Ahpc Alkykhydroxide

Msa Modulator

Keywords:

well as personal life. A warmest thank you to my

ACKNOWLEDGMENT

Steve Jobs once said "Great things are not done by one person. They're done by a team of people". As I read these lines, I am realized that a thesis is far from being a solitary task. I could never have completed this journey without the support of a large number of people whose kindness, exchanges and advice have allowed me to learn and grow.

First of all, I would like to acknowledge INSERM, Region de Bretagne and Pharmacy Faculty of University of Rennes 1 for providing me with the financial means to complete this project. Next, I would like to express my gratitude to the jury members of my thesis defense: Dr. Francis Repoila, Dr. Maude Guillier, Dr. David Lalaouna, Dr. Eric Guedon, Pr.

Olga Soutourina and Pr. Reynald Gillet, who so generously took time out of their schedules to supervise my work. I am also thankful to Dr. Philippe Bouloc for the constructive comments and recommendations for my paper, and to Dr. Tatiana Rochat and Pr. Olga Soutourina for being on my thesis committee and providing me invaluable feedback on my work.

Further, I wish to express my special regards to Pr. Brice Felden for believing in me and for giving me the opportunity to pursue my PhD in his laboratory. His passion for scientific research is a source of inspiration and his absence will be missed by many people.

I would also like to thank Dr. Vincent Cattoir for carrying on the responsibility of guiding the team and for supporting me throughout my last year of PhD.

I would like to express my deepest gratitude to Dr. Svetlana Chabelskaya, who has guided me positively throughout this project with your motto: "Everything will be alright!". I could not ask for a better supervisor. Your patient support, guidance, human qualities, and overall insights in the research field have made this an inspiring experience for me.

I would like to express my appreciation to my "partner in crime", Dr. Cintia D. Gonzalez.

You have been there since the beginning of my PhD journey. I will miss all the over working hours together, our discussions and our laughter. My thesis would not be the same without you.

I am also thankful to Valérie Bordeau, Nathalie Ramage and Hélène Le Pabic for lending me your ear, for the comforting words you always seem to be able to find and all the laughter that have brightened up my days.

ACKNOWLEDGMENT

Many thanks to my former colleagues, Camille Riffaud, Julie Bronsard and Simon Raynaud for welcoming me into the lab and for supporting me during my thesis. All the nights out in the bars with you all and with Steven Crété and Julie Legros were memorable. I hope that we can reunite one day to reminisce our experiences together.

I would also like to acknowledge all the past and present members of the lab: Annick Lefèvre, Anita Quelavoine, Astrid Rouillon, Anne Moreau, David Luque Paz, Gaëtan Pascreau, Guillaume Ménard, Irène Nicholas, Killian Le Neindre, Lorraine Adam, Loren Desjoies, Marie-Laure Pinel-Marie, Matthieu Revest, Marc Hallier, Mohammed Sassi, Noëlla Amiot, Pierre-Yves Donnio, Régine Brielle, Sophie Reissier, Thomas Vernay, Tony Mauro, Yoann Augagneur, for their precious advice and support.

I would like to wish good luck to the new arrivals: Chloé Silard, Emiline Ostyn, Kévin Huguet, Marie Suriray, Maëliss Germain, and Laurence Fermon. "May the Odds be ever in your favors". And all the best for your PhD journey ending, Charlotte Oriol.

I. Discovery and characterization of Staphylococcus aureus

The history of bacteriology attributes constantly the name Staphylococcus aureus to Anton J. Rosenbach, a German surgeon of Göttingen (Figure 1). However, it was Robert Koch, a German physician-scientist (reviewed in [START_REF] Blevins | Robert Koch and the 'golden Age' of Bacteriology[END_REF], who first discovered Staphylococcus aureus as the major cause of suppuration in 1878. Not too long after, in 1880, Alexander Ogston, a Scottish surgeon, extended the studies of these micrococci in acute abscesses and found Streptococcus as the other cause of wound infection [START_REF] Ogston | Report upon Micro-Organisms in Surgical Diseases[END_REF]. In 1882, Ogston named the clustered micrococci "staphylococci", from Greek staphyl for "bunch of grapes". Two years later, Anton J.

Rosenbach successfully isolated two strains of staphylococci and named them based on the form and the color of their colonies: Staphylococcus aureus, aurum from Latin for "golden" and Staphylococcus albus, albus from Latin for "white" (later known as epidermidis) [START_REF] Newsom | Ogston's Coccus[END_REF]. This color was later shown to be due to the synthesis of an orange-yellow pigment, staphyloxanthin (STX), which is involved in resistance to oxidative stress, protection against lysis by host neutrophils and in animal model pathogenesis [START_REF] Clauditz | Staphyloxanthin Plays a Role in the Fitness of Staphylococcus Aureus and Its Ability to Cope with Oxidative Stress[END_REF].

Staphylococcus aureus is a bacterium with a high infectious power, of the genus Staphylococcus and of the family of Staphylococcaceae. For more taxonomic precision, the genus Staphylococcus belongs to the reign of the Prokaryotes, to the division of Firmicutes, class of Bacilli, order of Bacillales. To date, there are 49 species and 27 sub species classified in the genus Staphylococcus. S. aureus is characterized to be a non-motile, non-spore-forming Gram-positive coccus, which grows into round, golden-yellow and smooth colonies with a diameter of 0.8 -1 µm, in blood agar [START_REF] Merghni | Antioxidant and Antiproliferative Potential of Biosurfactants Isolated from Lactobacillus Casei and Their Anti-Biofilm Effect in Oral Staphylococcus Aureus Strains[END_REF]) (Figure 2). This bacterium has a relatively porous outer cell wall composed of peptidoglycans, teichoic acids, and lipoteichoic acids [START_REF] Vatansever | Antimicrobial Strategies Centered around Reactive Oxygen Species -Bactericidal Antibiotics, Photodynamic Therapy, and Beyond[END_REF], which is favorable for antibiotics treatments, compared to Gram-negative bacteria [START_REF] Segalla | Photophysical, Photochemical and Antibacterial Photosensitizing Properties of a Novel Octacationic Zn(II)-Phthalocyanine[END_REF].

