isolates of S. aureus evolved a dysfunctional agr activity during nosocomial infections (HA-acquired) [START_REF] Fowler | Persistent bacteremia due to methicillin-resistant Staphylococcus aureus infection is associated with agr dysfunction and low-level in vitro resistance to thrombininduced platelet microbicidal protein[END_REF]). In fact, 15-60% of S. aureus isolates from nosocomial infections have been reported to have lost agr activity [START_REF] Shopsin | Prevalence of agr dysfunction among colonizing Staphylococcus aureus strains[END_REF][START_REF] Traber | agr function in clinical Staphylococcus aureus isolates[END_REF]. In contrast, agr-negative community-associated S. aureus strains (CA-acquired) have been rarely identified. Of note, Mwangi et al. identified a mutation in agr which was acquired during exposure to prolonged antibiotic treatment [START_REF] Mwangi | Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing[END_REF]. Paulander et al. showed that agr-negative strains have a fitness advantage over agr-positive strains in the presence of sublethal concentrations of some antibiotics [START_REF] Paulander | Antibiotic-mediated selection of quorum-sensing-negative Staphylococcus aureus[END_REF]. Accordingly, the exposure to antibiotics in the hospital setting may select for the loss of function of agr and could explain the frequent identification of agr mutants from nosocomial infections. Overall, these selected studies reveal a trend whereby antibiotic treatment of hospital infections enhances the selection of mutations which mediate antibiotic resistance. In parallel, as expected during chronic infections, a transition towards a reduced virulence, persistent infection type is observed in agrnegative strains.

SCVs.

A prototypical reduced virulence state has been recognized by the study of small colony variants (SCVs), which is often associated with chronic osteomyelitis and cystic fibrosis lung infections [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF][START_REF] Kahl | Impact of Staphylococcus aureus on the pathogenesis of chronic cystic fibrosis lung disease[END_REF]. By definition, SCVs form small colonies on nutrient agar due to their slow growth and reduced metabolism. SCVs also show reduced sensitivity to antimicrobial killing [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF]. Furthermore, it is well established that SCVs express less toxins, but more surface proteins which could favor long term colonization and persistence [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF]Tuchscherr, Heitmann et al. 2010).

In most of the cases, spontaneous SCV are unstable and SCV phenotype is readily reversible after growth in nutrient-rich conditions [START_REF] Tuchscherr | Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection[END_REF], suggesting that the emergence of SCV may be a phenotypic acclimation without genotypic alterations. Indeed, it was shown that S. aureus phenotype switching occurs via a constitutive mechanism which is linked to replication [START_REF] Edwards | Phenotype switching is a natural consequence of Staphylococcus aureus replication[END_REF]. A large-scale reversible intra-chromosomal rearrangement is a possible mechanism for emergence of SCV [START_REF] Cui | Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria[END_REF].

Besides, stable SCVs have also been identified such as SCV mutants auxotrophic for thymidine, menadione or hemin because of mutations arising in thyA, menD or hemB genes respectively [START_REF] Von Eiff | A sitedirected Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly[END_REF][START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF][START_REF] Chatterjee | In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of Staphylococcus aureus[END_REF][START_REF] Lannergard | Identification of the genetic basis for clinical menadione-auxotrophic small-colony variant isolates of Staphylococcus aureus[END_REF].

Evolution of S. aureus in individual with cystic fibrosis

The cystic fibrosis disease

Cystic fibrosis (CF) is a fatal autosomal recessive disease that affects many organs including the lungs, pancreas, and sweat glands. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, encoding a chloride ion channel [START_REF] Riordan | Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA[END_REF]. The mutation of the Cl -channel causes a dysfunction that can be more or less severe resulting in an accumulation of thick dehydrated mucus layer on all mucociliary surfaces in the body [START_REF] Cheung | Misfolding of the cystic fibrosis transmembrane conductance regulator and disease[END_REF]. If both CFTR genes are defective (homozygous mutation), there will be no functioning CFTR protein, and the patient will exhibit the symptoms of CF [START_REF] Derichs | Targeting a genetic defect: cystic fibrosis transmembrane conductance regulator modulators in cystic fibrosis[END_REF]. A single defective CFTR gene (heterozygous mutation) is generally asymptomatic [START_REF] Cheung | Misfolding of the cystic fibrosis transmembrane conductance regulator and disease[END_REF]. There are more than 1,000 possible changes that can occur in CFTR to cause cystic fibrosis, but approximately 70% of all patients with cystic fibrosis have the same mutation (∆F508), corresponding to a deletion of a single amino acid (a phenylalanine residue at site 508) [START_REF] Zielenski | Genotype and phenotype in cystic fibrosis[END_REF]. CF was not recognized as a separate disease until 1938. Before this time, it was grouped together with Celiac disease [START_REF] Andersen | Cystic fibrosis of the pancreas and its relation to celiac disease: a clinical and pathologic study[END_REF]. CF is the most common autosomal recessive lethal disease in the Caucasian population.

The incidence varies by race and ethnicity and is estimated to be 1:3,200 in whites, 1:15,000 in people of African descent, 1:35,000 in people of Asian descent, and 1:9,200 to 1:13,500 in Hispanics [START_REF] Hamosh | Comparison of the clinical manifestations of cystic fibrosis in black and white patients[END_REF][START_REF] Rohlfs | Cystic fibrosis carrier testing in an ethnically diverse US population[END_REF]. In France, one newborn out of 4,200 has cystic fibrosis while 2 million people carry a mutation of the CFTR gene [START_REF] Farrell | The prevalence of cystic fibrosis in the European Union[END_REF]. CF is usually symptomatic early in life, sometimes just after birth [START_REF] Paranjape | Cystic fibrosis[END_REF]. CF is a pleiotropic disease (Paranjape and Mogayzel 2014) (Figure 17). Major manifestations impact the respiratory system, pancreas and, more rarely, the intestine or liver.

The dysfunction of respiratory system represents the most common cause of mortality in CF patients [START_REF] Tesse | Association of beta-defensin-1 gene polymorphisms with Pseudomonas aeruginosa airway colonization in cystic fibrosis[END_REF].

In CF, the airways are filled with thick, sticky mucus, making breathing difficult for the patient (Figure 18). Besides, the thick mucus is also an ideal breeding ground for bacteria and fungi [START_REF] Paranjape | Cystic fibrosis[END_REF]. The manifestation of lung disease among CF patients is variable. Most of CF patients do not show symptoms while they are newborns but signs of lung disease, such as wheezing, coughing, and difficulty in breathing, can be seen in infants around six months of age. When the patients get older, coughing becomes a daily occurrence, usually accompanied by sputa. During later stages of the disease, it is common to find blood-streaked sputa.

Microbiology of cystic fibrosis

As previously mentioned above, the thick mucus in the CF airways provides a perfect home for various bacterial infections. Shortly after birth, initial infection by bacterial pathogens occurs. The impaired function of the mucociliary transport prevents the clearing of the lungs.

The antimicrobial peptides located in the airways become inefficient, allowing infections to take place. Because of these infections, the CF airway displays a prolonged primary inflammatory response.

CF patients followed in cystic fibrosis treatment centers have systematic collections of sputa for microbiological analysis (identification of pathogens and antibiotic susceptibility testing).

A wide range of microorganisms is observed associated with pulmonary infections in CF patients. We will briefly review below the most commonly found bacteria and fungi identified in the sputa of CF patient.

Infants and young children are most likely to be infected with S. aureus and Haemophilus influenzae. Among older children and adults, Pseudomonas aeruginosa is more likely to be found within the airways (Foundation 2017)(Figure 19).

H. influenzae is the most common microorganism found in the lower airways at one year of age [START_REF] Gibson | Pathophysiology and management of pulmonary infections in cystic fibrosis[END_REF]. H. influenzae strains infecting CF patients are non-typeable, which means that the vaccine against H. influenzae type b has no affect in preventing these infections. Currently, the role of this microorganism in the progressive pulmonary infection and inflammation in CF affected patients has not yet been clearly demonstrated, though it has been proven to be pathogenic in non-CF patients [START_REF] Gibson | Pathophysiology and management of pulmonary infections in cystic fibrosis[END_REF].

During childhood, S. aureus is the most common pathogen found in CF children. S. aureus is often the first organism cultured from the respiratory tract of young children with CF [START_REF] Armstrong | Lower airway inflammation in infants and young children with cystic fibrosis[END_REF]. S. aureus has gained a lot of attention due to the rise of methicillin resistant S. aureus (MRSA). Once MRSA has established itself within the CF airways, it is almost impossible to eradicate through antimicrobial therapy. Studies have investigated how MRSA affects CF patients in comparison to methicillin sensitive S. aureus (MSSA). CF patients infected with MRSA as the primary pathogen have lower lung function compared to those infected by MSSA. Meanwhile, patients with MRSA also show a higher rate of hospitalization and use of antibiotics [START_REF] Ren | Presence of methicillin resistant Staphylococcus aureus in respiratory cultures from cystic fibrosis patients is associated with lower lung function[END_REF]. P. aeruginosa is the most significant microorganism associated with CF. About 70% of CF patients will eventually be infected by this pathogen, resulting in poor clinical outcomes [START_REF] Foundation | 2017 Patient Registry Annual Data Report[END_REF]. The source of P. aeruginosa is currently unknown, but the fact that most CF patients harbor different genotypes of the bacteria suggests that the infection comes from the environment [START_REF] Gibson | Pathophysiology and management of pulmonary infections in cystic fibrosis[END_REF]. Typically, the infection is not initially chronic.

Instead, there is a period of recurrent, intermittent colonization by the pathogen until the patient eventually develops a chronic infection. This usually occurs during the late twenties or early thirties in patients. In a chronic infection, there is a continuous growth of P. aeruginosa that leads to an increased inflammation. This inflammatory response along with the damages done by the bacteria itself is a major cause of lung function alteration [START_REF] Folkesson | Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective[END_REF].

Burkholderia cepacia complex (BCC) can also plays a role in CF lung function alteration. It can be found in various environments, including agricultural crops and aqueous solutions such as detergent solutions and intravenous fluids commonly found in hospitals. CF patients had a shorter life expectancy when infected by BCC. Now that life expectancy is greater among CF patients, BCC can be found in about 10% of adult CF patients [START_REF] Gautam | Burkholderia cepacia complex: beyond pseudomonas and acinetobacter[END_REF]. A pulmonary infection with BCC can persist for months or even years. The infected CF patients may develop bacteremia and die within a few months [START_REF] Mahenthiralingam | Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology[END_REF].

However, patients with CF may be infected with BCC without a corresponding alteration in clinical status. Overall, in common with many other opportunistic pathogens, clinical outcomes may vary within CF patients infected with the same BCC strain [START_REF] Gautam | Burkholderia cepacia complex: beyond pseudomonas and acinetobacter[END_REF].

Stenotrophomonas maltophilia and Achromobacter spp. are more commonly observed than BCC among CF patients with advanced lung disease but are usually less virulent. There is no significant correlation between these infections and outcomes in CF patients [START_REF] Goss | Detecting Stenotrophomonas maltophilia does not reduce survival of patients with cystic fibrosis[END_REF][START_REF] Tan | Alcaligenes infection in cystic fibrosis[END_REF].

Finally, fungal colonization and infection of the CF airway in late disease progression is not surprising because these patients receive frequent broad-spectrum antibiotic therapy [START_REF] Bargon | Prophylactic antibiotic therapy is associated with an increased prevalence of Aspergillus colonization in adult cystic fibrosis patients[END_REF]. Candida spp. are the most frequent colonizers, isolated from almost 50 to 75% of patients with CF [START_REF] Burns | Microbiology of sputum from patients at cystic fibrosis centers in the United States[END_REF][START_REF] Bakare | Prevalence of Aspergillus fumigatus and other fungal species in the sputum of adult patients with cystic fibrosis[END_REF], but colonization usually does not lead to infection.

Evolution of S. aureus in CF patient

CF airways are an especially suitable model for an in vivo study on bacterial evolution and adaptation because within the airways, and especially in the mucus, bacteria are under enormous selective pressure. In this peculiar environment, S. aureus has to cope with the presence of polymorphonuclear neutrophils, alveolar macrophages, numerous antibiotic treatments, and competition with other bacteria [START_REF] Worlitzsch | Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients[END_REF][START_REF] Denamur | Evolution of mutation rates in bacteria[END_REF][START_REF] Cohen | Cystic fibrosis: a mucosal immunodeficiency syndrome[END_REF]. Despite these pressures, the same clone of S. aureus is able to persist in airway CF patients for months or even years [START_REF] Kahl | Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study[END_REF]. The fact that S. aureus can persist for a long time supports the notion that S. aureus is able to adapt to-and to survive in-the CF airways environment.

There are already several known mechanisms revealing adaptive processes of S. aureus in CF airways including: i) phage insertions and excisions (Goerke, Matias y Papenberg et al.

2004

), ii) changes in the protein A gene (spa) repeat region [START_REF] Kahl | Variation of the polymorphic region X of the protein A gene during persistent airway infection of cystic fibrosis patients reflects two independent mechanisms of genetic change in Staphylococcus aureus[END_REF] as well as, iii) the emergence of SCVs [START_REF] Kahl | Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis[END_REF]. In particular, the thymidinedependent SCVs (TD-SCVs) are observed after prior use of trimethoprim-sulfamethoxazole (SXT) treatment, and genome analyses have shown that random mutations in the highly conserved thymidylate synthase encoding gene thyA were involved in the formation of the TD-SCV phenotype [START_REF] Besier | The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates[END_REF][START_REF] Chatterjee | In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of Staphylococcus aureus[END_REF].

McAdam et al. investigated the within-host evolution of S. aureus during chronic infection of CF patient, via genome analysis of sequential isolates of S. aureus obtained from the lung of a patient over a 26-month period and carried out phenotypic analysis of the strains [START_REF] Mcadam | Adaptive evolution of Staphylococcus aureus during chronic endobronchial infection of a cystic fibrosis patient[END_REF]. The majority of the single nucleotide polymorphisms (SNPs) identified in the core genome were specific for each isolate. Furthermore, there was considerable variation in growth rate (leading to small and large colony variants), in hemolytic activity and antibiotic sensitivity. Genetic polymorphisms such as point mutations, insertions and deletions were identified in various loci, including in the genes encoding the global regulators CodY and SigB, which could affect growth and virulence phenotype. In addition, in a late isolate, a mutation in the fusA gene (encoding elongation factor G, EF-G) correlated with the resistance to fusidic acid [START_REF] Nagaev | Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus[END_REF]. Finally, consistent with the observations of Goerke et al., phage deletion and/or acquisition was also observed [START_REF] Goerke | Increased frequency of genomic alterations in Staphylococcus aureus during chronic infection is in part due to phage mobilization[END_REF].

Overall this study, performed on only a small number of isolates, indicated a profound impact on the evolution of S. aureus during chronic infection in CF patients.

A longitudinal study of more patients chronically infected with S. aureus is needed to further characterize the genomic alterations occurring during chronic infection. Besides genomic studies, proteomic, transcriptomic and metabolomic analyses need to be implemented to comprehensively examine the diversification of infecting populations and the relevance for the pathogenesis of chronic infections in CF.

Objectives

In this thesis work, we addressed the different adaptation strategies of S. aureus during chronic infections. Relapse of S. aureus infection after a well-conducted antibiotic treatment constitutes a major health issue. One hypothesis is that relapse may result from a lack of access of the antibiotic to the site of infection, especially to the intracellular niche.

1) The time course of S. aureus intracellular persistence is still poorly characterized. The first objective of my thesis was to better characterize the dynamics of intracellular persistence of the CA-MRSA strain S. aureus USA300-LAC in endothelial cells in vitro. For this, we quantitatively and qualitatively followed the behavior of intracellular bacteria, by using a combination of confocal and electron microscopy and live-cell imaging. Our results highlighted the heterogeneity of S. aureus behavior during cell infection and suggest that intracellular survival is a selective pressure that selects transient slow growing bacteria that are able to persist inside cell cytosol for several days and evade the cells by a mechanism yet to be determined (Chapter I).

2) S. aureus can dynamically switch phenotypes from a highly aggressive and cytotoxic phenotype to a metabolically inactive phenotype associated with decrease TCA activity. The second objective of my thesis was to evaluate the role of pentose phosphate pathway (PPP) during the intracellular persistence of S. aureus, by focusing on one of the key enzyme of this pathway, transketolase (TKT) (Chapter II). Reprogramming of the TCA cycle notably involves the glycolysis/gluconeogenesis and pentose phosphate pathway (PPP). The contribution of glycolysis/gluconeogenesis to intracellular persistence of S. aureus has been already studied. In contrast, the precise role of the PPP is unexplored. The activity of the PPP has been shown to be frequently increased in Gram-positive pathogens in response to environmental stresses and in infection models or in some hospital-acquired isolates.

3) S. aureus is the most common pathogen found in children with cystic fibrosis (CF) and S. aureus is often the first organism cultured from the respiratory tract of young children with CF. However, very few studies have addressed the adaptations S. aureus has undergone in this context. The third objective of my thesis was to evaluate the "within host adaptation" of S. aureus clinical isolates in this pathogenesis model. By studying serial isolates, we showed that during long-term lung infection, S. aureus adaptation occurs through genomic modifications that accumulate over time and lead to major metabolic modifications and protein expression changes. We also revealed that persistence of S. aureus was associated with convergent phenotypic evolution responsible for an increased ability to form biofilm as well as to survive within host cells (Chapter III). The Gram-positive human pathogen Staphylococcus aureus is a leading cause of severe bacterial infections. Recent studies have shown that various cell types could readily internalize S. aureus and infected cells have been proposed to serve as vehicle for the systemic dissemination of the pathogen. Here we focused on the intracellular behavior of the Community-Associated Methicillin-Resistant S. aureus strain USA300. Supporting earlier observations, we found that wild-type S. aureus strain USA300 persisted for longer period within endothelial cells than within macrophages and that a mutant displaying the small colony variant phenotype ( hemDBL) had increased intracellular persistence. Time-lapse microscopy revealed that initial persistence of wildtype bacteria in endothelial cells corresponded to distinct single cell events, ranging from

Part II Results

Intracellular

INTRODUCTION

The gram-positive bacterial pathogen, Staphylococcus aureus, can colonize multiple anatomic sites within its human host, including nares, respiratory tract and skin. Under defined conditions, S. aureus is also capable of systemic dissemination and can cause skin and soft tissue infections, pneumonia as well as sepsis, endocarditis, bones and joints infections [START_REF] Thomer | Pathogenesis of Staphylococcus aureus Bloodstream Infections[END_REF]. The rates of infections caused by staphylococci, both community-and hospital-acquired strains, are regularly escalating [START_REF] Laupland | Population-based epidemiology and microbiology of community-onset bloodstream infections[END_REF]. However, treatment of these infections is becoming increasingly difficult due to the prevalence of multidrug-resistant strains. In particular, S. aureus USA300, an epidemic community-associated methicillin-resistant strain (CA-MRSA), has now emerged as the predominant cause of methicillin-resistant S. aureus (MRSA) infections in the United States and is continuously spreading around the world (DeLeo et al., 2010).Initially classified as strict extracellular pathogen, S. aureus is now considered as a nonclassical facultative intracellular pathogen (Sendi and Proctor, 2009). Indeed, numerous cell types can ingest S. aureus and the bacterium is able to persist within these cells for quite variable periods of time [START_REF] Fraunholz | Intracellular Staphylococcus aureus: livein and let die[END_REF]Strobel et al., 2016).

Relapse of S. aureus infection after a well-conducted antibiotic treatment constitutes a major health issue. One hypothesis is that relapse may result from a lack of access of the antibiotic to the site of infection, especially to the intracellular niche. The molecular mechanisms underlying S. aureus virulence have been extensively studied and have been shown to be mediated by a multitude of virulence attributes, including adhesins and toxins that are regulated by complex networks of regulatory systems [START_REF] Somerville | At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci[END_REF]Felden et al., 2011;[START_REF] Ibarra | Global analysis of transcriptional regulators in Staphylococcus aureus[END_REF]Foster et al., 2014). Important contributions have been made over the past 10 years regarding the physiological and metabolic status of intracellular S. aureus (Sendi and Proctor, 2009;Tuchscherr et al., 2011;Proctor et al., 2014;Thammavongsa et al., 2015). However, the time course of S. aureus intracellular persistence is still poorly characterized.

The aim of the present work was to better characterize the dynamic stability of intracellular persistence of the CA-MRSA strain S. aureus USA300-LAC. We focused on endothelial cells that have been shown to readily internalize staphylococci in vitro (Strobel et al., 2016). We quantitatively and qualitatively followed the behavior of intracellular bacteria, by using a combination of confocal and electron microscopy and live-cell imaging. Our results highlight the heterogeneity of S. aureus behavior during cell infection and suggest that intracellular survival is a selective pressure that selects transient slow growing bacteria that are able to persist inside cell cytosol for several days and evade the cells by a mechanism yet to be determined.

MATERIALS AND METHODS

Strains and Culture Conditions

The epidemic clone S. aureus USA300-LAC (designated USA300-WT) was provided by the Biodefense and Emerging Infections Research Resources (BEI). The GFP-expressing strain (designated USA300-GFP) was generated by curing the p03 plasmid from USA300-WT and introducing the pCN57-GFP recombinant plasmid (obtained from the BEI) by electroporation (electroporator settings: 2,450 V, 100 , 25 µF, time constant = 2.3-2.5 ms). Growth curve were performed in BHI broth. Internalization rate and survival inside the EA.hy926 endothelial cell line were similar between the GFP strain and the parental strain.