As for growth environment characterization, S. aureus is halophilic (well growth in medium containing high salt concentration, approximate 5 -7% of NaCl), mesophilic (optimal growth temperature around 37°C) and neutrophil (pH ~ 7). However, these bacteria are capable to adapt to different growth conditions, such as temperature ranges from 15 to 45 °C or pH ranging from 4 to 10 [START_REF] Valero | Modelling the Growth Boundaries of Staphylococcus Aureus: Effect of Temperature, PH and Water Activity[END_REF].

S. aureus is a facultative anaerobic bacterium with the ability to reduce nitrate to nitrite and to ferment mannitol (in contrast to S. epidermidis). The bacterium has the capacity of producing catalase, which decomposes hydrogen peroxide to water and oxygen. It also produces coagulase that converts fibrinogen to fibrin. This process induces human plasma coagulation, inhibits the phagocytosis, in contrast to S. epidermidis and S. saprophyticus (Cheng et al. 2010), and not only help distinguish S. aureus from other coagulase-negative bacteria but an essential criterion to look for potential pathogen strains. Moreover, the majority of S. aureus strains are urease-positive (decomposes urea to ammonia); this enzyme is essential for environmental adaptation of certain bacterial pathogens by increasing pH under acid stress and nitrogen limitation [START_REF] Cotter | Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low PH[END_REF][START_REF] Zhou | Urease Is an Essential Component of the Acid Response Network of Staphylococcus Aureus and Is Required for a Persistent Murine Kidney Infection[END_REF].

II. Genome of Staphylococcus aureus

The first S. aureus genomes to be completed were those of N315 and Mu50 in 2001 [START_REF] Kuroda | Whole Genome Sequencing of Meticillin-Resistant Staphylococcus Aureus[END_REF]. Within the past two decade, genomes sequences of approximately 500 Staphylococcus strains have been completed and annotated (Gill 2009).

The genomes are presented in circular chromosomes of approximately 2.8 million base pairs with low GC composition (32%) and encode approximately 2700 coding sequences (CDSs). Most of CDSs in the S. aureus genome have a function assigned to them, based on significant homology with genes in other species (Lindsay and Holden 2006). To encompass the gene repertoire of a species, the concept of pan-genome has been defined as the sum of the core genome and the accessory genome [START_REF] Tettelin | Comparative Genomics: The Bacterial Pan-Genome[END_REF].

The pan-genome of S. aureus gathers approximately 7,500 genes of which approximately 1,500 belong to the core genome. Conjugation occurs by direct contact between two bacteria: plasmids form a mating bridge across the bacteria and DNA is exchanged [START_REF] Furuya | Antimicrobial-Resistant Bacteria in the Community Setting[END_REF].

A. The core genome represents approximatively 75% of the chromosome and is highly conservative in all strains sequenced. Thus, this brings together all the genes conserved within a species encoding the basic functions for the growth of the bacteria such as the metabolism of the bacteria, the protein synthesis, and the replication of nucleic acids [START_REF] Bossi | Competing Endogenous RNAs: A Target-Centric View of Small RNA Regulation in Bacteria[END_REF][START_REF] Lindsay | Staphylococcus Aureus: Superbug, Super Genome?[END_REF]).

B. Mobile genetic elements (MGEs)

The rest of the genome is variable, composed of so-called accessory genes that bacteria acquire by horizontal transfer of MGEs, with a G+C content different from core genome, because they are obtained from different isolates of S. aureus or other low G+C bacteria [START_REF] Lawrence | Amelioration of Bacterial Genomes: Rates of Change and Exchange[END_REF]. These genes are usually acquired from other cells or the surrounding environment in three ways: the transformation, the conjugation, and the transduction (Figure 3). Transformation was the first type of horizontal transfer discovered, by which free DNA in the environment is taken up by a competent bacterium.

The conjugation is the transfer of genetic material through direct contact between the so-called donor bacteria and a receptor bacterium [START_REF] Malachowa | Mobile Genetic Elements of Staphylococcus Aureus[END_REF].

However, these two types of transfer remain a minority in S. aureus. The third type of transfer is the transduction, which is probably the most used horizontal transfer by S. aureus. It is carried out through bacteriophages (Lindsay 2014).

MGEs account for approximately 10-20% of a S. aureus chromosome, including plasmids, transposons, bacteriophages, staphylococcal pathogenicity islands, and staphylococcal cassette chromosomes. They notably have a role in virulence, in resistance to antibiotics, in the pathogenicity and the adaptability in different environmental conditions of S. aureus (reviewed in [START_REF] Lindsay | Staphylococcus Aureus: Superbug, Super Genome?[END_REF]).

Plasmids

Discovered since 1960s, they are classified in three classes based on their sizes and the ability to conjugate (Richard P. [START_REF] Novick | PT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator[END_REF][START_REF] Paulsen | Multidrug Resistance Proteins QacA and QacB from Staphylococcus Aureus: Membrane Topology and Identification of Residues Involved in Substrate Specificity[END_REF]. Plasmids from class I have a size ranging between 1 and 5 kb with high copies number (15-20 copies per cell) and are classified in four subgroups based on the replication origin. They usually Genes encoding resistance determinants shown in green, cassette chromosome recombinase in red, transposases in yellow, arginine deiminase pathway (arc) cluster in orange, oligopeptide permease (opp-3) cluster in purple, and other genes in black. In USA300, same region contains two genetic elements-type I ACME and type IV SCCmec element. Various genetic elements are demarcated by sets of direct-repeat (DR) and inverted-repeat (IR) sequences characteristic of SCC elements that integrated precisely into orfX. DR sequences indicated by black arrows and IR sequences by red arrows [START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF].

encode for single antibiotic resistance gene such as pT181, a 4.4 kb plasmid encoding for tetracycline resistance gene (Richard P. [START_REF] Novick | PT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator[END_REF]. Class II plasmids are up to 40 kb and carry multiple resistance genes in combination with resistance to penicillin and heavy metals, aminoglycosides and/or fusidic acid. Class III plasmids are large (40-60 kb), not only multiresistant but also conjugative, due to tra gene which allows conjugative transfer between bacterial isolates [START_REF] Thomas | Identification and Cloning of the Conjugative Transfer Region of Staphylococcus Aureus Plasmid PGO1[END_REF]). In addition, some virulence genes also are reported to be carried on plasmids such as exfoliate toxin B (etb) for scalded skin syndrome in pRW001 plasmid [START_REF] Alibayov | Staphylococcus Aureus Mobile Genetic Elements[END_REF][START_REF] Jackson | Cloning and Expression of the Exfoliative Toxin B Gene from Staphylococcus Aureus[END_REF]).

Staphylococcal cassette chromosome

Staphylococcal cassette chromosome (SSC) are 3-60 kb genomic cassette that usually are inserted into the orfX gene (T. [START_REF] Ito | Cloning and Nucleotide Sequence Determination of the Entire Mec DNA of Pre-Methicillin-Resistant Staphylococcus Aureus N315[END_REF]. SCC are transferred less than other MGEs because they are more stable than other. The most well studied SSC is SCCmec that carry methicillin resistance gene (T. [START_REF] Ito | Cloning and Nucleotide Sequence Determination of the Entire Mec DNA of Pre-Methicillin-Resistant Staphylococcus Aureus N315[END_REF][START_REF] Katayama | Identification in Methicillin-Susceptible[END_REF]. To date, eight types of SCCmec have been identified (A-E or I-V) based on the organization of mec gene and associated genes within SCCmec complex (Gill 2009;[START_REF] Ito | Structural Comparison of Three Types of Staphylococcal Cassette Chromosome[END_REF][START_REF] Katayama | Identification in Methicillin-Susceptible[END_REF][START_REF] Malachowa | Mobile Genetic Elements of Staphylococcus Aureus[END_REF].

Another non-mec SCC contains fitness and/or survival determinants such as far for fusidic acid resistance [START_REF] Holden | Complete Genomes of Two Clinical Staphylococcus Aureus Strains: Evidence for the Evolution of Virulence and Drug Resistance[END_REF] or kanamycin and erythromycin [START_REF] Hiramatsu | Molecular Genetics of Methicillin-Resistant Staphylococcus Aureus[END_REF], etc. No SSC elements encoding for virulence genes has been found and studied until now.

ACME and COMER

The arginine catabolic mobile element (ACME) and the copper and mercury resistance (COMER), both have been identified first in USA300 S. aureus and in ATCC12228 S. epidermidis strains [START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF][START_REF] Planet | Emergence of the Epidemic Methicillin-Resistant[END_REF] (Figure 4). They are identified as an element associated to the end of SCCmec element [START_REF] Almebairik | Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus Epidermidis Correlates With Rate of Excision[END_REF][START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF]. Moreover, they are suggested to enhance the fitness of S. aureus [START_REF] Almebairik | Genomic Stability of Composite SCCmec ACME and COMER-Like Genetic Elements in Staphylococcus Epidermidis Correlates With Rate of Excision[END_REF][START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF][START_REF] Zapotoczna | Mobile-Genetic-Element-Encoded Hypertolerance to Copper Phagocytes[END_REF], and to promote survival on skin which facilitates persistence, spread, and causes skin infection (Foster and Geoghegan 2014).

Transposons

Transposons (Tns) are short mobile genetic elements that can be integrated into another mobile element such as plasmids or chromosomes or through bacteriophage-based transposition to move between bacterial strains and can transfer genes involved in contingency functions.

Transposons are divided into two main groups: retrotransposons often found in eukaryotes and DNA transposons found in both eukaryotes and prokaryotes. The bacterial transposons belong to the DNA transposons (DNA Tns) and the Tn family [START_REF] Babakhani | Transposons: The Agents of Antibiotic Resistance in Bacteria[END_REF]. In addition, DNA Tns are also divided into four main groups: Insertion sequence (IS), composite Tns, non-composite Tns (Tn3 family) and transposable phage Mu [START_REF] Babakhani | Transposons: The Agents of Antibiotic Resistance in Bacteria[END_REF]. IS are transposons that encode for only the transposase. However, they can also act as a simple transposon, which represents a transposase followed by a resistance gene; or as a composite transposon, when two IS copies flank an unrelated piece of DNA (normally resistance gene) and allow the transfer of DNA to another location.

Transposons are typically 3-60 kb of size, carry resistance determinants and usually reside in the chromosome, in plasmids or within SCCmec elements (like IS431) [START_REF] Malachowa | Mobile Genetic Elements of Staphylococcus Aureus[END_REF]. Those MGEs represent presumably a particularly important selective advantage for the bacteria. Resistance to penicillin, erythromycin, tetracycline aminoglycoside and vancomycin have been found on transposons, such as Tn551, which is one of the first transposon found in S. aureus encoding for erythromycin resistance gene (S. A. [START_REF] Khan | Terminal Nucleotide Sequences of Tn551, a Transposon Specifying Erythromycin Resistance in Staphylococcus Aureus: Homology with Tn3[END_REF] or Tn554 for erythromycin and streptomycin resistance [START_REF] Murphy | Transposon Tn554: Complete Nucleotide Sequence and Isolation of Transposition-Defective and Antibiotic-Sensitive Mutants[END_REF]. Recent reports characterized a novel transposon carrying several resistance determinants, Tn6349, which is a 48 kb composite transposon inserted into a ΦN315-like prophage (D' Andrea et al. 2019).

Bacteriophages

In S. aureus, phages are approximately 45 kb and are integrated commonly into the chromosome (called prophage) as another piece of DNA. These lysogenic phages are mostly quiescent and are replicated as part of bacterial chromosome and passed down to daughter cells during cell division. However, under certain stress conditions, they excise or replicate the phage genome and produce phage progeny, which leads to the host cell lysis [START_REF] Iandolo | Comparative Analysis of the Genomes of the Temperate Bacteriophages Φ11, Φ12 and Φ13 of Staphylococcus Aureus 8325[END_REF][START_REF] Kwan | The Complete Genomes and Proteomes of 27 Staphylococcus Aureus Bacteriophages[END_REF][START_REF] Vybiral | Complete Nucleotide Sequence and Molecular Characterization of Two Lytic Staphylococcus Aureus Phages: 44AHJD and P68[END_REF]). Among all phages, temperate phages are the most numerous groups of the Siphoviridae family and belong to Cluster B phages, based on shared genes content (H. [START_REF] Oliveira | Staphylococci Phages Display Vast Genomic Diversity and Evolutionary Relationships[END_REF]). However, S.

aureus Siphoviridae classification is a matter of discussion (reviewed in [START_REF] Ingmer | Temperate Phages of Staphylococcus Aureus[END_REF]), some represent generalized transducing phages (Φ11, Φ52A…) [START_REF] Fillol-Salom | Bacteriophages Benefit from Generalized Transduction[END_REF], whereas others associate with Staphylococcal pathogenicity islands (SaPIs) (ΦSa1, ΦSa2, ΦSa3, ΦSa4, ΦCOL, etc.) (reviewed in (Gill 2009)).