Construction of the S. aureus hemDBL Triple Deletion Mutant

We simultaneously inactivated the three consecutive genes hemD hemB and hemL of the heme biosynthesis locus in wild-type S. aureus USA300 strain and substituted them by the kanamycine resistance gene nptII fused with pGro promoter. For this, we used the pMAD-temperature-sensitive shuttle vector system (Arnaud et al., 2004). Briefly, the recombinant plasmid pMAD-hemDBL was constructed by overlap PCR. First, the two regions (upstream 690 bp, downstream 458 bp) flanking hemB and the nptII gene (fused with pGro promoter 1,091 bp) were amplified by PCR using the following pairs of primers: i) Primers p1 and p2 amplified the region upstream (hemBUp) of the start codon of the hemB coding sequence (p1 5 ′ -CGGAATTCCCGGTTGAGTTAGGCA AAACAGTGAG-3 ′ and p2 5 ′ -TTTAGCTCGACTAATCCATAC AAGGTCCGTGCTGTTTGTTCTCC-3 ′ ); primers p3 and p4 amplified the region downstream (hemBDown) of the hemB stop codon (p3 5 ′ -CCTTCTTGACGAGTTCTTCTGAGCCTGGTG GTGTAAATAGTCCAG-3 ′ and P4 5 ′ -CGGGATCCCACCAGG AGAATCCGGCAATCC-3 ′ ); iii) primers p5 and p6 amplified the nptII gene (p5 5 ′ -TTGTATGGATTAGTCGAGCTAAA-3 ′ and p6 5 ′ -TCAGAAGAACTCGTCAAGAAGG-3 ′ ).

The region hemBUp-npt-hemBDown (2,239 bp) was then amplified by two-step overlap PCR. A first overlap PCR was realized to amplify the region hemBUp-npt, using the hemBUp and npt PCR fragments. The amplified product was cloned in pMiniT 2.0 (using the PCR Cloning Kit New England BioLabs). A second overlap PCR was then realized for the amplification of region hemBUp-npt-hemBDown, using hemBUp-npt and hemBDown PCR fragments. The resulting PCR product was cloned in pMiniT 2.0 (to yield recombinant plasmid pMiniT/hemBUp-npt-hemBDown). This recombinant plasmid was digested with BamHI and EcoRI (New England BioLabs) and the BamHI-EcoRI hemBUp-npt-hemBDown DNA fragment was finally subcloned into BamHI-EcoRI-digested pMAD. All PCR reactions were realized using Phusion High-Fidelity DNA Polymerase (ThermoScientific) and PCR products were purified using QIAquick PCR purification kit (Qiagen).

The pMAD-hemDBL plasmid was first introduced into E. coli DH5α and then transferred to S. aureus RN4220 prior to electroporation into S. aureus USA300. A standard two-step allelic exchange procedure was used (Arnaud et al., 2004) to create the chromosomal S. aureus USA300 hemDBL mutant. The hemDBL mutant strain was finally checked for loss of the corresponding wild-type genes by PCR sequencing (GATC Biotech) using specific primers (p7 5 ′ -CGGAATTCGTCGGAG GCAAAGGCTTATTTG-3 ′ and p8 5 ′ -CCGACTCTGAAACCA GTCATTACTTC-3 ′ ).

Cell Lines

To study S. aureus interaction with endothelial cells we used the EA.hy926 cell line (ATCC R CRL-2922 TM ), originally derived from human umbilical vein. EA.hy926 cells were grown in Dulbecco's modified Eagle high glucose medium (DMEM, Dominique Dutscher TM ) supplemented with 10% fetal bovine serum (FBS, Eurobio TM ). Culture media contained 1% penicillin/streptomycin and 1% amphotericin B. Antibiotics were removed from culture medium prior to infection.

The human monocytic cell line THP-1 (ATCC R TIB-202 TM ) was grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 5% FBS. Two days before infection, phorbol myristate acetate 200 ng mL -1 (PMA) was added to the culture medium to induce monocytes differentiation into macrophages.

All cells were incubated in a humidified 5% CO2 atmosphere at 37 • C.

In vitro Infection

Four days prior to infection cells were seeded in plastic 12-well plates in culture medium without antibiotic. At time of infection each well contained a monolayer of ∼5 × 10 5 cells. Overnight cultures of S. aureus in BHI were diluted in BHI broth to an optical density 600 nm (OD 600 ) of 0.05. Bacteria were grown at 37 • C until the culture reached the initial-middle log phase (OD 600 = 0.6), and then diluted in 1mL infection medium. Bacteria were added at a multiplicity of infection (MOI) of 1 and placed in a humidified 5% CO2 atmosphere at 37 • C for 1 h.

One hour after infection, each well was washed three times with 1 mL of phosphate buffer saline (PBS) containing 300 µg.mL -1 gentamicin, to remove extracellular bacteria. Cells were then incubated with 1 mL of cell culture medium containing 50 µg.mL -1 gentamicin. Infected cells were kept in a humidified 5% CO2 atmosphere at 37 • C. Importantly, gentamicin is an antibiotic with a high bactericidal effect on S. aureus USA300 (CMI = 2 µg.mL -1 ) and a very poor penetration inside eukaryotic cells.

At chosen times, the culture medium containing gentamicin was removed from infected cells and wells were washed three times with antibiotic free PBS. Cells were lysed with 1mL of distilled water during 15 minutes (min) and mechanically detached and serial dilutions were plated on Trypticase Soy Agar plates. Colony forming units were numerated after 24 h (h) at 37 • C.

Cytochalasin D Assay

We used Cytochalasin D, a fungal metabolite that binds to actin and reversibly modifies its polymerization, to inhibit S. aureus internalization into EA.hy926 endothelial cells. One hour before infection, cytochalasin D (Sigma) 50 µg.mL -1 was added to cell culture medium and maintained during the first hour of infection.

Re-Seeding of S. aureus EA.hy926 were infected as described above. At day 7 and 10 after infection, extracellular and intracellular bacterial load was assessed (Figure 2A). Cell monolayers were washed 3 times with PBS containing gentamicin 300 µg.mL -1 and 3 times with antibiotic free PBS. After 8 or 24 h of incubation in antibioticfree medium the supernatant was collected, centrifugated and the pellet was resuspended in PBS before being plated on TSA for bacterial count. Then, cells were lysed and plated on TSA for intracellular bacteria count. Cells that were infected for 24 h were washed after 8 h and then incubated 16 additional hours in antibiotic-free medium.

Microscopy

Immunofluorescence Microscopy

EA.hy926 cells were grown to confluence on 12-mm diameter glass coverslips coated with 5 µg per cm 2 of rat tail collagen type I. Immunofluorescence assays were performed 1 h post infection by USA300-GFP and the day of experiment. Cells were fixed in PFA 4% in PBS for 20 min and permeabilized for 5 min with PBS containing 0.1% Triton X-100. Cells were incubated with DAPI 1 µg.mL -1 and Alexa-633 conjugated phalloidin 1:50 in PBS containing 1% bovine serum albumin (BSA) for 1 h. After several washes, coverslips were mounted in mowiol. Image acquisition was performed on confocal microscope (Leica TM TCS SP5). Images were collected and processed using Image J software. Each image shown corresponds to the Z-projected maximal intensity signal for each fluorochrome.

Time Lapse Microscopy

EA.hy926 cells were grown to confluence on Ibidi TM µslide 8wells and infected with USA300-GFP at a MOI of 1 as described above. Cells were incubated in cell culture medium containing 2.5 µg.mL -1 propidium iodide and 50 µg.mL -1 gentamicin. Image acquisition of infected cells was performed with an Apotome CO2 microscope (Zeiss TM ). One image was acquired every 20 min during 64 h. Images were collected and films were processed using Image J software. Each image corresponds to the Zprojected maximal intensity signal for GFP and to the projected median intensity for bright light.

The mutant strain USA300 hemDBL was imaged using the IncuCyte TM technology. EA.hy926 cells were grown to confluence in ImageLock 96-well plates (Essen BioScience Inc. Ann Arbor, MI, USA) and infected with the mutant strain USA300 hemDBL-GFP (at a MOI of 1). Cytotox Red reagent (Essen Bioscience) was added to the medium (final concentration of 125 nM). Plates were incubated and monitored at 37 • C for 6 days in an IncuCyte TM (Essen BioScience), an incubator equipped with a fully automated phase contrast microscope. Images were taken every 20 min. The microscope had a 20 X objective. Time-lapse video were generated by using Image J software.

Transmission Electron Microscopy

EA.hy926 were grown and infected with USA300-WT as previously described. One hour after infection and at day 3 and 7 infected cells were fixed with glutaraldehyde 3% in phosphate buffer during 1 h. Cells were then post-fixed with osmium tetroxide to stabilize lipids and enhance contrast. Sample were then dehydrated with sequential ethanol baths (from 25 to 100%) and embedded in Epon 812 resin with a 48 h polymerization time at 60 • C. Embedded samples were sliced in 90 nm thick pieces with an ultramicrotome and laid down on a copper observation grid. Image acquisitions were performed with a JEOL 1011 transmission electron microscope (tungsten filament) at the Institut Cochin (Paris, France).

Statistics

In vitro experiments were at least repeated twice in triplicates. Data were analyzed using GraphPad Prism software. Statistical significance was assessed using Student t-test. In figures all results correspond to mean ± SEM.

RESULTS

Persistence of Intracellular S. aureus In vitro

Whereas several earlier studies have described the capacity of S. aureus to persist inside human cells, including non-professional phagocytes such as endothelial cells, other studies clearly showed the extreme propensity of this pathogen to rapidly kill infected cells [START_REF] Loffler | Staphylococcus aureus persistence in non-professional phagocytes[END_REF]Jubrail et al., 2016;Strobel et al., 2016). These apparent discrepancies are likely due the heterogeneity of protocols used to infect cells. Moreover, the vast majority of the studies related to S. aureus intracellular persistence have focused on early time points (generally up to 24 h post infection).

In this work, our aim was to follow the behavior of S. aureus USA300-LAC (referred as to USA300 in this work) when internalized into endothelial cells and to understand how this fast growing extracellular pathogen may survive in the cytosol of these cells. We decided to follow the outcome of intracellular S. aureus internalized during the first hours of the infection. Therefore, we adapted a gentamicin protection assay as follows: (i) after 1 h, infected cells were first washed three times with a solution containing 300 µg.mL -1 gentamicin (to eliminate all extracellular bacteria); and (ii) the experiments were pursued in the constant presence of 50 µg.mL -1 gentamicin in the culture media, to ensure elimination of extracellular staphylococci continuously escaping from lysed infected cells. The antibiotic gentamicin cannot penetrate into eukaryotic cells and has been shown to be poorly effective against intracellular S. aureus [START_REF] Imbuluzqueta | Cellular pharmacokinetics and intracellular activity against Listeria monocytogenes and Staphylococcus aureus of chemically modified and nanoencapsulated gentamicin[END_REF][START_REF] Mohamed | Intracellular proliferation of S. aureus in osteoblasts and effects of rifampicin and gentamicin on S. aureus intracellular proliferation and survival[END_REF].

We first compared the ability of S. aureus USA300 to survive inside the Ea.Hy296 endothelial cell line and THP-1 monocytic cell line, for 10 days following infection at a multiplicity of infection (MOI) of 1 (Figure 1A). The number of viable intracellular bacteria (i.e., able to form colonies on solid medium) recovered in human endothelial cell lines remained stable during the first 24 h after the infection. Then, bacterial intracellular persistence started to progressively decrease from day 2 but was still recorded up to 10 days after infection. No viable (i.e., culturable) bacteria were recovered after 14 days. In contrast, THP-1 macrophages had almost completely eliminated bacteria at day 7.

To confirm that surviving S. aureus were indeed a consequence of bacterial internalization inside endothelial cells in our assay, we inhibited bacterial uptake at day 0, using cytochalasin D (Figure 1B). A severe 1,000-fold decrease in bacterial uptake was observed in EA.hy926 cells pretreated with cytochalasin D compared to that of untreated cells. As a result, no bacterium was recovered from treated cells 2 days after infection. We next focused on the characterization of S. aureus intracellular persistence, using endothelial EA.hy926 cells as model.

Interaction between S. aureus and Infected Host Cells

We next performed confocal and transmission electron microscopy (TEM) assays (Figure 2). Confocal microscopy analysis (Figure 2A) indicated that intracellular S. aureus actively replicate inside endothelial cells during the first 3 days of infection. After day 3, a progressive reduction in the number of GFP-expressing intracellular bacteria was observed concomitant with an increase in the number of bacteria only stained by DAPI that are likely corresponding to dead bacteria. To ascertain the fate of bacteria and that of their intracellular location inside endothelial cell we used TEM (Figure 2B). Image analysis confirmed the intracellular location of S. aureus. At day 0, a majority of intracellular bacteria were alive, according to their high cytoplasmic density and envelope integrity. After 3 days of infection, most of the intracellular bacteria appeared to be seriously damaged; only 23% of intracellular bacteria were still morphologically intact. As previously described, less than 5% of intracellular bacteria were surrounded by a visible vacuolar membrane (Grosz et al., 2014). After 7 days, only few intracellular bacteria were morphologically living. Confocal microscopy is consistent with this observation since dead bacteria were stained with DAPI but no longer express GFP.

S. aureus USA300 Uses Multiple Intracellular Survival Strategies

To follow the fate of single S. aureus USA300 after entry into endothelial cells, we next used time lapse-microscopy on EA.hy926 cells infected by USA300-GFP. As above, gentamicin was present throughout the experiment to prevent extracellular bacterial growth and new infection. One image was captured every 20 min during 64 h, starting 1 h after infection (Videos S1; Supplementary Materials). At the single cell level, three distinct types of behaviors were observed: (i) in the majority of cases, S. aureus actively proliferated during the first 48 h, leading to host cell death and to bacterial release in the extracellular medium (as illustrated in the screen captures of Figure 3A, Videos S2, S3); (ii) in some cases, however, S. aureus remained inside the host cell without replicating throughout the observation period Figure 3B; or (iii) after a transient phase of bacterial multiplication, host cell could control and partly eliminate intracellular S. aureus (Figure 3C, Video S4). Interestingly, at day 7 we did not detect intracellular replication anymore (Video S6).

Altogether these results indicate that the infection of endothelial cells by S. aureus is not as straightforward as suggested by colony forming units counts (CFU). In some circumstances S. aureus can proliferate in the cytosol of endothelial cells, leading to cell lysis, while endothelial cells may also limit bacteria proliferation leading to the emergence of persistent bacteria.

Inactivation of the Heme Biosynthetic Pathway Improves Intracellular Persistence

S. aureus small colony variants (SCVs) constitute a slow growing subpopulation which can be recovered from patients who present persisting or relapsing infections, as it may be observed in cystic fibrosis [START_REF] Goerke | Adaptation of Staphylococcus aureus to the cystic fibrosis lung[END_REF]. SCV phenotype (colony that exhibits significantly reduced size) can be either transient (i.e., reversible), mostly due to transcription regulation mechanisms, or permanent due to a variety of genetic defects, such as those leading to an impaired electron transport chain (due to mutations inhibiting heme or menadione biosynthesis) and/or resulting in thymidine auxotrophy [START_REF] Loffler | Staphylococcus aureus persistence in non-professional phagocytes[END_REF]. Laboratory engineered mutants that mimic the SCV phenotype have been largely used to study S. aureus persistence and notably hemB mutants (inactivated heme biosynthetic pathway). For example, early work of von [START_REF] Von Eiff | A sitedirected Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly[END_REF] have shown that a hemB mutant of S. aureus (NCTC 8325 strain) was able to persist longer than wild-type strain (over a 48 h period) within cultured bovine aortic endothelial cells, likely due to reduced cytotoxicity. More recently, one of the rare studies that addressed long term S. aureus persistence (Tuchscherr et al., 2011), reported that staphylococcal strains could survive within cultured host cells for up to 4 weeks and suggested that SCV formation was directly linked to chronic infection.

These observations prompted us to count the proportion of SCVs in wild type S. aureus that infected endothelial cells (Figure 4A). Ten days after infection, only 30-40% of CFUs obtained after cell lysis showed a SCV phenotype suggesting that surviving internalized S. aureus only transiently slowdown their metabolism and growth. Since SCVs are known to better survive in human cells we decided to follow the fate of internalized SCVs. We thus engineered a SCV mutant by inactivating the heme utilization locus ( hemDBL) in wild-type S. aureus USA300 and USA300-GFP. Then we infected endothelial cells and recovered viable CFUs in infected cells (Figure 4B). While the CFUs count in Figure 4B were similar between wild type and hemDBL mutant strains up to day 3, the hemDBL mutant persisted more than the wild type parental strain at days 7 and 10. We then imaged the growth of our SCV mutant over a 7 day-course, using time lapse-microscopy (Figure 4C, Video S5). Neither proliferation inside endothelial cells nor cell detachment was noticed during the time of infection. This is consistent with previous reports, which indicate that SCVs induce less cell death after invasion of human cells (Tuchscherr et al., 2010(Tuchscherr et al., , 2011)). Interestingly this result recapitulated what was observed with wild type strain USA300-GFP between day 5 and day 10 of endothelial infection, suggesting that surviving wild type USA300 between day 5 and day 10 may be potent persister SCVs.

S. aureus Can Escape from Infected Cells

We next tested whether S. aureus USA300 could escape from infected EA.hy926 cells and re-seed the extracellular medium after prolonged intracellular persistence, upon removal of antibiotic treatment. For this, we set up the infectious scheme depicted in Figure 5A. Briefly, 7 and 10 days after infection, we removed gentamicin from the culture media (see Materials and Methods). Cells were then washed several times and incubated 8 h in antibiotic-free medium and supernatant were tested for the presence of S. aureus. No bacteria were detected in the cell culture media 8 h after removal of antibiotics, indicated that no detectable S. aureus survived in the extracellular media nor were released by cells early after removal of gentamicin. Cells were then incubated for 16 additional hours before counting extracellular and intracellular bacteria. At days 7 and 10, the number of S. aureus was dramatically increased in the supernatants of 5 out of 9 wells, and 7 out of 12 wells, respectively (Figures 5B,C). Bacterial multiplication in cell culture supernatant was associated with an increase in the number of intracellular CFUs of the corresponding well. Considering that we never observed an increase in the intracellular CFU count in the presence of gentamicin at day 7 and 10, these data tend to suggest that S. aureus USA300 that have been internalized for 7-10 days in endothelial cells are still able to evade the cells. However, as mentioned above we did not detect proliferation of intracellular wild type bacteria by time-lapse microscopy at day 7 (Video S6).

To confirm that single bacteria did not escape from the intracellular compartment by cell lysis (due to overwhelming intracellular proliferation as observed at day 1-3), we also tested the relapse of the hemDBL mutant 10 days after infection (Figures 5D,E). As for the wild type parental strain, S aureus hemDBL mutant bacteria proliferated in the supernatant to reach ≃10 6 CFU in 5 wells out of 6. This proliferation was also associated with an increase in the intracellular CFU count.

Altogether, these data are compatible with the idea that single S. aureus USA300 bacteria are: (i) able evade endothelial cells after 10 days of intracellular persistence (either actively or passively, after cell extrusion or cell death), (ii) proliferate in the extracellular medium, and (iii) eventually infect neighboring cells, in the absence of antibiotics.

DISCUSSION

Having confirmed the increased intracellular persistence of S. aureus USA300 in endothelial cells as compared to macrophages, we show here that the initial persistence of wild-type S. aureus in endothelial cells corresponds to distinct simultaneous situations at the single cell level. Indeed, time-lapse microscopy revealed that, although in most cases, rapid bacterial proliferation leading to cell lysis was observed; in some cells, a transient bacterial multiplication ultimately controlled by the host, as well as a prolonged bacterial persistence without apparent morphological cellular damage, could also be observed. This dynamic view of S. aureus intracellular persistence provides a complementary and necessary assessment of the intracellular lifestyle of the bacterium that should contribute to better understand the in vivo situation than any classical snapshot analysis.

Persistence in Endothelial Cells Corresponds to Different Single Cell Events

Two main outcomes of host cell invasion by S. aureus are generally described: (i) rapid host cell lysis, triggered by the secretion of toxins and other pro-inflammatory factors, which induce strong inflammatory and cytotoxic effects; (ii) persistence within morphologically-intact host cells for extended periods of time, where secretion of virulence factors is either downregulated or not expressed (Proctor et al., 2014). Of note, these persisting bacteria generally correspond to so-called "small colony variants" (SCV). The association between S. aureus SCVs and persistent infection was first reported by Proctor and coworkers in 1995 [START_REF] Proctor | Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus[END_REF] and recent studies have shown that the impaired growth of S. aureus SCVs was due to decreased metabolic activity (Amato et al., 2014). Since a number of other pathogenic bacteria species have been shown to persist within their host as SCVs (Kahl et al., 2016), it may be considered as a major survival strategy in chronic bacterial infections. Summarizing the prolific literature addressing S. aureus intracellular persistence is quite complicated and may even be misleading. Indeed the comparison of results obtained in one experimental setup with those obtained in another one is quite often impossible. Of interest, recent studies have revealed that the general stress transcription Sigma B (SigB) was a major player of the dynamic switch between full virulence and long-term persistent phenotypes in wild-type strains. SigB by down-regulating the quorum sensing agr system, silences toxins production and improves persistence within the host cell (Tuchscherr et al., 2015).