Most S. aureus strains, like MW2, MSSA476, MRSA252, NCTC8325 or USA300, carry one to four prophages [START_REF] Baba | Genome and Virulence Determinants of High Virulence Community-Acquired MRSA[END_REF][START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF][START_REF] Holden | Complete Genomes of Two Clinical Staphylococcus Aureus Strains: Evidence for the Evolution of Virulence and Drug Resistance[END_REF][START_REF] Iandolo | Comparative Analysis of the Genomes of the Temperate Bacteriophages Φ11, Φ12 and Φ13 of Staphylococcus Aureus 8325[END_REF]. The phage genome is circularized and can integrate into the bacterial host attachment site (attB) via the phage attachment site (attP). The integration reaction produces prophage flanked by the new attachment sites, attL and attR, which correspond to hybrid sites containing half of attP and half of attB. Excision of the prophage occurs between attL and attR to regenerate attP on the excised phage genome and attB on the host chromosome (Figure 5). Both integration and excision require integrase, the enzyme that mediates the site-specific DNA recombination [START_REF] Carroll | Novel Organization of the Site-specific Integration and Excision Recombination Functions of the Staphylococcus Aureus Serotype F Virulence-converting Phages Φ13 and Φ42[END_REF][START_REF] Lee | Integration of Staphylococcal Phage L54a Occurs by Site-Specific Recombination: Structural Analysis of the Attachment Sites[END_REF]). Since each phage carry a different integrase gene (int) that specifies its insertion site in the S. aureus chromosome, the phage immune event inside the bacteria, when two phages of the same family cannot be inserted in the same bacterial cell, is commonly observed [START_REF] Fogg | New Applications for Phage Integrases[END_REF]. Recent studies found that some chromosomal integration site could be heterogeneous, which means that several phages of different strains could share the same attR and attL sites sequences, but some of them also possess a unique integration-site sequence, such as the case of φSa2 in CA-MRSA strains [START_REF] Coombs | Diversity of Bacteriophages Encoding Panton-Valentine Leukocidin in Temporally and Geographically Related Staphylococcus Aureus[END_REF]).

Many S. aureus bacteriophages contain virulence genes such as sak (staphylokinase A) (Collen 1998;Jin et al., 2004), chp (chemotaxis inhibitory protein) (Haas et al., 2004) or lukSF-PV (Panton-Valentine leucocidin) [START_REF] Kaneko | Identification of ORF636 in Phage ΦSLT Carrying Panton-Valentine Leukocidin Genes, Acting as an Adhesion Protein for a Poly(Glycerophosphate) Chain of Lipoteichoic Acid on the Cell Surface of Staphylococcus Aureus[END_REF]. Small RNAs also have been discovered in staphylococcal phages, such as SprD, a sRNA located in Φ315 that enhances the virulence of S. aureus (Chabelskaya, Gaillot, and Felden 2010a;[START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF]; or SprX1 found in Φ12 bacteriophage in S. aureus [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF]. Furthermore, staphylococcal phage phi12 (or Φ12) is also known as bacteriophage Sa2 (or ΦSa2) and was originally isolated from NCTC8325 S. aureus strain beside Φ11 and Φ13 [START_REF] Ye | Sequence Analysis and Comparison of Int and Xis Genes from Staphylococcal Bacteriophages L54a and Φ11[END_REF]. Φ12 is demonstrated to be integrated in a specific unknown gene named SAOUHC_01583 through its attB site, whereas φ11 was describe by [START_REF] Iandolo | Comparative Analysis of the Genomes of the Temperate Bacteriophages Φ11, Φ12 and Φ13 of Staphylococcus Aureus 8325[END_REF] an attB site within the intergenic region of NCTC8325 S. aureus chromosome [START_REF] Iandolo | Comparative Analysis of the Genomes of the Temperate Bacteriophages Φ11, Φ12 and Φ13 of Staphylococcus Aureus 8325[END_REF][START_REF] Xia | Phages of Staphylococcus Aureus and Their Impact on Host Evolution[END_REF]. However, there are hardly characterization studies of Φ12 comparing to the other two phages (Φ11 and Φ13) [START_REF] Iandolo | Comparative Analysis of the Genomes of the Temperate Bacteriophages Φ11, Φ12 and Φ13 of Staphylococcus Aureus 8325[END_REF][START_REF] Xia | Phages of Staphylococcus Aureus and Their Impact on Host Evolution[END_REF].

Staphylococcal pathogenicity islands

The staphylococcal pathogenicity islands (SaPIs) are highly mobile 15 kb genomic islands.

Since they do not possess the structural genes for phage head, tail, tape measure etc., they encode an integrase gene, which allow themselves to be incorporated into temperate bacteriophage (also called "helper phage") such like Φ11, Φ12 and 80α lysogens, for excision, replication, packaging and mobilizing among S. aureus strains [START_REF] Lindsay | The Gene for Toxic Shock Toxin Is Carried by a Family of Mobile Pathogenicity Islands in Staphylococcus Aureus[END_REF][START_REF] Ram | Staphylococcal Pathogenicity Island Interference with Helper Phage Reproduction Is a Paradigm of Molecular Parasitism[END_REF]).

In the sequenced S. aureus genomes, ten SaPIs have been identified: seven found in human S. aureus isolates (SaPI 1-5) and three from bovine isolates (SaPIbov1-3) (reviewed in (Gill 2009). SaPIs form a coherent family with highly conserved core genes including two open reading frames encoding transcriptional regulatory proteins and a region encoding integrase, Rep protein, and terminase (Figure 6). In addition, SaPIs integrate into the S. aureus chromosome through the chromosomal attachment site (att). There are five known att sites to date and they are specific for SaPIs and no other mobile genetic elements [START_REF] Lindsay | The Gene for Toxic Shock Toxin Is Carried by a Family of Mobile Pathogenicity Islands in Staphylococcus Aureus[END_REF][START_REF] Novick | The Phage-Related Chromosomal Islands of Gram-Positive Bacteria[END_REF].

SaPIs encode a number of virulence determinants, which explain their important role in the pathogenesis and evolution of the bacteria. Several superantigens, toxic shock syndrome toxin (tsst-1) and enterotoxins are often found encoded in SaPIs [START_REF] Lindsay | Staphylococcus Aureus: Superbug, Super Genome?[END_REF]; for example, SaPI1, the first SaPI found in S. aureus COL strain, encodes for enterotoxin B, Q, K (seb, seq, sek) and putative B-lactamase protein (ear) [START_REF] Gill | Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus Aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus Epidermidis Strain[END_REF].

Moreover, recent studies discovered the presence of small regulatory RNAs (sRNAs) in SaPIs. SprC, a sRNA, was first found in SaPIn3 [START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF]) in N315 S. aureus strain and was shown to reduce the virulence of the bacteria and its colonization in host cells [START_REF] Pabic | A Bacterial Regulatory RNA Attenuates Virulence, Spread and Human Host Cell Phagocytosis[END_REF]. Another case of sRNA is SprX1, which is encoded in SaPI within Φ12 [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF], is implicated in both antibiotic resistance [START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF]) and the virulence of S. aureus [START_REF] Bronwlee | The Small RNA SprX Regulates the Autolysin Regulator WalR in Staphylococcus Aureus[END_REF][START_REF] Kathirvel | Enhancement of the Pathogenicity of Staphylococcus Aureus Strain Newman by a Small Noncoding RNA SprX1[END_REF].

III. Multiple antibiotics resistance of Staphylococcus aureus

A. The discovery and use of antibiotics to prevent and/or treat diseases and infections go way back from approximately 1000 years ago [START_REF] Harrison | A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity[END_REF]. However, not until the 20 th century that antibiotics have drastically changed the medicine field with salvarsan as the first antibiotic deployed in 1910 (reviewed in (Hutchings, Truman, and Wilkinson 2019; Kathrin I. Mohr 2016)), followed by the golden age of antibiotics with the discovery of Penicillin by Alexander [START_REF] Fleming | On the Antibacterial Action of Culture of a Penicillium, with Special Reference to Their Use in the Isolation of B. Influenza[END_REF]. Subsequently new antibiotics have been discovered rapidly as well as the production of synthetic substances within numerous laboratories of pharmaceutical companies. The insertion of antibiotics into clinical uses was possibly the greatest medical breakthrough of the 20 th century (Figure 7). To date, the term "antibiotic" was defined as a chemical substance of natural or synthetic origin that inhibits or kills pathogenic bacteria [START_REF] Waksman | What Is an Antibiotic or an Antibiotic Substance?[END_REF]. To inactivate virulent microorganisms, these substances operate on different levels: B. Regarding of the classification, based on different criteria such as the chemical structure, the origin, the range of activity, the effects of their activities, antibiotics are divided into eight major groups (reviewed in [START_REF] Begum | A Review on Antibiotic Resistance and Way of Combating Antimicrobial Resistance[END_REF]; Figure 8; Julie thesis):

• Beta-lactams, with four subgroups (Penicillins, Cephalosporins, Monobactams and Carbapenems), target the synthesis of proteins needed for bacteria cell wall (Ebimieowei Etebu; Ibemologi Arikekpar 2016).

• Macrolides aim at protein synthesis and the bacterial ribosome (Ebimieowei Etebu; Ibemologi Arikekpar 2016).

• Tetracyclines also target ribosome and disrupt the protein synthesis [START_REF] Fuoco | Classification Framework and Chemical Biology of Tetracycline-Structure-Based Drugs[END_REF]).

• Quinolones interfere with DNA replication and transcription (Domagala 2018).

• Aminoglycopeptides inhibit the protein synthesis by binding to one of the subunits of Ribosome (Ebimieowei Etebu; Ibemologi Arikekpar 2016).

• Sulfonamides target the bacterial metabolic (reviewed in (Kathrin I. [START_REF] Mohr | History of Antibiotics Research[END_REF]).

• Glycopeptides hinder the bacterial cell wall peptidoglycan synthesis of Grampositive bacteria (reviewed in (Kathrin I. [START_REF] Mohr | History of Antibiotics Research[END_REF]).

• Oxazolidinones are synthetic substances that inhibit bacterial protein synthesis [START_REF] Pandit | Current Updates on Oxazolidinone and Its Significance[END_REF].

C. Antibiotic resistance in S. aureus

Although antibiotics made many medical procedures possible, the misuse of these substances has resulted in a situation where multidrug-resistant pathogens have become a severe menace to human health worldwide. Antibiotic resistance is now, one of the three most crucial community health threats of the 21 st century, according to The World Health Organization [START_REF] Woolhouse | Global Disease Burden Due to Antibiotic Resistance -State of the Evidence[END_REF]. Most pathogens are developing multiantibiotics resistance, including Staphylococcus aureus, such as the case of Methicillin Resistant Staphylococcus aureus (MRSA) [START_REF] Enright | The Evolutionary History of Methicillin-Resistant Staphylococcus Aureus (MRSA)[END_REF][START_REF] Kourtis | Vital Signs: Epidemiology and Recent Trends in Methicillin-Resistant and in Methicillin-Susceptible Staphylococcus Aureus Bloodstream Infections -United States[END_REF]. N315 is a methicillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. Statistical data demonstrated that MRSA was reported in more than 25% of cases in hospital in Europe, 34% of isolates S. aureus from patients in the United States, and 47% in China [START_REF] Wang | Antimicrobial Susceptibility and Molecular Typing of Methicillin-Resistant Staphylococcus Aureus in Retail Foods in Shaanxi, China[END_REF] S. aureus is able to gain the capacity of resistance to multiple antibiotics through the horizontal transfer of MGEs, which may carry antibiotic-resistance genes (ARGs) [START_REF] Felden | Bacterial Adaptation to Antibiotics through Regulatory RNAs[END_REF][START_REF] Haaber | Transfer of Antibiotic Resistance in Staphylococcus Aureus[END_REF][START_REF] Partridge | Mobile Genetic Elements Associated with Antimicrobial Resistance[END_REF]. For instance, the genome of MRSA strains has been demonstrated to carry SSCmec mobile genetic elements containing the mecA gene that confer resistance to methicillin and all other β-lactam antibiotics [START_REF] Katayama | Identification in Methicillin-Susceptible[END_REF].

Although the rise in bacterial diseases that are resistant to almost all known antibiotics is worrying, recent research have led to the discovery of many new molecules with remarkable biological activities [START_REF] Rutledge | Discovery of Microbial Natural Products by Activation of Silent Biosynthetic Gene Clusters[END_REF].