Here, we thought to use video microscopy to follow the behavior of individual S. aureus USA300-GFP infected endothelial cells over a seven day-period. This technique has been very recently used by Jubrail et al. (2016) to visualize the fate of GFP-labeled S. aureus strain Newman in infected THP-1 macrophages, over a 72 h-period. This experiment showed that intracellular bacteria re-emerged from macrophage from 24 to 72 h. This leads to a constant reseeding of the extracellular compartment. Our experiment in endothelial cells revealed that, whereas most infected cells were unable to restrict bacterial growth and were lysed within the first 48 h, some cells were able to resolve the infection. In these cells, morphologically intact bacteria were observed at least for 7 days.

Thus, upon entry of S. aureus into endothelial cells, simultaneous single cellular fates coexist which could not have been anticipated from classical confocal microscopy analyses or quantification of viable intracellular bacteria at fixed time points.

Persistence and Exit of S. aureus from Human Cells

Our results and those of others indicate that, within the first hours after internalization, bacteria tend to actively multiply inside human cells (and ultimately lyse them) whereas infected cells attempt to restrict their proliferation. This selective pressure on S. aureus may favor the emergence of transient persisters which are able to survive into host cells for several days, without multiplication, before re-emerging in the extracellular compartment where they can rapidly proliferate and infect neighboring cells.

Of note, after several days of intracellular persistence, S. aureus is no longer capable of proliferation. Thus, the evasion of SCVs in the supernatant of infected cells (Figure 5D) in the absence of antibiotic, suggests that single non-proliferative bacteria are able to evade cells by an unknown, possibly active, process yet to be determined. 17, 54-58. doi: 10.1016/j.tim.2008.11.004 Somerville, G. A., and Proctor, R. A. (2009). At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci. Microbiol. Mol. Biol. Rev. 73, 233-248. doi: 10.1128/MMBR.00005-09 Strobel, M., Pfortner, H., Tuchscherr, L., Volker, U., Schmidt, F., Kramko, N., et al. (2016). Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain. Clin. Microbiol. Infect. 22, 799-809. doi: 10.1016/j.cmi.2016.0 6.020 Thammavongsa, V., Kim, H. K., Missiakas, D., and Schneewind, O. (2015).

Staphylococcal manipulation of host immune responses. Nat. Rev. Microbiol. [START_REF] Tuchscherr | Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections[END_REF]. Sigma Factor SigB Is crucial to mediate Staphylococcus aureus adaptation during chronic infections. PLoS Pathog. 11:e1004870. doi: 10.1371/journal.ppat.1004870 Tuchscherr, L., Heitmann, V., [START_REF] Tuchscherr | Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence[END_REF]. Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence. J. Infect. Dis. 202, 1031-1040. doi: 10.1086/656047 Tuchscherr, L., Medina, E., Hussain, M., Volker, W., Heitmann, V., Niemann, S., et al. (2011). Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. EMBO Mol. Med. 3, 129-141. doi: 10.1002/emmm.201000115 von Eiff, C., Heilmann, C., Proctor, R. A., Woltz, C., Peters, G., and[START_REF] Von Eiff | A sitedirected Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly[END_REF]. A site-directed Staphylococcus aureus hemB mutant is a smallcolony variant which persists intracellularly. J. Bacteriol. 179, 4706-4712. doi: 10.1128Bacteriol. 179, 4706-4712. doi: 10. /jb.179.15.4706-4712.1997 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Rollin, Tan, Tros, Dupuis, Nassif, Charbit and Coureuil. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Paper 1 Intracellular survival of Staphylococcus aureus in endothelial cells: a matter of growth or persistence

Contributions by Xin TAN

Methods

-Generation of Erythromycin-sensitive USA300 wild-type strain and the GFP-expressing USA300 wild type strain.

-Construction of USA300 ∆hemDBL strain and the GFP-expressing USA300 ∆hemDBL strain.

-In vitro infection assay for USA300 ∆hemDBL strain and wild-type strain in endothelial cells.

-Time lapse microscopy for USA300 ∆hemDBL strain.

Results

Figures 3 and Figure 4 Brief Abstract

Inactivation of the Staphylococcus aureus transketolase activity leads to impaired early intracellular proliferation and kidneys colonization, associated with reduced Sigma B and RpiRc transcription. The data suggest that TKT -or a functional PPP-strongly influences S.

aureus intracellular proliferation.

Abstract

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans.

The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. Here, we focused on one key PPP enzyme, transketolase. We showed that inactivation of the unique gene encoding transketolase activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared to wild-type strain in early intracellular proliferation and in the ability to colonise kidneys. Transcriptional activity of the two master regulators Sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription, suggests that TKT -or a functional PPP-strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.

Background

Staphylococcus aureus is a leading cause of severe skin and soft tissue infections, pneumonia, endocarditis and bone and joint infections [1,2]. S. aureus invades and survives within eukaryotic host cells [3][4][5][6][7][8][9]. This is likely to play an important role in chronicity and treatment failures [10][11][12]. To evade host defences and silently persist within human cells [13,14], S. aureus can dynamically switch phenotypes from a highly aggressive and cytotoxic phenotype to a metabolically inactive phenotype associated with decrease TCA activity [15][16][17][18]. Reprogramming of the TCA cycle notably involves the glycolysis/gluconeogenesis and pentose phosphate pathway (PPP) [19].

The contribution of glycolysis/gluconeogenesis to intracellular persistence of S.

aureus has been recently studied [20][21][22][23]. In contrast, the precise role of the PPP is unexplored. The PPP is important for producing nucleotide precursors for DNA synthesis and repair as well as precursors for aromatic amino acids or peptidoglycan synthesis. In addition, the oxidative branch of the PPP contributes to bacterial tolerance to oxidative stress by generating the redox molecule NADPH/H + , which is the substrate for other reducing agents such as glutathione. The activity of the PPP has been shown to be frequently increased in Gram-positive pathogens in response to environmental stresses and in infection models [24][25][26] or in some hospital-acquired isolates [27]. While it has been suggested that the transcriptional regulator RpiRc might control the PPP [28][29][30], no gene regulation has been yet associated with any change in its activity in S. aureus.

Here, we study the role of PPP during the intracellular persistence of S. aureus by focusing on one of the key enzyme of this pathway, transketolase (TKT). tkt genes are found in a wide range of organisms including bacteria, plants and mammals, underlining the ubiquitous role of this enzyme in biology. We show here that tkt inactivation in S. aureus USA300 leads to deregulation of whole cell metabolism. This correlates with impaired proliferation of intracellular staphylococci in vitro and kidney colonisation defect in infected mice.

Unexpectedly, we found that the tkt defective mutant was unable to regulate the two master regulators of virulence RpirC and SigB, leading to a dramatic increase of RNAIII during intracellular invasion of human cells. Altogether our data suggest a major role of the PPP in the control of S. aureus virulence.

Methods

Strains, Cells, Culture conditions and Infection.

S. aureus USA300-LAC (designated USA300-WT) was provided by the Biodefense and

Emerging Infections Research Resources (BEI). The GFP-expressing strain, the tkt mutant strain and the complemented strain were constructed and cultured as described in supplementary methods. Strains and plasmids used are summarized in supplementary methods.

S. aureus were grown in brain heart infusion (BHI) or chemically defined medium (CDM)

supplemented with ribose or fructose or glycerol when it was required [31].

EA.hy926 endothelial cells (ATCC CRL-2922) were grown in DMEM 10 % FBS (5 % CO2 at 37 °C ). When indicated, cells were incubated with no glucose DMEM supplemented with glucose, ribose or fructose at a concentration of 25mM. EA.hy926 expressing nuclear mKate2 were transduced with the IncuCyte ® NucLight Red Lentivirus.

Cells were infected at a multiplicity of infection (MOI) of 1 for 1 h and washed three times with 100 µL of phosphate buffer saline (PBS) containing 300 µg/ml gentamicin to remove extracellular bacteria. Cells were then incubated in PBS-gentamicin (50µg/ml). Gentamicin is an antibiotic with a high bactericidal effect on S. aureus USA300 (CMI=2 µg/ml) and a very poor penetration inside eukaryotic cells. Its use was necessary to abolish extracellular proliferation.

LAMP-1 colocalization assay in EA.hy926 infected cells is described in the supplementary methods.

Time lapse microscopy

EA.hy926 cells expressing nuclear mKate2 were seeded in ImageLock 96-well plates (Essen BioScience Inc). Cells were infected with WT and tkt mutant of the GFP-expressing USA300 strain. Plates were incubated and monitored at 5% CO2 and 37°C for 24 hours in the fully automated microscope Incucyte ® S3 (Essen BioScience). The detailed analysis is further described in the supplementary methods.

Transcriptional analyses

The bacteria were recovered from lysed cells or planktonic overnight culture. Nucleic acids were released by resuspending bacteria in TE buffer containing lysostaphin. RNAs recovery, reverse transcription and PCR were performed as described in the supplementary methods.

We used the "The Relative Standard Curve Method" for analyzing the qRT-PCR data.

Metabolomic analyses

Metabolite profiling of S. aureus isolates grown to stationary phase in BHI Broth was performed by liquid chromatography-mass spectrometry (LC-MS) as described in the supplementary methods [9,32,33].

Transketolase activity assay

TKT activity was analysed on bacteria grown to stationary phase in BHI as described in the supplementary methods [34]. TKT activity was expressed as units per microgram of total protein. One unit of enzyme was defined as the amount of enzyme that oxidized 1 µmol of NADH per minute.

In vivo infection

Mice were infected intravenously (iv) in the tail vein as described in the supplementary methods. For bacterial burden determination, mice were euthanized after 4, 24, 48 and 72 hours. Sequential dilutions of homogenized preparations from spleen and kidney were spread onto BHI agar plates.

Statistics

Results

Transketolase inactivation in S. aureus USA300 S. aureus genomes possess a unique transketolase-encoding gene, designated tkt, which is highly conserved within species. Transketolase (TKT) is an enzyme of the nonoxidative branch of the PPP involved in two main reversible enzymatic reactions: i) Fructose-6-P + Glyceraldehyde-3-P <-> Erythrose-4-P + Xylulose-5-P; and ii) Sedoheptulose-7-P + Glyceraldehyde-3-P <-> Ribose-5-P + Xylulose-5-P (Supplementary Figure S2A). Several TKT structures have been solved [35] and allow the modelisation of the S. aureus TKT (Supplementary Figure S1B). The protein of S. aureus is 662 amino acid long and harbours three domains (Supplementary Figure S1A). Of note, although ubiquitously expressed in Eukaryotes and Bacteria, S. aureus and Homo sapiens TKT proteins share only 22.4% amino acid identity (Supplementary Figure S1C). We constructed a chromosomal deletion of the tkt gene in S. aureus strain USA300 (∆tkt strain), and generated a complemented strain (designated Cp-tkt) by expressing the wild-type tkt allele preceded by its own promoter in the ∆tkt strain. Gene inactivation was confirmed by quantification of the transketolase activity (Supplementary Figure S2).

The ∆tkt mutant showed only a slight growth decrease compared to the WT strain in complete BHI Broth or in Dulbecco's Modified Eagle Medium (DMEM) (Figure 1A). In contrast, the ∆tkt mutant strain was unable to grow in chemically defined medium [31] supplemented with ribose (which can be converted to Ribose-5-phospate by ribokinase) (Supplementary Figure S2C). In all assays, functional complementation was observed in the Cp-tkt strain.

We next performed a whole cell metabolomic analysis on WT and ∆tkt strains grown for 24h in BHI Broth (i.e., when identical numbers of cells were recorded) to evaluate the global impact of tkt deletion on bacterial metabolism (Figure 1B and Supplementary Figure S3 and Table S1). Remarkably, we observed a massive accumulation of ribose-5-phosphate (R5P; 56-fold increase) concomitant with a decrease in ribose content (4-fold decrease) in the ∆tkt mutant compared to WT. This accumulation was associated with an increased amount of metabolites derived from Ribose-5P, such as inosine-5-monophosphate (IMP), xanthosine-5-monophosphate (XMP) and hypoxanthine. We also observed a 2.5-fold decrease in D-sedo-heptulose-7P amount in the ∆tkt mutant compared to WT accompanied by a significant decrease in tyrosine, histidine and tryptophan amounts (5, 10 and 12 fold respectively), three aromatic amino-acids produced from the PPP. The glutamate pathway seemed to be significantly more active in the ∆tkt strain compared to WT as the amounts of glutamate, proline and ornithine were all increased. The higher amounts of glutamate, possibly fuelling the TCA cycle at the level of α-ketoglutarate, could be responsible for the increased expression of some TCA cycle metabolites (i.e., fumarate, malate, succinate and α-ketoglutarate) in the ∆tkt strain. Of note, ATP content was not altered in ∆tkt strain.

These results demonstrate that deregulation of the PPP by tkt inactivation has direct effects on the whole cell metabolism, including aromatic amino acid production, TCA activity and pathways involved in adenine, guanine or peptidoglycan synthesis.

Tkt is essential for intracellular proliferation of S. aureus

Intracellular staphylococci exist as two different populations: bacteria that actively proliferate during the first 24 hrs after invasion, leading to their release in extracellular milieu as a consequence of host cell death, and bacteria that do not replicate. These latter staphylococci can persist within host-cell cytoplasm for several days [3][4][5][6][7][8][9]36]. Here we addressed the role of TKT in these two phenotypes in EA.hy926 human endothelial cells by using: i) time lapse-microscopy, to evaluate early intracellular proliferation of S. aureus and ii) colony forming units counting, to monitor the survival kinetics of internalized bacteria over a ten days period.

Time-lapse video microscopy. EA.hy926 cells expressing mKate2 nuclear fluorescent protein were infected by GFP-expressing bacteria. Gentamicin was used throughout the experiment to eliminate extracellular bacteria and prevent re-infection (see Methods), allowing us to solely focus on the fate of intracellular persistent bacteria. Cell infection was monitored using an IncuCyte ® S3 imaging system and images were captured every 30 minutes. Bacterial entry into endothelial cells was similar for WT and ∆tkt strains, showing 0.3~0.5 GFP expressing particles recorded per cell (Figure 2A-B). Active proliferation of WT S. aureus inside EA.hy926 cells was confirmed after 24 hours of infection (Figure 2A-B and Movie 1) with an average number of GFP expressing particles rising from 0.5 to 2 per cells.

At the single cell level, WT S. aureus showed active intracellular multiplication, ultimately leading to cell death and bacterial release in the extracellular medium (Figure 2A-B and Movie 1). Thanks to the permanent presence of gentamicin in the medium bacterial release did not lead to proliferation in the extracellular medium. In contrast, the ∆tkt mutant strain showed limited proliferation during the first 24 hours (Figure 2A-B and Movie 2). Finally, using LAMP-1 colocalization assay (Lysosome-associated membrane protein 1), we confirmed that the intracellular ∆tkt strain growth defect was not due to a decreased phagosomal escape (Supplementary Figure S4).

Long-term intracellular survival. Using the same infection conditions as for time-lapse experiment, we monitored survival of internalised Δtkt mutant compared to that of the WT strain at three, seven or ten days after infection by CFU counting. The Δtkt strain behaved likes the WT strain throughout the infection period, ultimately leading to its complete elimination after 10 days (Figure 2C).

Thus, the role of TKT during S. aureus host cell infection seems to be essential for bacteria proliferation and restricted to early time points.

Tkt inactivation is associated with an altered rpiRc and sigB expression.

S. aureus is known to sense its environment and to regulate virulence factors depending on the availability of carbon sources. For example, recent studies have shown that the transcriptional regulator RpiRc could sense metabolic shifts, especially in the PPP, and control expression of RNAIII [29,30]. RNAIII is a key effector in the quorum-sensing system that regulates the expression of a large number of virulence factors [37]. RpiRc is also linked to the alternative Sigma B factor (SigB) that has pleiotropic roles in gene regulation and is a master regulator of intracellular survival [29,38,39].

These data prompted us to follow the expression profile of the master regulator SigB and RpiRc in the WT and the tkt mutant strains during endothelial cells invasion (Figure 3).

EA.hy296 cells were infected as described above to solely focus on intracellular bacteria.

Between day 1 and day 7, sigB and rpiRc transcription increased in the WT strain (by 4-fold and 3-fold, respectively) (Figure 3). In sharp contrast, as early as one day after infection, both genes were significantly down-regulated in the ∆tkt mutant strain compared to WT (8fold and 7-fold decrease for sigB and rpirC expression, respectively). Consistent with reports demonstrating inhibition of RNAIII expression by RpirC and SigB [18], we observed a dramatic increase in the expression of RNAIII in the ∆tkt mutant strain (Figure 3), suggesting that the tkt mutant strain is unable to control rpiRc and sigB expression during intracellular survival. Functional complementation always restored the wild type phenotype (Supplementary Figure S5). To further confirm these data, we monitored the expression of two major regulators of SigB activity, RsbU and RsbW [40]. RsbW, which is co-expressed with sigB, is known to sequestrate and inhibit SigB. RsbU, whose expression is under the control of SigA, is a phosphatase involved in the dephosphorylation of RsbV. In turn, dephosphorylated RsbV (dRsbV) is able to sequestrate RsbW, allowing the release of active SigB. RsbU is thus considered as an activator of SigB, allowing a fine-tuning of SigB activity.

We found that the transcription of both rsbW and rsbU genes was down-regulated in the tkt mutant during infection of endothelial cells. The down-regulation of rsbW is consistent with the down-regulation of its co-expressed gene sigB (Figure 3). The concomitant down-regulation of rsbU suggests a global inhibition of the sigB pathway in the tkt mutant strain. Finally, we followed expression of asp23, a sigB-dependent locus [41].

The recorded down-regulation of asp23 transcription in the tkt mutant strain confirmed the inhibition of SigB-dependent transcription. All together these transcriptional analyses suggest that the metabolic deregulation observed in tkt strain is sufficient to promote a dramatic reprogramming of the RpiRc and SigB pathways when bacteria invade human cells.

Tkt inactivation impairs proliferation of S. aureus in the kidney of infected mice.

The ability of internalized S. aureus to readily proliferate inside host cell cytoplasm may be seen as an effective mechanism to avoid host innate immunity, ultimately allowing an increased colonization of the infected site. In this respect, it has been previously shown in the mouse model that after intravenous (IV) injection, although rapidly cleared from the bloodstream, bacteria were able to proliferate in the kidney, leading to abscesses formation within the first days of infection [42].

Here, we monitored the kinetics of in vivo proliferation of WT, tkt and Cp-tkt strains in kidneys and spleens of BalB/c mice (Figure 4) after IV infection with 10 7 bacteria per mouse. As previously reported, S. aureus poorly proliferates in the spleen of infected mice.

We therefore used the spleen as a non proliferative control. The bacterial burden in kidneys and spleens was quantified by plating CFUs at 4 h, 24 h, 48 h and 72 h after infection. We observed no significant differences between the bacterial counts in the spleens of mice infected either with WT, the Δtkt mutant or the Cp-tkt strain. In each case, a progressive reduction of the bacterial burden was recorded (ranging from 2.5x10 5 , 1.1x10 5 and 1.5x10 5 at 4 h; down to 3x10 3 , 7.3x10 2 and 1.2x10 2 at 72 h, for WT, ∆tkt and Cp-tkt respectively). In contrast, in kidneys, whereas the WT and the Cp-tkt strains readily proliferated (from 3.9x10 4 ± 8.6x10 3 WT and 2.1x10 4 ± 4.6x10 2 Cp-tkt S. aureus per organ at 4 hrs to 7.7x10 7 ± 3.3x10 7

WT and 3.6x10 7 ± 1.3x10 7 Cp-tkt S. aureus per organ at 72 hrs), multiplication of the Δtkt mutant was severely impaired at all time-points tested, with a 10 4 -fold reduction of ∆tkt counts at 24h compared to WT counts (Figure 4). This result confirms the importance of TKT -and most likely of the PPP-in bacterial proliferation in vivo and especially during the first 24 hours after infection. This result is consistent with the previously described role of SigB in intracellular growth in vivo [43].

The SigB pathway may be controlled by modulating carbon source availability.

Inside the cytoplasm of host cells, the Δtkt mutant is likely to deal simultaneously with PPP blocking and altered levels of carbon-based nutrients compared to the outside compartment. The above results suggested that the PPP might take control of rpiRc and sigB expression when bacteria invade endothelial cells. We thus hypothesized that PPPassociated carbon sources may possibly influence rpiRc and/or sigB expression in vitro. We therefore grew WT USA300 in CDM supplemented either with glucose (CDM-glucose), ribose (CDM-ribose) or fructose (CDM-fructose) a carbon source possibly fuelling the PPP)

and quantified by qRT-PCR the transcription of sigB, rpiRc, rsbU/W and asp23 genes.

Remarkably, we found that the regulation of the whole sigB operon was impacted by the carbon source used in CDM (Figure 5). Indeed, expression of sigB, rsbU/W and rpiRc decreased in CDM-fructose or CDM-ribose, compared to CDM-glucose. In contrast, asp23 expression was only decreased by 2-fold in CDM-fructose and increased by 3-fold in CDMribose, suggesting that in this growth conditions, asp23 expression might be regulated by other factor than sigB (Figure 5). Hence, the availability of carbon sources, and especially those related to PPP, may be sensed by the bacteria and affect the regulation of rpiRc and sigB expression.