IV. Virulence of Staphylococcus aureus

A. A commensal bacterium

An estimated 20% to 30% of the human population are long-term carriers S. aureus which can be found as part of the normal skin flora, in the nostrils, and as a normal inhabitant of the lower reproductive tract of women. According to different studies in late 1990s, three types of carriage individuals were observed between diverse human populations [START_REF] Kluytmans | Nasal Carriage of Staphylococcus Aureus: Epidemiology, Underlying Mechanisms, and Associated Risks[END_REF]VandenBergh et al. 1999;[START_REF] Williams | Healthy Carriage of Staphylococcus Aureus: Its Prevalence and Importance[END_REF]. • Community-acquired infections are defined as infections manifesting and diagnosed within 48 hours of admission in patients, without any previous encounter with healthcare such as pimples, boils, sties.

• Hospital-acquired infections, also known as a nosocomial infection, are diseases obtained in a hospital or other health care facility such as bacteremia or pneumonia…

For decades, S. aureus has been predominately a nosocomial pathogen and is a leading cause of mortality and morbidity in hospitals. It is the second most frequently isolated species during nosocomial infection in France after Escherichia coli [START_REF] Colomb-Cotinat | Estimating the Morbidity and Mortality Associated with Infections Due to Multidrug-Resistant Bacteria (MDRB), France, 2012[END_REF], and the second cause of Food-borne diseases (FBD) behind Salmonella sp [START_REF] Le Loir | Staphylococcus Aureus and Food Poisoning[END_REF]. However, the community S. aureus infections are in rise. S. aureus is only second to S. epidermidis in causing primary bacteremia in hospital patients [START_REF] Otto | Staphylococcus Epidermidis -the 'Accidental' Pathogen[END_REF]. The important clinical S. aureus infections cover a wide range of illnesses, from minor skin infections (pimples, abscesses), to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome [START_REF] Archer | Staphylococcus Aureus : A Well-Armed Pathogen[END_REF]Tong et al. 2015) (Figure 9).

C. Virulence factors

The process of S. aureus infections involves several stages: colonization, local infection, systemic dissemination and/or sepsis, metastatic infections and finally, toxinosis [START_REF] Bronner | Regulation of Virulence Determinants in Staphylococcus Aureus: Complexity and Applications[END_REF]Foster and Geoghegan 2014;[START_REF] Gnanamani | Staphylococcus Aureus: Overview of Bacteriology, Clinical Diseases, Epidemiology, Antibiotic Resistance and Therapeutic Approach[END_REF]. Hence, during the bacterial journey from the initial point of entry to its eventual last stop, S. aureus encounters different conditions changing in the host cells. To colonize and/or invade these different environments, S. aureus employs a wide range virulence factors that mediate attachment of the bacterium to the substrate, evasion of the host immune shield, tissue invasion, causing sepsis and elicit toxin-mediated syndromes. Although these factors can be classified based on their localizations in bacterial cell or their mechanisms, here, we organized based on their origins: cell surface factors and secreted factors (as proposed in [START_REF] Chavakis | The Anti-Inflammatory Activities of Staphylococcus Aureus[END_REF][START_REF] Costa | Microbial pathogens and strategies for combating them: science[END_REF]Foster 2019b). All the proteins with their specific ligands and putative functions are described succinctly in Table 1. [START_REF] Barie | Immunization against Staphylococcus Aureus Infections[END_REF]Foster et al. 2014): Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), NEAT motif family, G5-E repeat proteins and three-helical bundle repeats (Spa) (Figure 10). Among all CWA proteins, MSCRAMMs represent the largest member of surface proteins. They consist of Fibronectin-binding proteins (FnBPA and FnBPB) [START_REF] Jonsson | Two Different Genes Encode Fibronectin Binding Proteins in Staphylococcus Aureus[END_REF][START_REF] Josse | Staphylococcal Adhesion and Host Cell Invasion: Fibronectin-Binding and Other Mechanisms[END_REF], Clumping factors (ClfA and ClfB) [START_REF] Josefsson | Three New Members of the Serine-Aspartate Repeat Protein Multigene Family of Staphylococcus Aureus[END_REF], Collagen-binding adhesin (Cna) [START_REF] Switalski | A Collagen Receptor on Staphylococcus Aureus Strains Isolated from Patients with Septic Arthritis Mediates Adhesion to Cartilage[END_REF], Serin-aspartic acid rich proteins (Sdr protein) [START_REF] Josefsson | Three New Members of the Serine-Aspartate Repeat Protein Multigene Family of Staphylococcus Aureus[END_REF](Josefsson et al. , 2001) ) and Elastin-binding protein (EbpS) [START_REF] Ghasemian | The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) Genes among Clinical Isolates of Staphylococcus Aureus from Hospitalized Children[END_REF].

They are composed of signals typical of proteins anchored to the wall: N-terminal with a signal peptide (S) which is implicated in the transport of the cytoplasmic proteins to the cell wall; and C-terminal containing a LPXTG domain required for attaching on the cell wall [START_REF] Joh | Role of Fibronectin-Binding MSCRAMMs in Bacterial Adherence and Entry into Mammalian Cells[END_REF]). In addition, the shared mechanism of MSCRAMMs for ligand binding is called "Dock, Lock and Latch" or DLL (reviewed in (Foster 2019a)). The ligand of interest binds to the "opened" form of MSCRAMM and leads to a structural changing of MSCRAMM to a "closed" form to capture the ligand. They primarily target ECM such as fibronectin, fibrinogen, collagenous tissues, and other surface proteins of the host cells, resulting in their implication in adhesion and invasion of host cells, immune evasion as well as biofilm formation. Another well-studied surface protein is protein A (Spa), a protein comprising three-helical bundle repeats, which target the constant domain of Immunoglobulin IgG and is involved in protecting staphylococcus from phagocytosis [START_REF] Deis | Suppression of Conformational Heterogeneity at a Protein-Protein Interface[END_REF].