Discussion

Inactivation of the tkt gene, encoding the unique transketolase of S. aureus USA300, led to a major decrease in early intracellular proliferation and impaired in vivo the multiplication in a kidney colonization model. Our results suggest that the intracellular proliferation defect of the ∆tkt mutant occurs through the inhibition of sigB and rpiRc expression and the concomitant increased RNAIII transcription, thus highlighting a novel connection between the PPP and these master regulators during cell invasion.

Our whole cell metabolomic analysis of planktonic-grown bacteria (Figure 1, Supplementary Figure S2 and Table S1) showed a huge increase in R5P intermediates in the ∆tkt mutant compared to WT that could be explained by the impaired entry of R5P into glycolysis and a concomitant significant decrease in SH7P relative concentration. In S.

aureus, ribose is imported by the RbsU transporter and converted to R5P by the RbsD/RbsK pathway. RbsR, the repressor of rbsUDK operon, is itself highly regulated by SigB [44].

Interestingly, S. aureus grown in CDM-ribose showed a decreased expression of sigB.

Hence, stress signals that modulate SigB activity are likely to be important effectors for controlling the quantity of RbsR, thereby affecting ribose uptake.

Significant changes in the relative concentrations of TCA cycle intermediates were also recorded in the ∆tkt mutant compared to WT (Supplementary Figure S3). The TCA cycle plays a central role in maintaining the bacterial metabolic status and has been repeatedly implicated in the regulation of staphylococcal virulence [45,[START_REF] Ding | Metabolic sensor governing bacterial virulence in Staphylococcus aureus[END_REF]. Hence, changes in TCA cycle activity are likely to induce alterations of the overall metabolome of the bacterium that, in turn, may modulate the activity of metabolite-responsive global regulators such as SigB or RpiR.

RpiRc, a RpiR family-member controlling enzymes involved in sugar catabolism in S.

aureus, has been shown to repress RNAIII [28], supporting the notion of a direct connection between the PPP and the regulation of S. aureus virulence. The expression of sigB has also been shown to play a crucial role during the intracellular life of bacteria possibly by downregulating pro-inflammatory virulence factors and increasing the expression of factors promoting persistence [18,39].

We propose (Figure 6) that in wild-type S. aureus, transketolase activity results in a positive regulation of rpiRc and sigB transcription and allows the repression of RNAIII.

Consequently, repression of toxins production favours intracellular growth. In contrast, in a ∆tkt mutant, rpiRc and sigB transcriptions are not activated, possibly due to metabolic changes (such as R5P accumulation) and RNAIII is not repressed. At this stage, it cannot be excluded that the enzyme itself might also directly affect transcription of these regulators.

TKT inhibitors are currently actively tested in cancer therapy [START_REF] Xu | Transketolase counteracts oxidative stress to drive cancer development[END_REF] and TKT could constitute an efficient target against tuberculosis [35] and malaria [START_REF] Thota | Drug Discovery and Development of Antimalarial Agents: Recent Advances[END_REF]. The present work, highlighting an unprecedentedly reported role of transketolase in S. aureus intracellular survival, suggests that modulation of the pentose phosphate pathway activity may also represent an interesting mean to fight S. aureus infections. B. Metabolomic analysis of the ∆tkt mutant strain. Quantitative metabolomic analysis was performed by ion chromatography and tandem mass spectrometry (IC-MS/MS) after overnight cultures, in BHI Broth, from WT USA300 and ∆tkt mutant strains (see Table S1).

Legends

The heatmap, obtained using MetaboAnalyst (https://www.metaboanalyst.ca), illustrates the metabolic shift between the two strains. Endothelial EA.hy926 cells expressing mKate2 nuclear restricted red fluorescent protein were infected with GFP expressing WT and tkt strains. One hour after infection, cells were washed several times with gentamicin containing medium to eliminate extracellular bacteria.

Gentamicin concentration of 50µg/ml was maintained throughout the experiment. Images were acquired every 30 minutes using Incucyte ® S3 live cell imaging system. A.

Representative images (see also Movie SigB dependent regulation of RNAIII in tkt context.

Movie 1: Intracellular proliferation of USA300 wild type strain. Endothelial EA.hy926 cells expressing a nuclear restricted Red Fluorescent Protein were infected with GFP expressing wild type strains. One hour after infection cells were washed several times with gentamicin containing medium to eliminate extracellular bacteria (see materials and methods section). Gentamicin concentration of 50µg/ml was maintained throughout the experiment.

Images were acquired every 30 minutes using Incucyte ® S3 live cell imaging system.

Movie 2: Intracellular proliferation of USA300 tkt mutant strain. Same protocol as above in movie 1.

recommendations, to obtain red nuclear labelling of living cells due to the expression of the red fluorescent protein mKate2 containing a nuclear localization signal.

Infection.

When fresh cultures reached OD600 of 0.6, bacteria were added to endothelial cells at a multiplicity of infection (MOI) of 1 and placed in a humidified 5 % CO2 atmosphere at 37 °C for 1 h. One hour after infection, each well was washed three times with 100 µL of phosphate buffer saline (PBS) containing 300 µg/mL gentamicin, to remove extracellular bacteria. Cells were then incubated with 100 µL of cell culture medium containing 50 µg.mL -1 gentamicin.

Infected cells were kept in a humidified 5 % CO2 atmosphere at 37 °C. Importantly, gentamicin is an antibiotic with a high bactericidal effect on S. aureus USA300 (CMI=2 µg.mL -1 ) and a very poor penetration inside eukaryotic cells. In our model the use of gentamicin in the extracellular medium was necessary to abolish extracellular proliferation.

Construction of the S. aureus ∆tkt chromosomal deletion mutant

We inactivated the tkt gene in wild-type S. aureus USA300 strain and substituted it by the kanamycin resistance gene nptII fused with pGro promoter. For this, we used the pMADtemperature-sensitive shuttle vector system [3]. Briefly, the recombinant plasmid pMAD-∆tkt was constructed by overlap PCR. First, the two regions (upstream 694bp, downstream 567bp) flanking tkt and the nptII gene (fused with pGro promoter 1,091bp) were amplified by PCR. The region (2,352 bp) was then amplified by two-step overlap PCR. The resulting PCR product was cloned in pMiniT 2.0 (to yield recombinant plasmid pMiniT/tktUp-npt-tktDown).

This recombinant plasmid was digested with BamHI and EcoRI (New England BioLabs) and the BamHI-EcoRI tktUp-npt-tktDown DNA fragment was finally subcloned into BamHI-EcoRIdigested pMAD. All PCR reactions were realized using Phusion High-Fidelity DNA Polymerase (Thermofisher Scientific) and PCR products were purified using QIAquick PCR purification kit (Qiagen). The pMAD-∆tkt plasmid was first introduced into S. aureus RN4220

prior to electroporation into S. aureus USA300. We used a standard two-step allelic exchange procedure [3] to create the chromosomal S. aureus USA300 ∆tkt strain. The ∆tkt strain was finally checked for loss of the corresponding wild-type genes by PCR sequencing (Eurofins Genomics) using specific primers (described at the end of these supplementary methods).

Construction of the complementation of S. aureus Cp-tkt strain

The plasmid pCN57-CPtkt was constructed by homologous recombination [4]. The pCN57 linear vector and tkt gene fragments were amplified by PCR. To allow recombination, overlapping region of 20pb was added at each ends. PCN57 was linearized using SphI (New England BioLabs). E. coli DH5α were chemically transfected with a mix of both PCR product (insert: vector stoichiometry 2:1, 100ng of vector) in 10 µl MilliQ water. The colonies containing plasmid pCN57-CP-tkt were checked by PCR and the construct was sequenced.

The pCN57-CP-tkt vector was then transfected into S. aureus RN4220 prior to electroporation into S. aureus USA300 ∆tkt. The ∆tkt-Cp-tkt (Cp-tkt) strain was finally checked for complementation of wild-type genes by PCR sequencing (Eurofins Genomics) using specific primers.

Time lapse microscopy

Four days prior to infection, NucLight Red labelled EA.hy926 cells were seeded in ImageLock 96-well plates (Essen BioScience Inc) in culture medium without antibiotic. Cells were infected with wild-type and mutant of the GFP-expressing USA300 strain as described above. Plates were incubated and monitored at 5% CO2 and 37°C for 24 hours in the fully automated microscope Incucyte ® S3 (Essen BioScience). Images were taken every 30 minutes with the 20X objective. The medium was changed once during the observation period. Analysis and time-lapse videos (from which images were extracted) were generated by using Incucyte ® S3 software.

Detailed analysis: Green items were analysed with a threshold of 1 GCU and using Top-hat segmentation mode. Red item were analysed with a threshold of 3 RCU and using Top-hat segmentation mode and edge sensitivity of -25. A spectral unmixing of 10% was applied to remove red signal from green signal. Green item counts were considered as an approximation of the number of intracellular bacteria, and red item counts considered as the number of EA-hy296 cells. (Green items/Red items) represents the number of intracellular bacteria per cell during the time of infection.

Transcriptional analyses

Intracellular bacteria RNA extraction: EA.hy926 cells were grown to confluence in 75 cm 2 flasks and infected with S. aureus as described above. At selected time points, the culture medium was removed and the flasks were washed twice with PBS without antibiotics. Cells were lysed with 5 mL of distilled water for 10 min and the surface of the flask was scraped to detach the cells. The contents were recovered and centrifuged for 5 min at 3000 g. The supernatant was carefully removed and the pellet (which contained cell debris and bacteria)

was resuspended in 100 μL of TE buffer containing 20 μg of lysostaphin and incubated for 5 minutes at 37 °C [5] to allow cell wall peptidoglycan degradation.

Planktonic bacteria RNA extraction: S. aureus was resuspended in CDM supplemented with glucose, ribose or fructose to an OD600 of 0.05 and grown overnight at 37°C . Bacteria were centrifuged at 12 000 g for 2 min and incubated in 50 μL of TE buffer containing 10 μg of lysostaphin for 5 minutes at 37 °C. The samples were resuspended in Trizol (Thermof isher Scientific) and treated with chloroform. RNAs were recovered using RNeasy Clean-up column (Qiagen) and DNA was digested using Turbo Dnase (Thermofisher Scientific). RNA concentration was measured using NanoDrop 1000 (Thermofisher Scientific). Reverse transcription was carried out with 500 ng of RNA and 0.5 μg of random primers (Promega). Luna Universal qPCR Master Mix (New England Biolabs) according to the manufacturer's recommendations. Transcript levels were analyzed using a 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the standard settings of the system software. The thermal cycling conditions were: 50°C for 2 min, 95°C for 10 min, follow ed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Gene specific primer sequences are presented at the end of these supplementary methods. Expression of the gene gyrA was chosen as a reference.

We used the "The Relative Standard Curve Method" for analyzing the qRT-PCR data.

Metabolomic analyses

Bacteria: Metabolite profiling of S. aureus isolates was performed by liquid chromatographymass spectrometry (LC-MS) as described [6,7]. Two independent experiments with three biological replicates were performed for each isolate. Briefly, S. aureus strains were grown in BHI Broth overnight to stationary phase and pelleted by centrifugation. Metabolic activity was blocked by immersion in liquid nitrogen for 10 sec. Metabolites were extracted using a solvent mixture of Methanol/ACN/H2O (50:30:20) at -20 °C (Extraction solution). Samples were vortexed for 5 min at 4°C, and then centrifuged at 16,000 g for 15 mi nutes at 4°C.

Endothelial cells: EA.hy926 endothelial cells were grown to confluence on a 12-well plate in no glucose DMEM supplemented with glucose or fructose or ribose (25 mM). One independent experiment with three biological replicates was performed for each condition. At each time points the cells were washed with cold PBS. Metabolism was blocked by plunging the plate into liquid nitrogen for 10 seconds. The cell metabolites were extracted from the samples by placing the cell culture plate on a rocking shaker at 4°C for 10 minutes. The extraction solution from each well was then collected and centrifuged at 16,000 g for 15 minutes at 4°C.

The supernatants were collected and analysed by LC-MS using SeQuant ZIC-pHilic column (Millipore) for the liquid chromatography separation [6]. The aqueous mobile-phase solvent was 20 mM ammonium carbonate plus 0.1% ammonium hydroxide solution and the organic mobile phase was acetonitrile. The metabolites were separated over a linear gradient from 80% organic to 80% aqueous phase for 15 min. The column temperature was 48 °C and the flow rate was 200 μl/min. The metabolites were detected across a mass range of 75-1,000 m/z using the Q-Exactive Plus mass spectrometer (Thermo) at a resolution of 35,000 (at 200 m/z) with electrospray ionization and polarity switching mode. Lock masses were used to insure mass accuracy below 5 ppm. The peak areas of different metabolites were determined using TraceFinder software (Thermo) using the exact mass of the singly charged ion and known retention time on the HPLC column. By this approach we were able to identify and measure: Glucose, Glucose-6P (G6P), UDP-glucose (UDP-Glc), Sedoheptulose Statistical analyses were performed using MetaboAnalyst 4.0 software [8,9]. The algorithm for heatmap clustering was based on the Pearson distance measure for similarity and the Ward linkage method for biotype clustering. Metabolites with similar abundance patterns were positioned closer together.

Transketolase activity assay

Bacteria were grown to stationary phase in BHI Broth and 1 ml of culture was harvested.

Bacteria were washed in PBS, pelleted by centrifugation, and resuspended in 300 µl of 50 mM Tris with 60 µg lysostaphin, pH 7.5. Samples were incubated at 37 °C for 5 min and then sonicated at 4 °C (30s on, 30s off, repeated 30 times) ( Diagenode). Samples were centrifuged at 12,000 g and 4 °C for 10 min to remove cell debris and protein concentration was determined using BCA assay with BSA as a standard (Thermo Fisher Scientific). TKT activity was analysed as described previously [10]. Briefly, each enzymatic reactions were performed at a final volume of 100 µl containing 20 µ l of supernatant, 50 mM Tris (pH 7.5), 0.24 mM MgCl2, 10 µM thiamine pyrophosphate, 0.5 mM NADH, 3 units of glycerol-3phosphate dehydrogenase, 10 units of triosephosphate isomerase, 0.5mM D-xylulose-5phosphate, and 0.5mM D-erythrose-4-phosphate (all reagents obtained from Sigma-Aldrich).

The reaction proceeded at 37 °C for 5 min, and the decrease in A340 nm was monitored with a microplate reader (Molecular Devices). Transketolase enzyme activity in bacteria lysates was expressed as units per microgram of total protein. One unit of enzyme was defined as the amount of enzyme that oxidized 1 µmol of NADH per minute.

LAMP-1 colocalization assay

EA.hy926 endothelial cells were grown to confluence on 8-well Slides (Lab Tek II) and infected with S. aureus as described above. For each point, cells were washed 3 times with PBS and fixed 20 min in PBS-4% paraformaldehyde at 4 °C . Fixation was stopped in PBS-NH4Cl 50 mM. Cells were permeabilized with PBS containing 0.1% saponin and 5% goat serum for 10 min on ice. Cells were incubated for 30 min with anti-LAMP-1 (Lysosomalassociated membrane protein 1) rabbit polyclonal antibody (1/200e, AbCam) diluted in PBS containing 5% goat serum (staining buffer). After washing, cells were incubated for 30 min with Alexa-568 conjugated donkey anti-rabbit secondary antibodies (1/400e, AbCam) diluted in staining buffer. After washing, 4,6-diamidino-2-phenylindole (DAPI) was added (1/10,000e) for 20 min and glass coverslips were mounted in Mowiol (Cityfluor Ltd.). Cells were examined using an X63 oil-immersion objective on an Apotome microscope (Zeiss TM ). Colocalization of GFP expressing bacteria with LAMP-1 staining was quantified by using Image J software (plug in: Coloc based Sur-vol _ Beta v6 ). Data were obtained from more than 200 infected cells for each condition and expressed as % of bacteria associated with LAMP-1 ± SD.

In vivo infection

For all experiments we used 6 to 10 weeks-old female BALB/c (Janvier Labs) with access to food and water ad libidum.

Infection. Overnight cultures of S. aureus in BHI Broth were diluted in BHI Broth to an OD600 of 0.05. Bacteria were grown at 37°C until the culture reached the initial -middle log phase (OD600=0.6) and resuspended in a sterile isotonic saline solution to an OD600 of 0.4.

Mice were infected with 10 7 bacteria intravenously (iv) in the tail vein. Inoculum titers were checked by spreading dilutions of the preparations on BHI agar plates. For bacterial burden determination, mice were euthanized after 4, 24, 48 and 72 hours. Sequential dilutions of homogenized preparations from spleen and kidney were spread onto BHI agar plates.

Mice infected with the complemented strain were treated with erythromycin to avoid loss of the complementing plasmid. For this, mice received one dose of erythromycin 16 hours before infection and 2 doses of erythromycin per day until they were sacrificed (500 µg of erythromycin per dose).

Statistics

Data were analyzed using GraphPad Prism software. Results are presented either with one representative experiment for clarity or as means ± standard deviation (SD). The number of biological and technical replicates is indicated in the legend of each figure. For two-sample comparisons statistical significance was measured using unpaired two-tail Student's t-test.

For comparisons between more than two groups, statistical significance was measured using one-way analysis of variance (ANOVA) with Dunnett's correction. P values of p<0.05 were considered to indicate statistical significance.

Ethics statement

All experimental procedures involving animals were conducted in accordance with guidelines established by the French and European regulations for the care and use of laboratory animals (Decree 87-848, 2001(Decree 87-848, -464, 2001(Decree 87-848, -486 and 2001-131 -131 Endothelial EA.hy926 cells were infected with USA300 WT, ∆tkt and Cp-tkt strains as described in figure 2. One, three or seven days after infection cells were washed and collected. RNA from intracellular S. aureus were prepared and gene expression of RNAIII, rpiRc and sigB was analysed by quantitative RT-PCR. Gene expression level was normalized by that of gyrA at days 1, 3 and 7 after infection of EA-hy296 cells. Data are shown as mean normalized expression ± SD of triplicate measurements from two independent experiments. (ANOVA, ns : not statistically different; * p<0.01, ** p<0.001). Experiments presented in Figure S5 were independent of those presented in Figure 3. -Time lapse microscopy and immunofluorescence microscopy for USA300 ∆tkt strain and wild-type strain.

Primers used for ∆tkt mutant construction and complementation

-Preparation of cell samples for metabolomic studies.

-In vivo infection assay.

-RT-qPCR.

-Transketolase activity assays. 1,2,3,4,5 and Figure S2,S3 and S4 Staphylococcus aureus and Pseudomonas aeruginosa are the most common pathogens that infect the lungs of patients with a chronic lung disease (CLD) including cystic fibrosis (CF) [1,2]. Furthermore, S. aureus is one of the earliest bacteria detected in infants with CF. However, very few studies have addressed the adaptations S. aureus has undergone in this context [3][4][5][6].

Results

Figure

Staphylococcus aureus has the ability to form bioilm [7][8][9] and to survive within a wide range of eukaryotic host cells [10][11][12][13][14][15][16][17][18][19]. hese abilities are likely to contribute to the persistence of S. aureus in airways of patients with CLDs despite appropriate antimicrobial treatments [20,21]. Staphylococcus aureus persistence is associated with a drastic decrease in metabolism [22], a decrease in the expression of virulence factors, and an increase in the expression of bacterial adhesins [23]. Such a proile is typical of small-colony variants (SCVs) that are deined by smallsized colonies [17,24,25]. In addition to SCVs, strains with normal colony morphology can exhibit similar patterns of "low toxicity" that allow them to persist intracellularly without being cleared by host cell defense mechanisms [23]. A low-toxicity pattern can be achieved either transiently, following changes in the expression of genes encoding toxins and/or regulators, or permanently by mutations in global regulators [26][27][28].

he alternative sigma B factor (SigB) has pleiotropic roles in gene regulation and has been shown to be a master regulator of intracellular persistence [29,30]. SigB was shown to downregulate the agr system, which is responsible for enhanced inlammatory activity. Hence, the expression of SigB during chronic persistence is linked to its capacity to downregulate proinlammatory virulence factors and to increase the expression of factors that promote persistence [28,30]. SigB enables the bacteria to downregulate its aggressive phenotype and to switch toward a silent SCV phenotype that allows for long-term intracellular persistence. By studying serial isolates, we show that during long-term lung infection, S. aureus adaptation occurs through genomic modiications that accumulate over time and lead to major metabolic modiications and protein expression changes. We also reveal that persistence of S. aureus is associated with convergent phenotypic evolution responsible for an increased ability to form bioilm as well as to survive within host cells. hese observations should be taken into account in therapeutic decisions aimed at eradicating S. aureus chronic infections by choosing drugs that speciically target bioilm-embedded and intracellular bacteria.

METHODS

Whole-genome sequencing was performed on an Illumina MiSeq instrument (2 × 150 bp), and the sequences were processed using the Nullarbor bioinformatic pipeline software v1.20 and RAST server. The sequences reported here are available at the National Center for Biotechnology Information's BioProject database under accession PRJNA446073.

Quantiication of bioilm formation was assessed with crystal violet staining in polystyrene 96-well plates. CF bronchial epithelial cell line CFBE41o-and primary nasal epithelial cell were infected with a multiplicity of infection of 100 using an inoculum taken from cultures of S. aureus grown in brainheart infusion (BHI) until the exponential growth phase. Infected cells were kept for 6 days in a medium that contained 50 μg/mL gentamicin to kill extracellular bacteria. E-tests were used to determine minimal inhibitory concentrations (MICs) to gentamicin. Gentamicin has a high bactericidal efect on S. aureus USA300-LAC (MIC = 2 μg/mL) and a very poor penetration inside eukaryotic cells. In addition, the measured MICs to gentamicin of all tested clinical strains were similar to that of USA300 reference strain (≤2 μg/mL). Hence, an accumulation of gentamicin within epithelial cells should impact equally all tested strains and is unlikely to explain diferences between isolates.

For proteomics, proteins were digested and analyzed using liquid chromatography (LC) coupled with tandem mass spectrometry (nanoLC-MS/MS). For metabolomics, metabolite proiling of S. aureus isolates was performed using LC-MS. he MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [31] partner repository with the dataset identiier PXD011281. he metabolomics raw data are provided in Supplementary Table S1. A full description of methods is available in the Supplementary Methods.

Statistical Analyses

Data were analyzed using R or GraphPad Prism software. Results are presented either with 1 representative experiment for clarity or as means ± standard deviation. The number of biological and technical replicates is indicated per figure.

For 2-sample comparisons, statistical signiicance was measured using unpaired 2-tail Student t test or Wilcoxon rank sum test as indicated in the igure legends. For comparisons between more than 2 groups, statistical signiicance was measured using 1-way analysis of variance with multiple comparisons (Dunnett's correction) performed, with each value compared to that of the reference strain. P values < .05 were considered to be statistically signiicance.

Ethics Statement

All experiments were performed in accordance with the guidelines and regulations described by the Declaration of Helsinki and the low Huriet-Serusclat on human research ethics, and informed consent was obtained for all participating patients. Serial isolates of S. aureus were obtained from airway secretions from 4 patients with chronic lung infection at the Necker-Enfants Malades University Hospital, Paris, France. Sputum sampling is part of routine standard care. The research procedure is validated by Ile de France 2 IRB (ID-RCB/Eudract: 2016 A00309-42).

RESULTS

Selection of S. aureus Sequential Isolates From Patients With Chronic Lung Infection

Three patients with CF (CF1, CF2, and CF3) and, for comparison purposes, 1 patient with non-CF CLD were chosen. For each patient, we selected 1 early and 1 late isolate separated by 3to 9-year intervals. By definition, in a pair of isolates that belong to the same clone, the "early isolate" corresponds to the isolate collected earlier than the second isolate that had been collected later and that corresponds to the "late isolate. " Whole-genome sequencing confirmed that each pair of isolates belonged to 4 distinct clones (Figure 1). Patient diseases and treatments are detailed in the Supplementary Methods.

S. aureus Clinical Isolates From CF Patients Evolved an Increased Persistence Ability Within CFBE-F508del Epithelial Cell Line

Numerous studies have shown that S. aureus has the ability to survive within human cells [10][11][12][13][14][15][16][17][18][19]. We subsequently aimed at determining if during the course of within-lung adaptation, S. aureus isolates have evolved a greater ability to persist within epithelial cells. We infected the bronchial CFBE epithelial cell line (F508del +/+ cystic fibrosis transmembrane conductance regulator [CFTR] mutation) with clinical isolates, the control strain USA300-LAC, and a stable SCV mutant altered in the haemin biosynthetic pathway (hereafter, named Δhem). As expected, wild-type bacteria were not able to persist, whereas the Δhem mutant was able to persist intracellularly during the whole course of the experiment (Supplementary Figure S1) [15]. All early and late clinical isolates were able to persist at least 2.6-fold, and up to 900-fold, more than the USA300-LAC reference strain at day 3 and day 6 post-infection (Figure 2A,2B). Furthermore, at day 3 and day 6 post-infection, all the late isolates recovered from CF patients exhibited an improved ability to persist intracellularly within CFBE-F508del epithelial cells compared to cognate early isolates (Figure 2A). Interestingly, the CLD_late isolate recovered from the non-CF patient did not exhibit an improved ability to persist within CFBE epithelial cells compared to CLD_early isolate (Figure 2B). These data suggest that S. aureus adaptation within CF lungs correlates with an improved ability to persist intracellularly in cells with a CFTR dysfunction.

Late S. aureus Isolate of CF3 Patient Exhibits an Increased Persistence Within Primary F508del Epithelial Own-patient Cells

To confirm the relevance of the results obtained with the bronchial CFBE epithelial cell line, we first assessed the persistence of CF3 isolates within primary epithelial cells isolated from the nose of a healthy donor (Figure 2C). In addition, we performed an infection assay with the CF3 primary epithelial own-patient cells (F508del +/+ CFTR mutation) to verify the specific within-patient adaptation of S. aureus recovered from long-term infection (Figure 2D). These experiments confirmed that the late isolate persistence ability is improved compared to the early isolate at day 3 and day 6 within both primary nasal epithelial cells retrieved from a healthy donor and from the CF3 patient.

S. aureus Clinical Isolates From Chronically Infected Patients Evolved High Biofilm Formation Ability

Assuming that isolates retrieved from chronic infections might have a high biofilm-forming capacity, we studied the biofilm formation ability of the pairs of isolates. Remarkably, all isolates displayed a greater capacity to form biofilms compared to that of the weak biofilm-producer USA300-LAC reference strain (P < .001; Supplementary Figure S2). Furthermore, for 3 chronically infected patients, the late isolates formed more biofilm than the early isolates, revealing that long-term adaptation within lungs had improved their biofilm formation capacity (P < .001; Figure 3).

Late S. aureus Clinical Isolates From Chronically Infected Patients Acquired Auxotrophies

Compared to that of USA300-LAC, all patients' early isolates and the CF1 late isolate displayed similar colony morphology on BHI agar plates and a wild-type growth in a liquid broth that mimics sputum (CF sputum medium [CFSM]; Supplementary Figures S3 and4A). In contrast, the late isolate of CF2 and CLD patients displayed a typical stable SCV phenotype with very small colonies on BHI agar (Supplementary Figure S4) and CF2_late, CF3_late, and CLD_late isolates exhibited a growth defect in CFSM broth (Figure 4B-4D). Thymidine-dependent SCVs are frequently isolated from patients treated with sulfamethoxazole-trimethoprim (SXT) [32]. Indeed, supplementation with thymidine restored most wild-type growth for CF2_late and CLD_late isolates (Figure 4B,4D). We used genomic data to determine the auxotrophy of CF3_late isolate and identified a frameshift in panB gene, which is involved in de novo biosynthesis of pantothenic acid (Table 1). Accordingly, growth of CF3_late isolate in the presence of pantothenate restored wild-type growth (Figure 4C). Thus, isolates from 3 of 4 patients with S. aureus chronic lung infection acquired auxotrophy during the course of the disease.

Late S. aureus Clinical Isolates From Chronically Infected Patients Acquired Antibiotic Resistance

Genome analysis evidenced mutations in thyA, gyrB, and rpsJ genes, which are likely to explain SXT, fluoroquinolones, and cyclines resistance, respectively (Table 1). However, in the case of point mutations, a causal relation with phenotypic changes has to be experimentally verified. Thus, isolates from 3 of 4 patients with S. aureus chronic lung infection acquired antibiotic resistance consistent with administration of the corresponding drugs during the course of the disease.

Genomic, Proteomic, and Metabolomic Modifications Associated With

S. aureus Adaptation During Chronic Lung Infection

In order to investigate the underlying genomic, proteomic, and metabolic modifications associated with the observed Branch length is proportional to the number of variant nucleotide sites within the core genes. For each patient, the isolate taken irst is termed "early" and the isolate taken later is termed "late." The dates of sampling and the ST of the isolates are indicated. "TA" and "PA" mean that the isolate is auxotrophic for thymidine or pantothenate, respectively. The name of the isolate is indicated in red when it is methicillin-resistant S. aureus. Reference strains included are PFESA1902 (ERR554197), st1307 (ERR158691), and PFESA1195 (ERR554722). Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease.

Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019 phenotypic changes, we compared genomes, proteomes, and metabolomes of late compared to early isolates. The differences in proteomic and metabolic profiles between early and late isolates of patients are highlighted by heat maps shown in Supplementary Figure S5. Genomes of all clinical isolates were de novo assembled, and coding DNA sequences (CDSs) were annotated. Most of the single-nucleotide polymorphisms were missense variants occurring in CDSs (Table 2). Nonsynonymous mutations acquired by late isolates were found mainly in genes involved in metabolic processes (Figure 5A) and, more speciically, in the following functional categories: "amino acid transport and metabolism" and "carbohydrate transport and metabolism". In addition, the largest category of proteins to be diferentially expressed for all pairs also comprised proteins related to metabolism processes (and more speciically to the "amino acid transport and metabolism" category) (Figure 5B). Concordant with genomic and proteomic results, the category "amino acids" was the most altered metabolite category in the late isolate of all patients compared to their cognate early isolates (Figure 5C). Of note, our proteomics data also indicated that there is no category in which the overall direction of change is the same in all patients (Figure 5B).

Many regulatory proteins were diferentially expressed. Indeed, proteins of the Agr, Rot, Sae, Sar, or Fur regulatory infected with the control strain USA300-LAC and clinical isolates from cystic ibrosis (CF) patients (A) or the chronic lung disease (CLD) patient (B). (C and D) Primary nasal epithelial cells retrieved from a healthy donor ("Primary WT") (C) and from the CF3 patient ("Primary CF3" with F508del +/+ CFTR mutation) (D) were infected with the control strain USA300-LAC and CF3 isolates. For all experiments, gentamicin was present throughout the experiment to prevent extracellular bacterial growth and new infection. Bacterial loads inside cells were evaluated by colony-forming unit (CFU) enumeration at 3 and 6 days after infection. Results are normalized with USA300-LAC strain as a reference and expressed as a fold change of CFUs. Results have been obtained from 2 independent experiments performed in triplicate for A, B, and C and 1 experiment for D. Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease; ns, not signiicant; WT, wild type. networks were diferently expressed in all late isolates. In CF1_ late isolate, the premature stop codon identiied in agrC (Table 1) is likely to profoundly impact the agr regulon and to explain a pleiotropic efect on the proteome (eg, downregulation of delta hemolysin and PSMb1 and upregulation of proteins encoded by spa, sbi, fnbA, rot, and coa genes). In addition, a missense mutation in saeR regulatory gene (Table 1) could also afect sae regulon and modify the proteome. In CF2_late isolate, the agr regulon is also downregulated, suggesting an evolution toward low virulent and highly adhesive properties. he metabolite proiling of CF2_late isolate showed a decrease in adenosine diphosphate, which is well correlated with the lack of hydroxyethylthiazole kinase expression found in proteomic analysis due to a frameshit in thiM gene (Table 1). In CF3_late isolate, frameshits in fakA and panB genes (Table 1) were associated with a lack of cognate protein expression in CF3_late isolate. In addition, adhesins encoded by sdrD and sasF were upregulated. Interestingly, an overproduction of penicillin-binding protein 2 encoded by mecA is correlated with the saeR mutation (Table 1) [33]. he metabolite proiling of CF3_late isolate revealed a drastic reduction in pantothenate, coenzyme A, and dephospho-coenzyme A, which is in line with the lack of expression of PanB and PanC proteins [34].

Of note, the non-CF control clone displays a diferent evolutionary trajectory. Indeed, AgrA and AgrC were upregulated in CLD_late isolate, suggesting that it has retained virulent properties.

SCVs have typically decreased ribosomal activity and DNA and amino acid synthesis. Indeed, we noticed that in CLD_late isolate, 2 ribosomal proteins as well as a ribosomal protein methyltransferase were less expressed compared to early isolate. In CF2_late, 1 ribosomal protein and a DNA topoisomerase were also less expressed compared to early isolate. Also, metabolic proiling suggests that all late isolates have evolved a reduced citric acid cycle activity compared to cognate early isolates.

DISCUSSION

Here, we show that during chronic lung infection, S. aureus adapts through the acquisition of common adaptive traits including antibiotic resistances, auxotrophies, reduced citric acid cycle activity, increased biofilm, and intracellular persistence abilities that occurred irrespective of the clone type. Of particular interest, we report mutations in 2 master regulatory systems, Agr and Sae, likely to impact multiple protein expression and metabolite amounts.

he agr-defective mutants, such as CF1_late isolate, have been shown to arise during chronic infections and are better adapted to persistence within the infected host [27,35,36].

Genetic alterations that directly or indirectly target SaeR regulon were identiied in the 3 CF patients. SaeR is directly involved in the regulation of more than 20 virulence factor genes [37] but is likely involved in the regulation of additional proteins by indirect mechanisms [38]. For instance, Cassat et al showed that 31 proteins, including SdrD, signiicantly increase in abundance upon inactivation of sae [38]. Furthermore, SaeRSdeicient bacteria are less infective in animal models [39].

In the 3 CF_late isolates, we observed an increase in the expression of the SdrD adhesin belonging to the SaeR regulon and involved in adhesion to human nasal epithelial cells and to human keratinocytes [38,40]. Our results suggest that SdrD is also important for long-term lung colonization.

In patients with chronic lung infections, SCV detection is most oten the consequence of a long-term SXT treatment [41]. Mutations in the thyA gene, as found in CF2 and CLD_late For each category, histograms represent the number of downand upregulated proteins from proteomic analysis using the threshold of <2 and >2, respectively. Only genes and proteins with functional annotation available are included. The "cellular processes and signaling" category encompasses regulatory proteins and proteins involved in cell wall and capsule synthesis. The "information storage and processing" category encompasses proteins involved in replication, translation, and repair processes. The "metabolism" category encompasses proteins involved in metabolism and transport. The "virulence" category encompasses exotoxins and proteins involved in adhesion, bioilm formation, and immunomodulation. (C) Categorization in 8 categories of an altered amount of metabolites in late compared to early isolates. isolates, are known to lead to stable clinical SCVs that are no longer susceptible to SXT and are thymidine-auxotrophic (TA-SCV) [32,41]. Since thymidine is assumed to be abundant during lung inlammation, TA-SCVs can still grow in the inlamed alveolar environment. In CF3 late isolate, we observed a pantothenate auxotrophy, which has been previously associated with persistence in Mycobacterium tuberculosis [42]. he acquisition of pantothenate auxotrophy suggests that pantothenate could also be present in CF lungs. hus, our data conirm that metabolic specialization is a common phenomenon among long-term colonizers [43].

Other striking traits of phenotypic convergent evolution of S. aureus identiied in this work were the increased ability to form bioilm and to persist in the intracellular niche. For CF2 and CF3 patients, the increased bioilm ability of late isolates could be linked to a mutation in the fakA gene, encoding fatty acid kinase A (FakA). Indeed, several studies showed that FakA-null strains were proicient in bioilm formation [8] and deicient in the expression of virulence factors controlled by the SaeRS system [44]. Overexpression of adhesins detected in proteomic analysis could also ultimately lead to increase bioilm formation in clinical isolates.

Numerous studies have demonstrated the ability of S. aureus to persist within host cells [10][11][12][13][14][15][16][17][18][19]. Strikingly, for the 3 CF patients, the S. aureus late isolates showed a greater ability to persist within CFBE-F508del epithelial cells compared to the early ones at day 3 and day 6 post-infection. Of note, the late isolate of the CLD patient did not present an improved ability to persist intracellularly within CFBE-F508del epithelial cells, possibly due to the fact that it has adapted to a non-CF patient. However, it is not possible to draw conclusions on lung adaptation of S. aureus among patients with non-CF lung dysfunction based on a single patient.

In a previous study, Ziebandt and coworkers [45] reported that the protein patterns recorded within each pair of S. aureus isolates were very similar, whereas the pattern recorded between pairs of isolates belonging to diferent clonal groups were highly variable. Strikingly, in our study, marked diferences in proteomic proiles between isolates belonging to the same clone were observed. hese proteomic changes are likely the consequence of within-host adaptation. he fact that each patient's isolates exhibited speciic proteomic changes could be correlated to host-speciic factors or to clone-speciic factors.

Our multi-omics approach allowed both conirmation of previously known mechanisms and identiication of novel candidate genes and pathways involved in the persistence ability of clinical isolates. We now provide evidence that the saeR/fakA regulon and the pantothenate pathway could also be promising therapeutic targets to ight persistent S. aureus infections.

all clinical strains was below or equal to 2 μg.mL -1 . Infected cells were kept in a humidified 5% CO2 atmosphere at 37°C for 6 days. To enumerate viable intracellular persisting bacteria, the culture medium containing gentamicin was removed from infected cells and wells were washed three times with antibiotic free PBS. Epithelial cells were lysed by scrapping with 1mL of distilled water during 15 minutes and serial dilutions of the cell lysates were plated on BHI agar. Colony forming units were numerated after 48 h at 37°C. Results shown are normalized with USA300-LAC strain as a reference and expressed as a fold change of CFUs.

Proteomic analyses

S. aureus isolates were grown in 50 ml of CFSM with addition of thymidine (100 μg/mL) in technical triplicates and collected after 19h of growth at 37°C with agitation (150 RPM). These growth conditions allow the bacteria to reach post-exponential growth phase as observed in the corresponding growth curves shown in Figure 4. Bacteria were lysed in PBS containing CaCl2 1 mM, MgCl2 1 mM and 10μg/mL lysostaphin.

Proteins were digested and analyzed by liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). Filter Aided Sample Preparation (FASP) was used for protein digestion [9,10]. Proteins were then reduced in 100 mM DTT for 30 min at 60°C, mixed with 8 M urea buffer, loaded on Microcon 30kDa centrifugal filters (Millipore) and alkylated with 50 mM iodoacetamide. Filters were washed twice with 8 M urea and twice with 50 mM ammonium bicarbonate. Following an overnight trypsin digestion at 37°C, samples were vacuum dried, and kept at -20 °C until MS analysis.

MS analysis was performed as previously published [10]. Dried peptides were resuspended in 10% ACN, 0.1% TFA. For each run, 5 μl (corresponding approximately to 1 µg of peptides) were injected in a nanoRSLC-Q Exactive PLUS MS (Dionex RSLC Ultimate 3000, Thermo Scientific, Waltham, MA). Peptides were separated on a 50 cm reversed-phase liquid chromatographic column (Pepmap C18, Dionex). Chromatography solvents were: (A) 0.1% formic acid in water, and (B) 80% acetonitrile with 0.08% formic acid. Peptides were eluted from the column with the following gradient: 5% to 40% of B for 120 min and then 40% to 80% of B in 1 min. At 125 min, the gradient was reversed to 5% to allow re-equilibration of the column for 20 min before the next injection. One blank with two 30 minutes linear gradients was run between triplicates to prevent sample carryover.

Peptides eluted from the column were analyzed by data dependent MS/MS, using the top-10 acquisition method. Briefly, the instrument settings were as follows: resolution was set to 70,000 for MS scans and 17,500 for the data dependent MS/MS scans in order to increase speed. The MS AGC target was set to 3.10 6 counts, whereas the MS/MS AGC target was set to 5.10 4 . The MS scan range was from 400 to 2000 m/z. MS and MS/MS scans were recorded in profile mode. Dynamic exclusion was set to 20 s duration. Each sample was analyzed in three technical triplicates by nanoLC/MS/MS. Raw MS files were processed with the MaxQuant software version 1.5.8.3 and searched with the Andromeda search engine against a custom protein database containing 3 457 protein sequences. To build this database we pooled protein sequences from both PROKKA and RAST annotations of all clinical isolates and the MAGE annotation of USA300 (NC_007793.1) [11]. Redundancy of the sequence set was reduced using CD-HIT v4.6 [12] with a 80% identity threshold. To search for parent mass and fragment ions, we set the mass deviation at 4.5 ppm and 20 ppm, respectively. The minimum peptide length was set to seven amino acids and strict specificity for trypsin cleavage was required, allowing up to two missed cleavage sites. Match between runs was allowed. Carbamidomethylation (Cys) was set as fixed modification, whereas oxidation (Met) and protein N-terminal acetylation were set as variable modifications. The false discovery rates (FDRs) at the protein and peptide level were set to 1%. Scores were calculated in MaxQuant as described previously [13]. The reverse and common contaminants hits were removed from MaxQuant output. Proteins were quantified according to the MaxQuant label-free algorithm using normalized label free quantification (LFQ) intensities. Protein quantification was obtained using at least two peptides per protein.

Statistical and bioinformatic analysis, including heatmaps and clustering, were performed with Perseus software (version 1.6.0.7) freely available at www.perseus-framework.org [14].

EggNOG annotations of all proteins were retrieved from webserver (last accessed 2017-12-15) [15]. Gene names were retrieved from AureoWiki [16] (last accessed 2017-12-15).

Extraction of annotations GO, Keywords, EggNOG and Kegg pathway were performed using Perseus.

For accurate quantification, we analysed each isolate separately (3 samples from early and 3 samples from late collection). We filtered the data to keep only proteins with at least 3 valid values in at least one group (late or early). Next, the data were imputed to fill missing data points by creating a Gaussian distribution of random numbers with a standard deviation of 33% relative to the standard deviation of the measured values and a 2.5 standard deviation downshift of the mean to simulate the distribution of low signal values. To discriminate differential protein amongst early and late for each isolate we performed an t-test, S0=0.1, and retained only proteins with an FDR<0.05. Hierarchical clustering of significantly different proteins was performed in Perseus on logarithmic LFQ intensities after z-score normalization of the data, using Euclidean distances and default parameters. We also performed a T Test, S0=0.1, FDR=0.001 to discriminate differential proteins between late and early.