SERAMs

SERAMs comprise Extracellular adherence protein (Eap) [START_REF] Cheng | Genetic Requirements for Staphylococcus Aureus Abscess Formation and Persistence in Host Tissues[END_REF][START_REF] Rhem | The Collagen-Binding Adhesin Is a Virulence Factor in Staphylococcus Aureus Keratitis[END_REF], von Willebrand factor binding protein (vWbp) [START_REF] Nilsson | A von Willebrand Factor-Binding Protein from Staphylococcus Lugdunensis[END_REF]), Coagulase (Coa) [START_REF] Kang | Collagen-Binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-Positive Bacteria Inhibit Complement Activation via the Classical Pathway[END_REF][START_REF] Sharp | Staphylococcus Aureus Surface Protein SdrE Binds Complement Regulator Factor H as an Immune Evasion Tactic[END_REF], ECM binding protein (Emp) (Josefsson et al. 2001), Extracellular fibrinogen binding protein (Efb) [START_REF] Shannon | Extracellular Fibrinogen Binding Protein, Efb, from Staphylococcus Aureus Binds to Platelets and Inhibits Platelet Aggregation[END_REF], and Second immunoglobulin-binding protein (Sbi) [START_REF] Wilke | Role of a Disintegrin and Metalloprotease 10 in Staphylococcus Aureus α-Hemolysin -Mediated Cellular Injury[END_REF]. They are structurally unrelated, unlike MSCRAMMs; however, they possess similar immunomodulatory functions. SERAMs mostly target fibrinogen, fibronectin and vibronectin and consequently contribute to host cell invasion and host colonization. Furthermore, they also target platelets and complements, which explain their importance in preventing the activation of complements and the phagocytosis during bacterial infection (reviewed in [START_REF] Chavakis | The Anti-Inflammatory Activities of Staphylococcus Aureus[END_REF]). [START_REF] Chavakis | The Anti-Inflammatory Activities of Staphylococcus Aureus[END_REF]D. Oliveira, Borges, and Simões 2018;[START_REF] Tam | Staphylococcus Aureus Secreted Toxins and Extracellular Enzymes[END_REF] 1.3. Other anti-inflammatory peptides Other anti-inflammatory peptides than SERAMs consist of chemotaxis inhibitory protein of S. aureus (CHIPS) [START_REF] Chavakis | Staphylococcus Aureus Extracellular Adherence Protein Serves as Anti-Inflammatory Factor by Inhibiting the Recruitment of Host Leukocytes[END_REF][START_REF] Clarke | Iron-Regulated Surface Determinant Protein A Mediates Adhesion of Staphylococcus Aureus to Human Corneocyte Envelope Proteins[END_REF][START_REF] Foster | Immune Evasion by Staphylococci[END_REF], Staphylococcal complement inhibitor (SCIN) [START_REF] Rooijakkers | Immune Evasion by a Staphylococcal Complement Inhibitor That Acts on C3 Convertases[END_REF][START_REF] Silverman | Confounding B-Cell Defences: Lessons from a Staphylococcal Superantigen[END_REF] or Formyl peptide receptor-like 1 inhibitory protein (FLIPr) [START_REF] Sinha | Fibronectin-Binding Protein Acts as Staphylococcus Aureus Invasin via Fibronectin Bridging to Integrin Α5β1[END_REF][START_REF] Stemerding | Staphylococcus Aureus Formyl Peptide Receptorlike 1 Inhibitor (FLIPr) and Its Homologue FLIPr-like Are Potent FcγR Antagonists That Inhibit IgG-Mediated Effector Functions[END_REF]. They are implicated in immune evasion by blocking chemotaxis and obstructing complements activation (reviewed in (Foster et al. 2014)) through binding to complements and chemoattractant receptors. Another non-proteinaceous factor is Staphyloxanthin (STX), a carotenoid pigment that contributes to the golden color of S.

aureus. The loss of STX has been demonstrated to make S. aureus susceptible to killing by ROS produced by neutrophils (G. Y. [START_REF] Liu | Staphylococcus Aureus Golden Pigment Impairs Neutrophil Killing and Promotes Virulence through Its Antioxidant Activity[END_REF][START_REF] Song | Inhibition of Staphyloxanthin Virulence Factor Biosynthesis in Staphylococcus Aureus: In Vitro, in Vivo, and Crystallographic Results[END_REF]).

Capsular polysaccharides (CPs)

CPs are produced by the majority of clinical isolates and serotypes 1, 5 and 8 are the most well studied to date [START_REF] Rausch | Coordination of Capsule Assembly and Cell Wall Biosynthesis in Staphylococcus Aureus[END_REF]. The high encapsulated serotype 1 (CP1) impedes opsonophagocytic killing of staph by preventing the binding between C3b complement with receptor on phagocytic cells. The serotypes 5 and 8 (CP5 and CP8) also protect S. aureus from phagocytic uptake and killing by human polymorphonuclear leukocytes, by masking the complement deposition C3b [START_REF] Cunnion | Availability of Complement Bound to Staphylococcus Aureus to Interact with Membrane Complement Receptors Influences Efficiency of Phagocytosis[END_REF]. Overall, their main function is to restraint phagocytosis by neutrophils, but they also involve in bacterial colonization and promote abscesses [START_REF] Rausch | Coordination of Capsule Assembly and Cell Wall Biosynthesis in Staphylococcus Aureus[END_REF] and they are the most important virulence factors under evaluation as vaccine antigens.

Cell wall components and wall components factors

Cell wall components include peptidoglycan, lipoteichoic acid (LTA), wall teichoic acid, etc., and are targets of Cationic antimicrobial molecules (CAMs), which disrupt bacterial membrane and plays a key role in innate immunity. Toll-like receptors 2 (TLR2) is a recognition receptor that recognizes Gram-positive cell wall molecules such as LTA or peptidoglycan and its activation results in the release of antimicrobial peptides [START_REF] Sieling | Toll-like Receptors: Mammalian 'taste Receptors' for a Smorgasbord of Microbial Invaders[END_REF], which leads to the induction of innate immune response. It has been Figure 11. Overview of S. aureus toxins and exoenzymes. Superantigens are proteins that cause T and B cell expansions, resulting in clonal deletion and massive cytokine production. Cytotoxins (Hla and leukocidins), cause cytokine production, hemolysis, and leukocyte cell death through targeting specific cell surface receptors. PSMs mediate cytolysis by inserting into the lipid bilayer of cell membranes. Enzymes (Hlb and the Ets), cause cytotoxicity on mammalian cells, resulting in cell death, inflammation, and tissue barrier disruptions. Other enzymes, (various proteases and nucleases), mediate host protein degradation, preventing important host immune system and defense molecules. Lipases and FAME work synergistically to degrade lipids in the environment for nutrients. Cofactors (Coa, vWbp, and Sak), bind and activate host zymogens in the coagulation system for clot formation. Altogether, these toxins and enzymes provide critical nutrients (i.e., iron and carbon) that are important for the growth and survival of the bacteria. They also target different levels of host immune defense, thus contributing to the virulence of S. aureus during infections (from Tam and Torres 2019).