Metabolomic analyses

Metabolite profiling of S. aureus isolates was performed by liquid chromatography-mass spectrometry (LC-MS) as described [17]. Two independent experiments with three biological replicates were performed for each isolate. Briefly, S. aureus isolates were grown in CFSM with addition of thymidine in the same conditions as for the proteomic analyses. Metabolic activity was blocked by immersion in liquid nitrogen for 10 sec.

Metabolites were extracted using a solvent mixture of Methanol/ACN/H2O (50:30:20) at -20 °C. Samples were vortexed for 5 min at 4°C, and then centrifuged at 16,000 g for 15 minutes at 4°C. The supernatants were collected and analysed by LC-MS using SeQuant ZIC-pHilic column (Millipore) for the liquid chromatography separation [17]. The aqueous mobile-phase solvent was 20 mM ammonium carbonate plus 0.1% ammonium hydroxide solution and the organic mobile phase was acetonitrile. The metabolites were separated over a linear gradient from 80% organic to 80% aqueous phase for 15 min. The column temperature was 48 °C and the flow rate was 200 μl/min. The metabolites were detected across a mass range of 75-1,000 m/z using the Q-Exactive Plus mass spectrometer (Thermo) at a resolution of 35,000 (at 200 m/z) with electrospray ionization and polarity switching mode. Lock masses were used to insure mass accuracy below 5 ppm. The peak areas of different metabolites were determined using TraceFinder software (Thermo) using the exact mass of the singly charged ion and known retention time on the HPLC column. By this approach we were able to identify and measure: Glucose, Glucose-6P (G6P), UDP-glucose (UDP-Glc), Sedoheptulose-7-P (SH-7P), Ribose 5-phosphate, Glyceraldehyde 3-phosphate, Lactate, Pyruvate, Phospho-Enol-Pyruvate (PEP), Citrate (Cit), cis-aconitate (cis-aco), Succinate (Suc), Fumarate (Fum), Discussion and perspectives S. aureus is generally considered as an extracellular pathogen. However, more and more studies have demonstrated that S. aureus was able to invade a variety of non-professional phagocytic cells (NPPCs) such as endothelial and epithelial cells, osteoblasts, fibroblasts, keratinocytes ... [START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF][START_REF] Moldovan | In or out: Phagosomal escape of Staphylococcus aureus[END_REF]. The intracellular environment could protect staphylococci from host's immune system and antibiotics and, thus, constitute a reservoir likely to play an important role for bacterial persistence in the host and relapse of infection. During this thesis, we addressed different aspects of S. aureus persistence, using different set up and models but with a common idea: understand the importance of metabolic adaptations during chronic persistence.

In the first article, we confirmed the increased intracellular persistence of S. aureus USA300 in endothelial cells as compared to macrophages. Furthermore, using time-lapse microscopy, we could show that the initial persistence of wild-type S. aureus in endothelial cells corresponded to distinct simultaneous situations at the single cell level, notably including a prolonged bacterial persistence without apparent morphological cellular damage. Our results and those of others indicate that, within the first hours after internalization, bacteria tend to actively multiply inside human cells (and ultimately lyse them) whereas infected cells attempt to restrict their proliferation. This selective pressure on S. aureus may favor the emergence of transient persisters which are able to survive into host cells for several days, without multiplication, before re-emerging in the extracellular compartment where they can rapidly proliferate and infect neighboring cells.

Two main outcomes of host cell invasion by S. aureus are generally described: (i) rapid host cell lysis, triggered by the secretion of toxins and other pro-inflammatory factors, which induce strong inflammatory and cytotoxic effects; (ii) persistence within morphologically-intact host cells for extended periods of time, where secretion of virulence factors is either down-regulated or not expressed (Proctor, Kriegeskorte et al. 2014). Of note, these persisting bacteria generally correspond to so-called "small colony variants" (SCV). The association between S. aureus SCVs and persistent infection was first reported by Proctor andco-workers in 1995 (Proctor, van Langevelde et al. 1995) and several studies have shown that the impaired growth of S. aureus SCVs was due to decreased metabolic activity (Amato, Fazen et al. 2014). Since a number of other pathogenic bacteria have been shown to persist within their host as SCVs (Kahl, Becker et al. 2016), it may be considered as a major survival strategy in chronic bacterial infections. However, summarizing the prolific literature addressing S. aureus intracellular persistence is quite complicated and may even be misleading. Indeed, the comparison of results obtained in one experimental setup with those obtained in another one is quite often impossible due to the different cell types and S. aureus strains which have been used. Our study reveals two major outcomes of intracellular S. aureus by Time-lapse microscopy. However, the molecular mechanism remains poorly understood. Next, we plan to investigate the transcriptional level of intracellular S. aureus on single cell level to better understand these distinct behaviors.

In the second article, we studied the role of the metabolic Pentose phosphate pathway (PPP) during the intracellular persistence of S. aureus, by focusing on transketolase (TKT), one key enzyme of this pathway. We showed that inactivation of the unique gene tkt, encoding transketolase in S. aureus USA300, conferred a slow-growth phenotype and was associated with important metabolomic alterations during planktonic growth. In infected cells, tkt inactivation also decreased bacterial intracellular persistence, most likely because of a drastic reduction of transcription of Sigma B-and RpiRc-encoding genes.

These data unraveled potential novel connections between the PPP and other regulatory circuits controlling chronic bacterial intracellular persistence. S. aureus SCVs have been particularly used as a model to study persistence [START_REF] Proctor | Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections[END_REF]Garzoni and Kelley 2009;Sendi and Proctor 2009;[START_REF] Mitchell | Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells[END_REF][START_REF] Tuchscherr | Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection[END_REF][START_REF] Kalinka | Staphylococcus aureus isolates from chronic osteomyelitis are characterized by high host cell invasion and intracellular adaptation, but still induce inflammation[END_REF][START_REF] Strobel | Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain[END_REF]. As recalled earlier, the SCV phenotype is generally associated with the formation of tiny colonies on plates, increased biofilm capacity and antibiotic resistance. Furthermore, supernatants from SCVs cultures generally show reduced cytotoxicity on cell cultures. The ∆tkt mutant of S. aureus USA300 shared the common properties of SCVs in planktonic conditions. The behavior of the ∆tkt mutant of S. aureus USA300 was also similar to a slow-growing strain (here ∆hemDBL) in time-lapse video microscopy, at the single cell level over a 24 hours-period. Somewhat unexpectedly, bacterial counts revealed that after seven days, the wild-type strain and the ∆tkt mutant showed similar intracellular survival decrease, ultimately leading to their complete elimination whereas the ∆hemDBL strain was still clearly detected even after ten days inside cells. Confocal microscopy analyses did not reveal any phagosomal escape defect of the ∆tkt mutant.

Hence, although non-proliferation is generally associated to prolonged persistence, the tkt mutant appeared to be a non-proliferative and non-persister strain. Among the most striking transcriptional changes recorded in the ∆tkt mutant compared to WT S. aureus USA300, was an increase in RNAIII transcription at day 1 and day 3 in infected cells. Simultaneously, the ∆tkt mutant showed a severe decreased transcription of rpiRc and sigB genes. In general, very low levels of RNAIII, either due to an inactivated agrBDCA operon or to an increased RNAIII degradation, constitute a hallmark of electron transport chain related SCVs (Felden, Vandenesch et al. 2011;[START_REF] Kriegeskorte | Small colony variants of Staphylococcus aureus reveal distinct protein profiles[END_REF][START_REF] Romilly | A non-coding RNA promotes bacterial persistence and decreases virulence by regulating a regulator in Staphylococcus aureus[END_REF][START_REF] Roux | CodYmediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals[END_REF]). The agr system constitutes one of the major and best-characterized players in S. aureus intracellular survival. Indeed, the up regulation of the agr locus, that triggers the expression of secreted virulence factors such as hemolysins and leukocidins, is unfavorable for intracellular persistence [START_REF] Tuchscherr | Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence[END_REF]. RpiRc, one of the three members of the transcriptional regulator RpiR family controlling enzymes involved in sugar catabolism in S. aureus, appears to function as a metabolite-responsive modulator of the agr system, that also negatively-controls RNAIII transcription [START_REF] Balasubramanian | Staphylococcus aureus Coordinates Leukocidin Expression and Pathogenesis by Sensing Metabolic Fluxes via RpiRc[END_REF]. Of note, RpiRc has been shown to repress RNAIII [START_REF] Zhu | RpiR homologues may link Staphylococcus aureus RNAIII synthesis and pentose phosphate pathway regulation[END_REF], supporting the notion of a direct connection between the PPP and the regulation of S. aureus virulence. The expression of SigB has also been shown to play a crucial role during persistence possibly by down-regulating pro-inflammatory virulence factors and increasing the expression of factors promoting persistence [START_REF] Tuchscherr | Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections[END_REF] and ∆sigB mutants are completely cleared by the host cells and fail to form SCVs (Tuchscherr and Loffler 2016). TKT inactivation which triggers the up-regulation of RNAIII and down-regulation of sigB and rpiRc appears thus to confer a non-persister phenotype during chronic infection of host cells.

A number of "Omics" approaches have been carried out to explain why S. aureus SCVs have a reduced metabolic activity and how these metabolic changes affect persistence [START_REF] Seggewiss | Reporter metabolite analysis of transcriptional profiles of a Staphylococcus aureus strain with normal phenotype and its isogenic hemB mutant displaying the small-colony-variant phenotype[END_REF][START_REF] Von Eiff | Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype[END_REF][START_REF] Kriegeskorte | Small colony variants of Staphylococcus aureus reveal distinct protein profiles[END_REF]. During its intracellular survival, a Δtkt mutant is likely to deal simultaneously with PPP blocking and altered levels of carbon-based nutrients. Our whole cell metabolomic analyses of planktonicgrown bacteria showed a huge increase in riobose-5-phosphate (R5P) intermediates in the ∆tkt mutant compared to wild-type that could be explained by the impaired entry of R5P into glycolysis and a concomitant significant decrease in sedoheptulose-7-phosphate (SH7P) relative concentration. We also observed significant changes in the relative concentrations of TCA cycle intermediates and TCA-related amino acids compared to WT strain. For example, we observed a decrease in citrate and cis-aconitase amounts, corresponding to the very first TCA intermediates, in Δtkt mutant compared to wild-type, which could account for a limited fueling of the TCA cycle at this stage. In contrast, we noted a significant increase in the relative concentrations of following TCA cycle intermediates such as α-ketoglutarate, succinate, fumarate and malate in Δtkt mutant that could be due to an increased import of proline and glutamine. The TCA cycle plays a central role in maintaining the bacterial metabolic status and has been repeatedly implicated in the regulation of staphylococcal virulence [START_REF] Somerville | Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival[END_REF][START_REF] Somerville | Synthesis and deformylation of Staphylococcus aureus delta-toxin are linked to tricarboxylic acid cycle activity[END_REF][START_REF] Ding | Metabolic sensor governing bacterial virulence in Staphylococcus aureus[END_REF]. Activity of TCA cycle enzymes, the amounts of biosynthetic intermediates and generated ATP can be affected by a number of conditions, including: i) nutrients availability, ii) a stressful environment (e.g., an intracellular environment), or iii) stress-inducing stimuli that alter carbon flux or here the PPP. These changes induce alterations of the overall metabolome of the bacterium that, in turn, may modulate the activity of metabolite-responsive global regulators such as SigB or RpiR.

We also found that bacteria grown in CDM supplemented with ribose or fructose had reduced sigB expression during stationary phase. We thus hypothesized that the modification of carbon source availability inside infected cells might be a suitable strategy to act on sigB expression and therefore impair S. aureus intracellular persistence. We were not able to modify sufficiently the carbon source availability in the cytoplasm of infected endothelial cell, using DMEM supplementation with either ribose or fructose, to abrogate S. aureus USA300 intracellular persistence. A modest reduction of S. aureus intracellular persistence was however recorded with both sugars. At any rate, impaired bacterial intracellular survival might also be due to a global modification of the endothelial cell physiology. For instance, fructose uptake by cells may be linked to oxidative stress, inflammation and autophagy [START_REF] Zhang | High Dietary Fructose: Direct or Indirect Dangerous Factors Disturbing Tissue and Organ Functions[END_REF]. Other carbon sources, non-toxic to the cell and with better efficacy on S. aureus intracellular survival control, could be the focus of future research. Ribose is present in the 100 µM range in human blood [START_REF] Gross | Serum levels of glucose, insulin, and C-peptide during longterm D-ribose administration in man[END_REF] and R5P concentration inside human lymphoblast is 70 µM [START_REF] Pilz | The influence of ribose 5-phosphate availability on purine synthesis of cultured human lymphoblasts and mitogen-stimulated lymphocytes[END_REF]. It is thus possible that the availability of ribose in the tissues may directly affects S. aureus pathogenesis by influencing bacterial growth. Our work highlighted an unprecedentedly reported role of transketolase in S. aureus intracellular persistence.

Modulation of the pentose phosphate pathway activity may represent an interesting mean to impair S. aureus survival during chronic infections.

In the third article, we showed that during chronic infection of patients with the chronic lung disease (CLD) including cystic fibrosis (CF), S. aureus adapts through the acquisition of common adaptive traits including antibiotic resistances, auxotrophies, reduced citric acid cycle activity, increased biofilm, and intracellular persistence abilities that occurred irrespective of the clone type. Of particular interest, we report mutations in two master regulatory systems, Agr and Sae, likely to impact multiple protein expression and metabolite amounts.

The agr-defective mutants, such as CF1_late isolate, have been shown to arise during chronic infections and are better adapted to persistence within the infected host [START_REF] Shopsin | Prevalence of agr dysfunction among colonizing Staphylococcus aureus strains[END_REF][START_REF] Painter | What role does the quorum-sensing accessory gene regulator system play during Staphylococcus aureus bacteremia?[END_REF][START_REF] Suligoy | Mutation of Agr Is Associated with the Adaptation of Staphylococcus aureus to the Host during Chronic Osteomyelitis[END_REF]. Genetic alterations, that directly or indirectly target SaeR regulon, were identified in the 3 CF patients.

SaeR is directly involved in the regulation of more than 20 virulence factor genes (Liu, Yeo et al. 2016) but is likely involved in the regulation of additional proteins by indirect mechanisms [START_REF] Cassat | A secreted bacterial protease tailors the Staphylococcus aureus virulence repertoire to modulate bone remodeling during osteomyelitis[END_REF]. For instance, Cassat et al showed that 31 proteins, including SdrD, significantly increase in abundance upon inactivation of sae [START_REF] Cassat | A secreted bacterial protease tailors the Staphylococcus aureus virulence repertoire to modulate bone remodeling during osteomyelitis[END_REF]. Furthermore, SaeRS-deficient bacteria are less infective in animal models [START_REF] Montgomery | Importance of the global regulators Agr and SaeRS in the pathogenesis of CA-MRSA USA300 infection[END_REF]). In the 3 CF_late isolates, we observed an increased expression of the SdrD adhesin belonging to the SaeR regulon [START_REF] Cassat | A secreted bacterial protease tailors the Staphylococcus aureus virulence repertoire to modulate bone remodeling during osteomyelitis[END_REF]Askarian, Ajayi et al. 2016), suggesting that SdrD could also be important for long-term lung colonization.

In patients with chronic lung infections, SCV detection is most often the consequence of a long-term SXT treatment [START_REF] Kriegeskorte | Thymidine-Dependent Staphylococcus aureus Small-Colony Variants Are Induced by Trimethoprim-Sulfamethoxazole (SXT) and Have Increased Fitness during SXT Challenge[END_REF]. Mutations in the thyA gene, as found in CF2 and CLD_late isolates, are known to lead to stable clinical SCVs that are no longer susceptible to SXT and are thymidine-auxotrophic (TA-SCV) [START_REF] Chatterjee | In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of Staphylococcus aureus[END_REF][START_REF] Kriegeskorte | Thymidine-Dependent Staphylococcus aureus Small-Colony Variants Are Induced by Trimethoprim-Sulfamethoxazole (SXT) and Have Increased Fitness during SXT Challenge[END_REF]. Since thymidine is assumed to be abundant during lung inflammation, TA-SCVs can still grow in the inflamed alveolar environment.

Altogether, our data confirm that metabolic specialization is a common phenomenon among long-term colonizers (La Rosa, Johansen et al. 2018). Other striking traits of phenotypic convergent evolution of S. aureus identified in this work were the increased ability of "adapted" isolates: i) to form biofilm and ii) to persist in the intracellular niche. Our multi-omics approach allowed both confirmation of previously known mechanisms and identification of novel candidate genes and pathways involved in the persistence ability of clinical isolates. We now provide evidence that the saeR/fakA regulon and the pantothenate pathway could also be promising therapeutic targets to fight persistent S. aureus infections.

The study of S. aureus adaptation in the CF model allowed us to better understand the importance of S. aureus adaptation during a chronic infection. However, our first study of S. aureus adaptation in CF patients was limited to samples collected from three patients. So, we plan to increase the number of patients in our cohort study and include bacterial isolates collected from patients since their very early period of life (at-birth) to follow the evolution of S. aureus from their primo-implantation in the lungs of CF patients.

The characteristic gel-like property of the mucus of CF patients is believed to be mainly due to the presence of high molecular weight, polydisperse glycoproteins, or mucins. Besides, the thick mucus is also an ideal breeding ground for bacteria [START_REF] Paranjape | Cystic fibrosis[END_REF]. How S. aureus interacts with the mucus components in CF patients is poorly understood.

Our laboratory has very recently initiated a collaboration with the team of C. Robbe (Institut Pasteur, Lille) who is expert on mucins. Preliminary observations of her group indicate an increased sialylation of mucins in CF airways both in vivo and ex vivo. Intriguingly, S. aureus USA300 strain appears to strongly adhere on mucins from CFTR -/-piglets airways while binding was lost after chemical desialylation of mucins. At this stage, one may hypothesize that increased sialylation function could be a convergent adaption for S. aureus in CF patient.

Further studies are underway to precise the role of sialylations on S. aureus persistence in the context of the CF disease.

  STAPHYLOCOCCUS AUREUS LUNG INFECTION CORRELATES WITH PROTEOGENOMIC AND METABOLIC ADAPTATIONS LEADING TO AN INCREASED INTRACELLULAR PERSISTENCE ................................................................................................... DISCUSSION AND PERSPECTIVES ................................................................. REFERENCE ..................................................................................................

Figure 1 :

 1 Figure 1: Entry mechanism of intracellular bacterial pathogens. A) Zipper mechanism, the example of L. monocytogenes; B) Trigger mechanism, the example of Salmonella; C) Looping mechanism, the example of F. tularensis. Adapted from Pizarro-Cerdá et al. 2016, Ribet et al. 2015.

Figure 2 :

 2 Figure 2: Model of Brucella intracellular trafficking in mammalian cells. A) the example of Brucella; B) the example of Salmonella; C) the example of Legionella. Taken from Celli 2015; Santos et al. 2016.

Figure 3 :

 3 Figure 3: Intracellular bacterial spread from cell-to-cell. Steps supporting the intracellular dissemination of Listeria monocytogenes. After escape from the vacuole into the cytosol, these bacteria polymerize actin at the bacterial pole, forming a comet-like tails on their surface, leads to the formation of bacteria-containing protrusions and cell-to-cell spread. Taken from Weddle et al. 2018.

Figure 4 :

 4 Figure 4: Model of apoptosis and pyroptosis inducing by intracellular bacteria. Apoptosis induced by F.tularensis is using the intrinsic pathway of activation: cytochrome c release from mitochondria and activation of the initiator caspase-9 and the effector capase-3. The bacterial molecule that is responsible for apoptotic induction remains unknown. Intracellular pathogens can induce proinflammatory pyroptosis via inflammasome activation by sensing of bacterial molecules, such as: flagellin of L. pneumophila and Salmonella spp. and chromosomal DNA in the case of F. tularensis. The inflammasome activates caspase-1, which leads to interleukin (IL)-1β and IL-18 activation and secretion. Cytokine secretion occurs initially through a caspase-1-dependent pore, and is then released upon necrotic cell lysis. Taken from Hybiske et al. 2008.

Figure 5 :

 5 Figure 5: Model of AIM2 inflammasome. AIM2 is a key sensor of pathogens that detects the presence of foreign DNA accumulating in the cytosol during the life cycle of intracellular pathogens. Inflammatory caspases

Figure 6 :

 6 Figure 6: Metabolic pathways of typical heterotrophic and prototrophic bacteria. The Central metabolic pathways (purple boxes) comprise: glycolysis and gluconeogenesis; the variant Entner-Doudoroff pathway; the pentose phosphate pathway; the tricarboxylic acid cycle; and various anapleurotic reactions that replenish arising metabolic gaps, such as the generation of oxaloacetate by pyruvate carboxylase. In particular, carbon metabolism driven by non-glycogenic carbon substrates, such as glycerol, pyruvate, acetate, fatty acids and succinate, requires anapleurotic reactions and gluconeogenesis. Major carbon substrates are in red boxes, and the metabolic fluxes during growth on glucose or glucose-6-phosphate (solid black arrows), glycerol or glycerol-3P (dashed black arrows) and acetate or fatty acids (red arrows) are also shown. Furthermore, the anabolic monomers (amino acids and fatty acids; blue boxes) and the major catabolic intermediates from which they derive (green arrows) are shown. Taken from Eisenreich et al. 2010.

Figure 7 :

 7 Figure 7: Simplified view of the intracellular metabolism of L. monocytogenes. Gray-framed yellow boxes and yellow arrows mark (by color intensity) the major carbon compounds taken up by the intracellular bacteria from

Figure 8 :

 8 Figure 8: Simplified view of the intracellular metabolism of Shigella flexneri. Solid arrows indicate reversible (double-headed arrows) and essentially irreversible (single-headed arrows) reactions of glycolysis and gluconeogenesis (GL/GN, red arrows), pentose phosphate pathway (PPP, blue arrows), Entner-Doudoroff pathway

Furthermore,

  Steeb et al. measured tissue colonization capabilities of the various S.Typhimurium mutants via competitive infections with mixtures of mutant and wildtype Salmonella in vivo assay[START_REF] Steeb | Parallel exploitation of diverse host nutrients enhances Salmonella virulence[END_REF]. Results indicated that no single major nutrient alone could support full Salmonella virulence. Instead, Salmonella depended on utilization of glycerol, fatty acids, N-acetylglucosamine, glucose, gluconate, arginine and lactate. These seven metabolites seemed to supply individual small nutritional contributions, only together enable normal Salmonella in vivo growth. See also Figure9.

Figure 9 :

 9 Figure 9: Simplified view of the intracellular metabolism of Salmonella enterica serovar Typhimurium. See legend of Figure 8 for further explanations and abbreviations. Taken from Eisenreich et al. 2015.

Figure 10 :

 10 Figure 10: Model of maturation of phagosome and trafficking of intracellular bacteria. Schematic drawing depicts the phagocytic responses of macrophages (MΦs) against bacterial invaders. In MΦs, upon phagosome closure, the maturing phagosome traverses an early and late phagosomal and a phagolysosomal stage paralleling endosomal maturation. Phagosome biogenesis is accompanied by continuous fusion and fission events, including fusion with trans-Golgi transport vesicles, endosomes, lysosomes, and autophagosomes. These interactions cause acquisition and loss of different stage-specific markers. A hallmark of phagosome biogenesis is acidification of the phagosomal lumen by the proton pumping vATPase. A low pH is a prerequisite for optimal enzymatic activity of most late endosomal/lysosomal hydrolases. Phagocytosis and fusion with the lysosomal azurophilic granules is often happening simultaneously. Taken from Weiss and Schaible 2015.

Figure 11 :

 11 Figure 11: A schematic of Nox2 NADPH oxidase assembly and activation. The Nox2 NADPH oxidasecomplex is comprised of the membranal proteins gp22 phox and gp91 phox (also known collectively as the cytochrome b558) and four regulatory cytosolic subunits: p40 phox , p47 phox , p67 phox and Rac2. This complex can produce a large quantity of ROS which are derived from initial production of superoxide (O2-. ). O2-. is extremely unstable and weakly bactericidal. It is subsequently transformed into hydrogen peroxide (H2O2) and then transforms into hydroxyl radical (OH-. ). The high ROS concentration generated within the phagosome are thought to kill pathogens directly due to their oxidizing capacity. Taken from[START_REF] Assari | Chronic Granulomatous Disease; fundamental stages in our understanding of CGD[END_REF]

Figure 12 :

 12 Figure 12: Model of autophagosome formation. Autophagy is stimulated during various pathological and physiological conditions such as starvation, growth factor deprivation, ER stress, and intracellular pathogen infection. Autophagy is initiated by the formation of double-membrane-bound vesicles, called autophagosomes, which sequester cytoplasmic material in a non-degradative compartment. Autophagy-related proteins (Atgs) are recruited to the isolation membrane, a source membrane of autophagosomes, and play a vital role in driving the membrane trafficking necessary for the generation of autophagosomes. Autophagosomes then fuse with lysosomes to become autolysosomes, leading to the degradation of the constituents trapped inside. Taken from Saitoh and Akira 2010.

Figure 13 :

 13 Figure 13: The model of Agr quorum-sensing accessory gene regulator system. From Painter et al. 2014. The agr operon consists of two divergent promoters, P2 and P3. Expression from P2 produces the components of a quorum-sensing system (AgrB, D, C, A) and expression from P3 produces the agr effector molecule RNAIII. RNAIII is responsible for the regulation of various virulence factors, leading to a switch from expression of cellsurface associated proteins to secreted toxins. A signal molecule, the autoinducing peptide (AIP), produced and secreted by the bacteria, accumulates outside of the cells. When the cell density increases and the AIP concentration reaches a threshold, it activates the agr response by increasing RNAIII expression. AgrB and AgrD

  infections and antibiotic treatment failure. It exits another operational definition for persistence: a subpopulation of microorganisms can persist under certain stressful condition, such as antibiotics treatment, starvation, carbon source limitation and internalization by host cells (Helaine and Kugelberg 2014), which is more widely used.

Figure 14 :

 14 Figure 14: Model of SigB activation and sigB operon structures in S. aureus. A) Model of SigB activation of S. aureus. The regulation of the σB activity is modulated by the rsbU, rsbV, and rsbW gene products. RsbW acts as an anti-σB factor. RsbV is an anti-anti-σB factor, which competes with σB for RsbW. RsbV is modulated by both the phosphatase RsbU and the phosphorylase RsbW. In S. aureus, σB is activated via the RsbU-dependent pathway (Pane-Farre, Jonas et al. 2009). RsbW inactivates σB through the complex RsbW-σB, and thus suppresses the aggregation of the σB subunit with the RNA polymerase (RNAP) core enzyme. During the exponential growth phase, RsbV is phosphorylated and inactivated by RsbW. Consequently, the σB factor is sequestered in a RsbW-σB complex. In response to environmental stress, RsbV is dephosphorylated by RsbU, leading to the formation of the highly specific RsbV-RsbW complex and the release of activated σB factor, which is able to bind to the RNAP core enzyme to form an active σB holoenzyme. B) sigB operon structures in S. aureus.

Figure 15 :

 15 Figure 15: Model of TCA cycle-dependent regulation. From Somerville et al. 2009. Several metaboliteresponsive regulators have been identified in S. aureus (e.g., CcpA, CcpE, CodY, and Rex) that are involved in the regulation of both metabolism and virulence. For examples, the carbon catabolite protein A (CcpA) responds to glucose-associated metabolic signals and inactivation of ccpA results in a lower level of RNAIII. The carbon catabolite protein E (CcpE) responds to citrate and inactivation of ccpE results in increased staphyloxanthin production and enhanced bacterial virulence in a mouse model of systemic infection. CodY responds to GTP and branched-chain amino acids and inactivation of codY results in derepressed synthesis of the agr locus enhanced bacterial virulence of USA300 strain in the mouse model.

Figure 16 :

 16 Figure 16: Model of S. aureus life cycle in professional and non-professional phagocytes. From Moldovan et al. 2019. Staphylococcus aureus is internalised by professional and non-professional phagocytes by phagocytosis.(1) Shortly after internalisation, S. aureus resides in Rab-5 positive early phagosomes, which quickly mature into

Figure 17 :

 17 Figure 17: Organs affected by cystic fibrosis. From Paranjape SM et al. 2014.

Figure 18 :

 18 Figure 18: Model of lung from normal people and cystic fibrosis patient. From Mayo Clinic website (https://www.mayoclinic.org).

Figure 19 :

 19 Figure 19: Prevalence of bacterial pathogens in cystic fibrosis. From Cystic Fibrosis Foundation Patient Registry (C) 2017 (USA).

  Survival of Staphylococcus aureus in Endothelial Cells: A Matter of Growth or Persistence Guillaume Rollin 1, 2 † , Xin Tan 1, 2 † , Fabiola Tros 1, 2 , Marion Dupuis 1, 2 , Xavier Nassif 1, 2, 3 , Alain Charbit 1, 2 * and Mathieu Coureuil 1, 2 * 1 Université Paris Descartes, Sorbonne Paris Cité, Bâtiment Leriche, Paris, France, 2 Institut National de la Santé et de la Recherche Médicale U1151 -Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Equipe 11: Pathogénie des Infections Systémiques, Paris, France, 3 Assistance Publique -Hôpitaux de Paris, Hôpital Necker Enfants Malades, Paris, France

FIGURE 1 |

 1 FIGURE 1 | S. aureus can invade and survive inside various cell types. (A) S. aureus survival inside non-professional-phagocyte cells: EA.hy926 endothelial cells derived from human umbilical vein and THP-1 monocyte-macrophages. **Wilcoxon-Mann-Whitney U-test (p < 0.05). (B) Endothelial cells EA.hy926 treatment with 50 µM cytochalasin D significantly reduced S. aureus uptake. *Student t-test (p < 0.01).

FIGURE 2 |

 2 FIGURE 2 | Observation of intracellular S. aureus. (A) EA.hy926 cells infected with USA300-GFP (green) observed in confocal microscopy (Z-project of max intensity) 1 h after infection and at day 3 and 7 after infection (DAPI: blue; phalloidin: red; USA300-GFP: green; scale bar = 20 µm). (B) EA.hy926 cells infected with USA300-WT observed in TEM 1 h after infection and at day 3 and 7. Arrows indicate living cells. Arrow heads indicate dying cells.

FIGURE 3 |

 3 FIGURE 3 | Behavior of intracellular wild type S. aureus. (A-C) Screenshots of time lapse microscopy (see Video S1-S4). EA-hy296 cells were infected by USA300-GFP and time lapse microscopy was processed for 64 h. Images were extracted at different time point. USA300-GFP: green; DNA of dying cells (propidium iodide): red. Time is in hours and minutes. Bar = 50 µm. White arrow heads point to multiplying USA300-GFP.

FIGURE 4 |

 4 FIGURE 4 | Persistence of S. aureus wild type and hemDBL mutant in endothelial cells. (A) Percent of SCVs of wild type S. aureus USA300-GFP recovered from endothelial cells at different day post infection. (B) USA300 S. aureus (dot) and hemDBL derivative (square) survival inside EA.hy926 endothelial cells. (C) Screenshots of time lapse microscopy (see Video S5). EA-hy296 cells were infected by hemDBL USA300-GFP and time lapse microscopy was processed for 8 days. Images were extracted at different time point. hemDBL USA300-GFP: green; DNA of dying cells (cytotox red reagent): red. Time is in hours and minutes. Bar = 50µm. *Student t-test (p < 0.01). White arrow heads point to non-growing hemDBL mutant bacteria.

FIGURE 5 |

 5 FIGURE 5 | Intracellular S. aureus may escape from infected host cells up to 10 days after infection. (A) Schematic representation of the experimental set-up (see Materials and Methods). Briefly, EA.hy926 cells were infected as described. At day 7 and 10 after infection cell monolayer was washed 3 times with PBS containing gentamicin 300 µg/mL and 3 times with antibiotic free PBS. After 8 h of incubation in antibiotic free medium the number of bacteria in the supernatant was assessed. The cells are then incubated 16 h more hours in antibiotic free medium and bacteria load in the supernatant and inside cells was assessed. (B-E) Mean of viable bacterial load 8 h (black) and 24 h (white) after incubation with antibiotic free medium, 7 or 10 days after infection. (B,D) Bacterial load in the supernatant. (C,E) Bacterial load inside cells. (B,C) USA300-GFP S. aureus. (D,E) hemDBL USA300-GFP. Ten CFU were considered as a detection threshold.

Figure 1 .

 1 Figure 1. Phenotypic characterization of the ∆tkt mutant A. Growth of USA300 wild type (WT), tkt mutant (tkt) and its complemented derivative (Cp-tkt). USA300 WT, ∆tkt and Cp-tkt strains were grown in BHI Broth or DMEM for 21 hours at 37°C. Optic densities were measured every hour from 0 to 8 hours and then

Figure 2 .

 2 Figure 2. Intracellular proliferation and persistence of S. aureus. A, B. Intracellular proliferation of USA300 wild type (WT) and tkt mutant (tkt) strains.

  1 and 2). Red: cell nuclei. Green: GFP expressing bacteria. For each line, a white circle was centred on a unique infected cell. Bar = 100µm. B. Quantitative analysis representing the number of green particles divided by the number of red nuclei during the first day after infection. Blue line: WT strain. Red line: tkt strain. Black line: non infected cells. C. Persistence of intracellular WT and tkt strains. Endothelial EA.hy926 cells were infected with WT and tkt strains as described above. Gentamicin was maintained throughout the experiment. Survival of S. aureus was assessed by plating and counting CFUs three, seven and ten days after infection. Results are shown as mean ± SD of triplicate measurements.

Figure 3 .

 3 Figure 3. Transcription profile of intracellular S. aureus.Endothelial EA.hy926 cells were infected with USA300 WT and tkt strain as described in figure 2. One, three or seven days after infection, cells were washed and collected. RNA

Figure 4 .

 4 Figure 4. Proliferation of S. aureus in the spleen and the kidney of infected mice. Bacterial loads in the spleen and the kidney of BalB/c mice infected intravenously with 10 7 bacteria of the WT, tkt and Cp-tkt strain. Bacterial counts are expressed in CFU/organ (WT and tkt n=10; Cp-tkt n=6). Data are shown as mean ± SD. Statistical significance was determined using the Kruskal Wallis test with Dunn's correction (ns: not statistically different, *** p<0.001, **** p<0.0001). No statistical significance between strains was observed in the

Figure 5 .

 5 Figure 5. Carbon sources influence the transcription profile of the SigB pathway.S. aureus USA300 WT strain was grown to stationary phase in CDM supplemented with

Figure 6 .

 6 Figure 6. Schematic depiction of the impact of TKT inactivation on sigB and rpiRc

  After denaturation at 70 °C for 5 min, 15 μL of the mix containing 4 μL M-MLV 5 X reaction buffer, 1 μL of 0.5 mM PCR Nucleotide Mix, 0.5 μL of RNasin Ribonuclease Inhibitor, and 1 μL M-MLV Reverse Transcriptase (Promega) was added. Samples were incubated 5 min at 25 °C, 1 h at 42 °C, then 15 min at 70 °C and chilled on ice. Quantitative RT-PCR was performed using FastStart SYBR Green qPCR Master Mix (ThermoFischer Scientific) or

  -7-P (SH-7P), Ribose 5-phosphate, Glyceraldehyde 3-phosphate, Lactate, Pyruvate, Phospho-Enol-Pyruvate (PEP), Citrate (Cit), cis-aconitate (cis-aco), Succinate (Suc), Fumarate (Fum), Malate (Mal), alpha ketoglutarate (aKG), dihydroxyacetone phosphate (DHAP), N-carbamoyl-L-aspartate, Nicotinamide adenine dinucleotide (NAD), NADH, Nicotinamide adenine dinucleotide phosphate (NADP), NADPH, Adenine, Adenosine, Adenosine Mono, Di and Triphosphate (AMP, ADP, ATP), Cytidine, Cytidine Mono, Di and Tri-phosphate (CMP, CDP, CTP), Uridine Mono, Di and Tri-phosphate (UMP, UDP, UTP), Hypoxanthine, Guanine, Guanaosine, Guanidine Mono and Di-phosphate (GMP, GDP), Inosine monophosphate (IMP), Pantothenate, Dephospho-Coenzyme A, Coenzyme A (CoA), Flavin adenine dinucleotide (FAD), Niacin, Nicotinamide, 3-hydroxybutyrate, Urate, 5-oxo-L-Proline, Acetylaspartate, Acetyl-glutamine, Alanine, Arginine, Argininosuccinate, Asparagine, Aspartate, Betaine, Citrulline, Cystathionine, Cystine, Glutamine, Glutamate, Glycine, Histidine, IsoLeucine, Leicine, Sarcosine, Lysine, Methionine, Ornithine, Phenylalanine, Proline, Serine, Taurine, S-Adenosyl-L-methionine, Threonine, Tryptophan, Tyrosine, Valine, Butyric acid, Carnitine, Decanoic acid, Lauric acid, Hexanoic acid, Linoleic acid, Myristic acid, Octanoic acid, Oleic acid, Palmitic acid.

Figure S1 .

 S1 Figure 1

Figure S2

  Figure S2

Figure S3 .

 S3 Figure S3

  Figure S4

Figure S5 .

 S5 Figure S5. Transcription profile of intracellular S. aureus.Endothelial EA.hy926 cells were infected with USA300 WT, ∆tkt and Cp-tkt strains as described in figure2. One, three or seven days after infection cells were washed and collected. RNA from intracellular S. aureus were prepared and gene expression of RNAIII, rpiRc and sigB was analysed by quantitative RT-PCR. Gene expression level was normalized by that of gyrA at days 1, 3 and 7 after infection of EA-hy296 cells. Data are shown as mean normalized expression ± SD of triplicate measurements from two independent experiments. (ANOVA, ns : not statistically different; * p<0.01, ** p<0.001). Experiments presented in FigureS5were independent of those presented in Figure3.

-

  Figure S5

  Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019 2 • CID 2019:XX (XX XXXX) • Tan et al

  Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019 Persistent infections in CF lungs • CID 2019:XX (XX XXXX) • 3

Figure 1 .

 1 Figure 1. Selection of 4 pairs of Staphylococcus aureus isolates belonging to 4 sequence types (STs) in 4 patients. Dendrogram generated by wgsa.net from the genomes of the 8 clinical isolates retrieved from the respiratory samples of 3 patients with cystic ibrosis (CF), 1 patient with non-CF chronic lung infection, and 4 reference genomes from public databases. Branch length is proportional to the number of variant nucleotide sites within the core genes. For each patient, the isolate taken irst is termed "early" and the isolate taken later is termed "late." The dates of sampling and the ST of the isolates are indicated. "TA" and "PA" mean that the isolate is auxotrophic for thymidine or pantothenate, respectively. The name of the isolate is indicated in red when it is methicillin-resistant S. aureus. Reference strains included are PFESA1902 (ERR554197), st1307 (ERR158691), and PFESA1195 (ERR554722). Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease.Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019

Figure 2 .

 2 Figure 2. Intracellular persistence of Staphylococcus aureus clinical isolates in cystic ibrosis bronchial epithelial (CFBE)-F508del epithelial cell line and within primary patient cells. (A and B) Bronchial CFBE epithelial cell line (CFBE-F508del homozygous for the F508del-cystic ibrosis transmembrane conductance regulator [CFTR] mutation) wasinfected with the control strain USA300-LAC and clinical isolates from cystic ibrosis (CF) patients (A) or the chronic lung disease (CLD) patient (B). (C and D) Primary nasal epithelial cells retrieved from a healthy donor ("Primary WT") (C) and from the CF3 patient ("Primary CF3" with F508del +/+ CFTR mutation) (D) were infected with the control strain USA300-LAC and CF3 isolates. For all experiments, gentamicin was present throughout the experiment to prevent extracellular bacterial growth and new infection. Bacterial loads inside cells were evaluated by colony-forming unit (CFU) enumeration at 3 and 6 days after infection. Results are normalized with USA300-LAC strain as a reference and expressed as a fold change of CFUs. Results have been obtained from 2 independent experiments performed in triplicate for A, B, and C and 1 experiment for D. Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease; ns, not signiicant; WT, wild type. Statistical analysis was performed by Wilcoxon rank sum test: *P < .05; **P < .01; P > .05. Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019

Figure 3 .

 3 Figure 3. Quantiication of bioilm formation of Staphylococcus aureus clinical isolates. Bioilm formation quantiication was performed using the crystal violet microtiter assay in brain-heart infusion medium with 1% glucose. Results shown are the mean ± standard deviation for 3 independent experiments performed in triplicate. Statistical signiicance was measured using a 2-tail Student t test when bioilm production of a late isolate was compared with bioilm production of a cognate early isolate from the same patient. Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease. ** indicates P < .001 and ns indicates P > .05.

Figure 4 .

 4 Figure 4. Growth of late Staphylococcus aureus clinical isolates in cystic ibrosis sputum medium. Growth curves were carried out in medium that mimics the respiratory luid of cystic ibrosis patients, with or without the addition of thymidine or pantothenate. The results shown correspond to a representative experiment. The orange and green curves correspond to bacterial growth in media supplemented with either thymidine or pantothenate; the red and blue curves correspond to bacterial growth in medium without thymidine or pantothenate. WT, USA300-LAC. Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease; Panto, pantothenate; Thy, thymidine; WT, wild type.

Figure 5 .

 5 Figure 5.Proteogenomic and metabolomic analysis of the 4 pairs of Staphylococcus aureus isolates. (A) Vertical histograms show the functional classiication of proteins encoded by genes with nonsynonymous mutations in the genomes of late isolates of S. aureus compared to early isolates. (B) Horizontal histograms show the functional classiication of differentially expressed annotated proteins in late compared to early isolates of each patient. For each category, histograms represent the number of downand upregulated proteins from proteomic analysis using the threshold of <2 and >2, respectively. Only genes and proteins with functional annotation available are included. The "cellular processes and signaling" category encompasses regulatory proteins and proteins involved in cell wall and capsule synthesis. The "information storage and processing" category encompasses proteins involved in replication, translation, and repair processes. The "metabolism" category encompasses proteins involved in metabolism and transport. The "virulence" category encompasses exotoxins and proteins involved in adhesion, bioilm formation, and immunomodulation. (C) Categorization in 8 categories of an altered amount of metabolites in late compared to early isolates. Metabolites were detected by carrying out 2 independent experiments performed in triplicate. Abbreviations: AA, amino acids; CF, cystic ibrosis; CLD, chronic lung disease; CoA, coenzyme A; Nucl, nucleotides; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle.Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019

  Malate (Mal), alpha ketoglutarate (aKG), dihydroxyacetone phosphate (DHAP), Ncarbamoyl-L-aspartate, Nicotinamide adenine dinucleotide (NAD), NADH, Nicotinamide adenine dinucleotide phosphate (NADP), NADPH, Adenine, Adenosine, Adenosine Mono, Di and Tri-phosphate (AMP, ADP, ATP), Cytidine, Cytidine Mono, Di and Tri-phosphate (CMP, CDP, CTP), Uridine Mono, Di and Tri-phosphate (UMP, UDP, UTP), Hypoxanthine, Guanine, Guanaosine, Guanidine Mono and Di-phosphate (GMP, GDP), Inosine monophosphate (IMP), Pantothenate, Dephospho-Coenzyme A, Coenzyme A (CoA), Flavin

Figure S2 .

 S2 Figure S2. Quantification of biofilm formation of S. aureus clinical isolates.Biofilm formation was monitored using the crystal violet microtiter assay in BHI medium with 1% glucose. USA300-LAC strain (WT) was used as a reference for weak biofilm production whereas its agrC derivative obtained from the Nebraska Transposon Mutant Library was used as a reference for strong biofilm production.Results shown are the mean ±SD for three independent experiments performed in triplicate. Statistical significance was measured using one-way ANOVA with multiple comparisons (Dunnett's correction) performed on the dataset as a whole, with each value compared to the WT. ** indicates p-value of <0.001.

Figure S3 .

 S3 Figure S3. Growth curves of early S. aureus clinical isolates in CFSM. Growth curves were carried out in CFSM. The results shown correspond to a representative experiment. WT, USA300-LAC.

Figure S4 .

 S4 Figure S4. Colony morphology of USA300-LAC (WT), CF2_late and CLD_late isolates. Bacteria were grown on BHI agar incubated 12h at 37°C.

Figure S5 .Figure 2 ,

 S52 Figure S5. Heatmaps showing comparison of protein and metabolite profiles of early/late S. aureus isolates. The isolates were cultured to the stationary phase in medium mimicking the respiratory fluid of cystic fibrosis patients (CFSM) with the addition of thymidine. A) Heatmap visualization and hierarchical clustering analysis of the proteomic profiling in the late isolate compared to the early isolate of each patient. One experiment with three biological replicates performed for each isolate is shown. Rows: proteins; columns: samples; color key indicates protein relative concentration value (yellow: lowest; blue: highest). B) Heatmap visualization and hierarchical clustering analysis of the metabolite profiling in the late isolate compared to the early isolate of each patient. The top 50 most changing compounds of one representative experiment with three biological replicates performed for each isolate are presented. Rows: metabolites; columns: samples; color key indicates metabolite relative concentration value (blue: lowest; red: highest).

Table 1 : Mechanisms of vacuolar escape by selected intracellular pathogens. Taken

 1

	Bacterial gene required	Shigella flexneri IpaB IpaC T3SS	Listeria monocytogenes LLO and type C phospholipases	Francisella tularensis T6SS, acid phosphatases,…
	Host factors required	Unknown	GILT activates reductase by a thiol LLO	Unknown
	Vacuole conditions required	Unknown	Acidic vacuole (pH 5.5)	Acidic vacuole
	Kinetics of escape	15-30 minutes	17 minutes	30-60 minutes

from Ray et al. 2009.

Table 2 : Virulence factors involved in the pathogenesis of S. aureus and respective putative functions. From

 2

	Costa Rita et al. 2013.

  [START_REF] Proctor | Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus[END_REF]. Persistent and relapsing infections associated with smallcolony variants of Staphylococcus aureus. Clin. Infect. Dis.20, 95-102. doi: 10.1093/clinids/20.1.95 Sendi, P., and Proctor, R. A. (2009). Staphylococcus aureus as an intracellular pathogen: the role of small colony variants. Trends Microbiol.

  . nptII: neomycin phosphotransferase gene, determing kanamycin resistance.

	pMiniT 2.0	Plasmid used to clone upstream and	New England Biolabs
		downstream regions from tkt gene and
		nptII, Amp R
						and European Directive
	2010/63/UE) and approved by the INSERM Ethics Committee (Authorization Number: 75-
	906).
	Strains and plasmids used in this study.
	Strain or plasmid	Relevant characteristic(s)		Source of reference
	Strains
	E.coli DH5α	Φ80' lacZ ∆M15 gyrA96 recA1 relA1	Invitrogen
		endA1 thi-1 hsdR17
	S.aureus USA300 LAC	MRSA containing phage ΦSA2usa	BEI Resources
		(PVL), Ery R
	USA300-Ery S	Erythromycin-sensitive derivative of	Laboratory collection
		USA300
	RN4220	Restriction-deficient transformation		Laboratory collection
	USA300 ∆tkt USA300 ∆tkt-CPtkt	recipient S. aureus tkt defective mutant, Kan R Complemented strain, USA300 ∆tkt	This study This study
		containing pCN57-CPtkt, Ery R Kan R
	USA300 LAC-gfp	Strain containing pCN57-GFP, Ery R	Laboratory collection
	USA300 ∆tkt-gfp	USA300 ∆tkt containing pCN57, Ery R	This study
		Kan R
	Plasmids
	pMAD	Plasmid with a thermosensitive origin,	Laboratory collection
		used for gene deletion; Amp R Ery R
	pMAD-∆tkt	Derivative of pMAD with upstream and	This study
		downstream sequences from tkt gene
		and nptII a , Amp R Ery R Kan R
	pCN57	Plasmid containing gfp expression	Laboratory collection
		cassette (constitutive expression),
		Ery R
	pXT02	Derivative	of	pCN57.	The	This study
		gfpexpression cassette was replaced
		by the sequence of the tkt gene with
		its own promoter.

a

  Protein model of S. aureus transketolase. Swiss-Model was used to predict the structure of S. aureus TKT (SAUSA300_1239) as a homodimer based on the template 3M49.1.A (Crystal Structure of Transketolase from Bacillus anthracis). The QMQE score and QMEAN were 0.83 and -0.59 respectively. C. Table showing coverage percentage between transketolase amino acid sequences and percentage of identical amino acids between aligned transketolase amino acid sequences. Protein sequences were retrieved from UniProt database and aligned using Clustal Omega.

	A	1 I		662 aa I
			Transketolase_N	Transket_pyr	Transketolase_C
		Phe 2A
				Leu 662A
		Protein ID TKT_HUMAN TKT_MOUSE TKT2_ECOLI TKT_HAEIN TKT1_VIBCH TKT_DICDI TKT1_YEAST TKT_TREPA TKT_MYCBO TKT_STRPN TKT_STAAS TKT_BACSU	% coverage % identity Species 100.0% 100.0% Homo sapiens 100.0% 94.7% Mus musculus 94.5% 24.1% Escherichia coli 94.2% 22.9% Haemophilus influenzae 94.4% 24.0% Vibrio cholerae 94.2% 22.6% Dictyostelium discoideum 94.5% 23.8% Saccharomyces cerevisiae 93.9% 21.9% Treponema pallidum 96.6% 20.6% Mycobacterium bovis 94.2% 23.0% Streptococcus pneumoniae 94.4% 22.4% Staphylococcus aureus 94.9% 22.7% Bacillus subtilis
	Figure S2. Transketolase proteins domain organization and structure. A. Conserved domains found in S. aureus transketolase. Transketolase_N (PF00456 domain): Transketolase, thiamine diphosphate binding domain; Transket_pyr (PF02779 domain): Transketolase, pyrimidine binding domain; Transketolase_C (PF02780): Transketolase, C-terminal domain (putative regulatory molecule binding site). B.

  Statistical analysis was performed by Wilcoxon rank sum test: *P < .05; **P < .01; P > .05. Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019

Table 2 . General Features of Detected Mutations

 2

	Variable	CF1_late	CF2_late	CF3_late	CLD_late
	Time since early isolate	2.8 years	6.7 years	9 years	4.4 years
	Total polymorphisms	21	34	79	30
	Single-nucleotide polymorphism	19	25	73	26
	Insertion-deletion	2	9	6	4
	Coding sequence	18	21	62	23
	Nonsynonymous mutation (%)	14 (77.8)	16 (47.1)	51 (64.6)	17 (56.7)
	Frameshift variant	2	4	4	2
	Missense variant	10	11	47	14
	Premature stop codon	1	1	0	1
	Other	1	0	0	0
	Synonymous mutation	4	5	11	6
	Intergenic	3	13	17	7

Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease.

Table 1 . Mutations Likely Linked to Phenotypic Changes in Clinical Isolates

 1

	Isolate	Gene	Product	Mutation	Putative Associated Phenotype
	CF1_late agrC Accessory gene regulator protein C	Premature stop codon (L193X) Downregulation of toxins and upregulation of adhesins
	CF1_late saeR Staphylococcus exoprotein expression protein R	Missense G179L	Upregulation of SdrD adhesin
	CF2_late thyA Thymidylate synthase	Premature stop codon (W88X) SXT resistance and thymidine auxotrophy
	CF2_late rpsJ 30S ribosomal protein S10 CF2_late thiM Hydroxyethylthiazole kinase CF2_late fakA Fatty acid kinase A CF3_late panB 3-methyl-2-oxobutanoate hydroxymethyltransferase Frameshift Missense K57M Frameshift Missense G187D CF3_late fakA Fatty acid kinase A Frameshift CF3_late gyrB DNA topoisomerase subunit B Missense F226S CF3_late saeR Staphylococcus exoprotein expression protein R Missense A190T CLD_late thyA Thymidylate synthase Missense P48R Abbreviations: CF, cystic ibrosis; CLD, chronic lung disease; SXT, sulfamethoxazole-trimethoprim.	Cyclines resistance Decrease in adenosine diphosphate Increased bioilm formation and intracellular persistency Pantothenate auxotrophy Increased bioilm formation and intracellular persistency Fluoroquinolones resistance Upregulation of SdrD adhesin SXT resistance and thymidine auxotrophy	Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019

Persistent infections in CF lungs • CID 2019:XX (XX XXXX) • 7

  Metabolites were detected by carrying out 2 independent experiments performed in triplicate. Abbreviations: AA, amino acids; CF, cystic ibrosis; CLD, chronic lung disease; CoA, coenzyme A; Nucl, nucleotides; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle. Downloaded from https://academic.oup.com/cid/advance-article-abstract/doi/10.1093/cid/ciz106/5308510 by SCD Paris Descartes user on 09 September 2019

Frontiers in Microbiology | www.frontiersin.org

July 2017 | Volume 8 | Article 1354

Acknowledgements

ACKNOWLEDGMENTS

We thank Elise Borezee-Durant for the gift of S. aureus strain RN4220 and helpful technical advises. We greatly acknowledge Corinne Cordier and Jerome Megret (Necker Institute Cytometry Facility), Nicolas Ugolin (Necker Institute Imaging Facility) and Alain Schmitt (Cochin Institute Electron Microscopy Facility) for their technical support. We also thank Diana Mihaela Dincã for kindly providing support with the live cell image analysis system IncuCyte TM . The following reagent was provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: Staphylococcus aureus, Strain USA300-0114, NR-46070; Escherichia coli-Staphylococcus aureus Shuttle Vector pNR-46158, Recombinant in Staphylococcus aureus, NR-46158. Acknowledgements We are grateful to the Cell imaging core facility of the "Structure Fédérative de Recherche" Necker INSERM US24/CNRS UMS3633 for his technical support. The following reagents were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: Staphylococcus aureus, Strain USA300-0114, NR-46070; Escherichia coli -Staphylococcus aureus Shuttle Vector pNR-46158, Recombinant in Staphylococcus aureus, NR-46158.

Many thanks to Pr. Xavier NASSIF, to accept me to work in his laboratory. Thanks to the financial support from China Scholarship Council and INSERM, These studies were supported by INSERM, CNRS and Université Paris Descartes Paris Cité Sorbonne. This work was supported by Vaincre La Mucoviscidose (grant RF20180502254). Xin Tan was funded by a scholarship from the China Scholarship Council (n° CSC NO. 201508500097). The funders had no role in study design, dat a collection and analysis, decision to publish, or preparation of the manuscript.

FUNDING

These studies were supported by INSERM, CNRS and Université Paris Descartes Paris Cité Sorbonne. GR was funded by a fellowship from the "Fondation pour la Recherche Médicale" (FRM: DEA20150633005). XT was funded by a scholarship from the China Scholarship Council (n • CSC NO. 201508500097). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abbreviations

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01354/full#supplementary-material

Chapter II: Transketolase of

Staphylococcus aureus is involved in the control of master regulators of stress response during infection

Ethics statement

All experimental procedures involving animals were conducted in accordance with guidelines established by the French and European regulations for the care and use of laboratory animals (Decree 87-848, 2001(Decree 87-848, -464, 2001(Decree 87-848, -486 and 2001-131 -131

Supplementary Methods

Strains, Cells, Culture conditions and Infection. S. aureus. The epidemic clone S. aureus USA300-LAC (designated USA300-WT) was provided by the Biodefense and Emerging Infections Research Resources (BEI). The GFPexpressing strain was generated by curing the p03 plasmid from USA300-WT and introducing the pCN57-GFP recombinant plasmid (obtained from the BEI) by electroporation as described before [1]. The tkt mutant strain and the complemented strain were constructed as described below. Strains and plasmids used are summarized at the end of these supplementary methods.

Before experiments bacteria were grown 24 hours at 37°C on BHI agar plates supplemen ted with antibiotics when required and then overnight in BHI Broth from an optical density 600 nm (OD600) of 0.05. The day of experiment, bacteria were resuspended in BHI broth to an OD600 of 0.05 and grown at 37°C until the culture reached the initial-middle log phase (OD600 of 0.6).

Growth curve were performed in brain heart infusion, DMEM and chemically defined medium (CDM) supplemented with ribose or fructose or glycerol when it was required [2].

Internalization rate and survival inside the EA.hy926 endothelial cell line were similar between the GFP strain and the parental strain. Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to beneit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Author contributions. X. T., M. C., X. N., A. C., and A. J. conceived and designed the study. X. T., E. R., M. D., D. E., F. T., J. M., and A. J. performed the experiments and analysis. I. N., C. C., I. C. G., A. F., and I. S. G. contributed with data and analysis. A. J., A. C., E. R., and X. T. wrote the manuscript, with contributions and comments from all authors.

Acknowledgments. he authors thank Aurélie Hatton, Charlotte Roy, and Zhicheng Zhou for their help with primary nasal epithelial cell cultures.

Disclaimer. he funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial support. his work was supported by a scholarship from the China Scholarship Council (CSC NO. 201508500097), provided to X. T.

Potential conlicts of interest. All authors: No reported conlicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conlicts of Interest. Conlicts that the editors consider relevant to the content of the manuscript have been disclosed.

Supplementary Methods

Patients and bacterial isolates

Serial isolates of S. aureus were obtained from airway secretions from four patients with chronic lung infection at the Necker-Enfants Malades University Hospital, Paris, France.

The CF1 patient (F508del +/+ CFTR mutation) (born in 2001) was treated repeatedly with the association of rifampicine and fusidic acid for methicillin-resistant S. aureus (MRSA) and ciprofloxacine, ceftazidime and meropenem together with inhaled tobramycin or colimycin for P. aeruginosa. We selected an early isolate (CF1_early) from 2012 and a late isolate (CF2_late) from 2015.

The CF2 patient (F508del/R347P CFTR mutation) (born in 1997) was treated with association of amoxicillin-clavulanate and trimethoprim-sulfamethoxazole (SXT) and also received occasionally minocycline and streptogramins during bronchial exacerbations. We selected an early isolate (CF2_early) from 2009 and a late isolate (CF2_late) from 2015.

The CF3 patient (F508del +/+ CFTR mutation) (born in 2002) displayed a coinfection with P. aeruginosa requiring repeated intravenous antibiotics including ceftazidime, amikacin and ciprofloxacin. We selected an early isolate (CF3_early) from 2008 and a late isolate (CF3_late) from 2017.

The CLD patient (born in 1997) was not CFTR mutated but exhibited a primary humoral immunodeficiency disorder with a mild IgG2 deficiency causing chronic bronchitis.

Bronchial exacerbations were treated with SXT association. We selected an early isolate (CLD_early) from 2009 and a late isolate (CLD_late) from 2013.

Nasal epithelial cell (HNE cells) were sampled by nasal brushing of both nostrils after local visualization of the nasal mucosa from one healthy volunteer and patient CF3.

The epidemic clone S. aureus USA300-LAC (designated WT) was provided by the Biodefense and Emerging Infections Research Resources (BEI). A derivative of USA300-LAC with a deletion in the haemin pathway (ΔhemDBL triple deletion mutant), involved in the electron transport system, has been previously constructed to obtain a strain with a stable SCV-phenotype [1].

S. aureus isolates were grown on brain heart infusion (BHI) broth and agar plates. Colony morphotypes were assessed after 24h of growth on BHI or Mueller-Hinton agar plates.

When indicated, S. aureus isolates were cultured in medium mimicking the respiratory fluid of cystic fibrosis patients (Cystic Fibrosis Sputum Medium or CFSM) [2] with or without the addition of thymidine (100 μg/mL) or pantothenate (50 μg/mL).

Whole-genome sequencing and analysis

Genomic DNA was extracted using DNeasy Blood and Tissue Kit with 10 μg/mL lysostaphin to facilitate cell lysis. Genomic libraries were prepared using a Nextera XT kit, multiplexed and sequenced on an Illumina MiSeq instrument (2x150 paired-end sequencing).

The short reads were processed using the Nullarbor bioinformatic pipeline software v1.20 (Available from: https://github.com/tseemann/nullarbor). Briefly, raw reads were trimmed using trimmomatic [3], de novo assembled using SPAdes 3.9.0 [4] and annotated using Prokka v1.11 [5]. Single nucleotide polymorphisms (SNPs) and small indels were assessed using Snippy v3.1 (https://github.com/tseemann/snippy). The Similar Genome Finder Service of the PATRIC 3.5.2 on-line database was used to identify the most similar publically available genome for each pair of clinical isolates (https://www.patricbrc.org/app/GenomeDistance). Snippy was used both to map reads of late isolates against the annotated assembly of the early isolate and to map reads of early and late isolates against the publically available closest reference genome for the four pairs. Snippy classifies variants by their predicted functional effect based on mapping to the annotated reference genome in synonymous, nonsynonymous or truncating for protein-coding sequences.

Assemblies generated by Nullarbor were uploaded in RAST server [6] and were also annotated through the RAST annotation pipeline with ClassicRAST annotation scheme. The 'Sequence Based Comparison Tool' of the RAST server, which is based on BLAST protein similarity search, was used to identify large insertions and deletions of each late isolate compared to early isolate as a reference.

Quantification of biofilm formation

To determine biofilm formation in static conditions, clinical isolates as well as reference strains were cultured in Brain Heart Infusion (BHI) medium in the presence of 1% glucose.

The reference strains include USA300-LAC that is a weak biofilm producer and its ΔagrC derivative that is a strong biofilm producer (obtained from the BEI Nebraska Transposon Mutant Library). The over-night cultures were diluted to an OD600nm of 0.05 in fresh BHI medium supplemented with 1% glucose. A total of 200 μl of each diluted culture were inoculated in two polystyrene 96-well plates in triplicate and incubated for 48 hours at 37 ° C without shaking. After 24 h of growth, the medium was renewed. After 48 h, one of the 96well plates was used to assess bacterial growth by taking into account both bacteria in suspension and adherent bacteria (resuspended mechanically) and measuring OD600nm. The other 96-well plate was used to quantify biofilm formation. The wells were washed three times with distilled H2O to remove unattached bacteria and the biofilm fixed at the bottom of the wells was stained with 100 μl 2 % crystal violet solution for 15 min. After staining, wells were washed three times with distilled H2O. Crystal violet was solubilized using 150 μl of acetic acid at 30 %. 100 μl of the solubilized dye was transferred to a new 96-well plate to determine the absorbance at 540 nm.

Antimicrobial susceptibility

Antimicrobial susceptibility of isolates was evaluated by disc diffusion method. Etests were used to determine minimal inhibitory concentrations to gentamicin. Susceptibility tests were carried out on BHI agar plates to allow the growth of auxotrophic isolates.

Cell culture experiments

Cystic Fibrosis Bronchial Epithelial cell line CFBE41o-that is an immortalized cell line homozygous for the delF508 mutation was generously provided by Dieter Gruenert (University of California, San Francisco) [7]. Cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% amphotericin B.

Primary nasal epithelial cell (HNE) were obtained as described in [8] and cultured in Dulbecco's Modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM:F12) supplemented with 10% FBS, 1%v/v non-essential amino acids, 90 μg/10 μg/ml Piperacillin/Tazobactam and 1% amphotericin B.

Prior infection assays, epithelial cells were seeded in uncoated wells for CFBE and wells coated with collagen for HNE and all antibiotics were removed from culture medium.

Epithelial cells were infected with a multiplicity of infection (MOI) of 100 using an inoculum taken from cultures of S. aureus grown in BHI until exponential growth phase. After 1h30, each well was washed three times with 1 mL of phosphate buffer saline (PBS) containing 300 μg/mL gentamicin, to remove extracellular bacteria. Washing and addition of fresh medium containing 50 μg/mL gentamycin were repeated at day 3. Gentamicin has a high bactericidal effect on S. aureus USA300 (minimum inhibitory concentrations (MIC) = 2 μg.mL -1 ) and a very poor penetration inside eukaryotic cells. In addition, the measured MIC of gentamicin of

Supplementary figures