shown recently that modification of teichoic acids and phospholipids with D-alanine and L-lysine in S. aureus reduces binding to CAMs, therefor enhances the virulence of the bacteria [START_REF] Weidenmaier | Role of Teichoic Acids in Staphylococcus Aureus Nasal Colonization, a Major Risk Factor in Nosocomial Infections[END_REF]. Moreover, wall components-modifying factors comprise Dlt operon (dltABCD) [START_REF] Peschel | Inactivation of the Dlt Operon in Staphylococcus Aureus Confers Sensitivity to Defensins, Protegrins, and Other Antimicrobial Peptides[END_REF]) and multiple peptide resistance factor F (MprF) [START_REF] Weidenmaier | Bacterial Resistance to Antimicrobial Host Defenses -An Emerging Target for Novel Antiinfective Strategies?[END_REF], have been demonstrated to be implicated in the modification of bacterial cell wall phospholipids (reviewed in [START_REF] Chavakis | The Anti-Inflammatory Activities of Staphylococcus Aureus[END_REF]). Their conservation in many Gram-positive pathogens besides S. aureus suggests that this pathway represents a common mechanism to achieve protection against CAMs.

2. At high density of Staph, the bacteria expresses a wide range of secreted factors such as extracellular enzymes, cytolytic toxins and superantigens. They play active roles in disarming host immunity by disrupting host cells and tissues and interfering with the host immune system to release nutrients and facilitate bacterial dissemination (Table 2 andFigure 11).

Extracellular enzymes or exoenzymes

Exoenzymes include catalase, hydrolases, staphylokinase (Sak), nucleases, and hyaluronidase, proteases (serine, cysteine (e.g. Staphopain) and metalloprotease (Aureolysin). Nearly all S. aureus strains secrete several extracellular enzymes whose functions are thought to be the disruption of host tissues and/or inactivation of host antimicrobial mechanisms, to acquire nutrients for bacterial growth and facilitate bacterial dissemination. For instance, proteases, consisting of serine-and cysteineproteases [START_REF] Singh | Interaction of Host and Staphylococcus Aureus Protease-System Regulates Virulence and Pathogenicity[END_REF][START_REF] Tam | Staphylococcus Aureus Secreted Toxins and Extracellular Enzymes[END_REF], are mainly involved in the maturation and activation of exoproteins of S. aureus [START_REF] Rzychon | Staphostatins: An Expanding New Group of Proteinase Inhibitors with a Unique Specificity for the Regulation of Staphopains, Staphylococcus Spp. Cysteine Proteinases[END_REF] but also allow the degradation of immunoglobulins and plasma proteins (L. [START_REF] Shaw | The Role and Regulation of the Extracellular Proteases of Staphylococcus Aureus[END_REF][START_REF] Sieprawska-Lupa | Degradation of Human Antimicrobial Peptide LL-37 by Staphylococcus Aureus-Derived Proteinases[END_REF]. Other enzymes such as Coagulase, Aureolysin or Staphylokinase have also been demonstrated to contribute to immune evasion of S. aureus, with a dual function since they allow the penetration of the bacteria through the skin [START_REF] Kwiecinski | Staphylokinase Control of Staphylococcus Aureus Biofilm Formation and Detachment through Host Plasminogen Activation[END_REF].

Figure 12. Overview of pore formation of several cytolytic toxins. (A) The α toxin is secreted as a monomer. By binding to the host ADAM-10 receptor, the monomers form a heptameric pre-pore. Then the pre-pore stems expand to form a pore piercing the membrane of the target cell. (B) Leukocidins are also secreted in the form of monomers. The S-subunit recognizes the target cell by binding to cellular receptors. These receptors are generally G protein-coupled receptors (GPCR). (C) LukAB, in the other hand, is secreted in the form of dimers and the S-subunit binds to the CD11b integrin. Upon binding to the receptor, the S-subunit dimerizes with the F-subunit, then this dimer oligomerizes with three other dimers to form an octameric pre-pore. Eventually, similar to the α toxin, the stems of this pre-pore expand to form a pore. (D) PSMs bind to the membrane in a nonspecific manner, leading to its disintegration, and then, PSMs aggregate to form a transient pore (Adapted from Tam and Torres 2019; D. Oliveira, Borges, and Simões 2018).

Cytolytic toxins or pore-forming toxins (PFTs)

S. aureus secretes a large number of cytolytic toxins that, although structurally diverse and with different target specificity, share a similar function on host cells (Figure 12). They are divided into two groups, based on the mechanism of regulation: receptor dependent PFTs and receptor independent PFTs. Among receptor dependent PFTs, the toxins targeting red blood cells are hemolysins while those targeting white blood cells are leukotoxins. They include hemolysins α, β and γ and several leucocidins including Panton-Valentine leukocidin (PVL) and leukotoxin E/D (LukE/D) (Otto 2014). The best-known PFT is α-hemolysin, produced by 80-90% of S. aureus strains and encoded by the hla gene. These toxins form β-barrel pores in the cytoplasmic membranes of target cells and cause leakage of the cell's content (when at low doses) and cell lysis (at high doses) [START_REF] Foster | Immune Evasion by Staphylococci[END_REF][START_REF] Song | Structure of Staphylococcal A-Hemolysin, a Heptameric Transmembrane Pore[END_REF]. The secretion of these toxins allows the bacteria to lyse a large number of host cell types such as erythrocytes, monocytes, macrophages, platelets, leukocytes, endothelial and epithelial cells by forming pores in the plasma membrane of target cells [START_REF] Bayer | Hyperproduction of Alpha-Toxin by Staphylococcus Aureus Results in Paradoxically Reduced Virulence in Experimental Endocarditis: A Host Defense Role for Platelet Microbicidal Proteins[END_REF][START_REF] Bhakdil | Alpha-Toxin of Staphylococcus Aureus[END_REF][START_REF] Finck-Barbançon | Pore Formation by a Two-Component Leukocidin from Staphyloccocus Aureus within the Membrane of Human Polymorphonuclear Leukocytes[END_REF][START_REF] Jayasinghe | The Leukocidin Pore: Evidence for an Octamer with Four LukF Subunits and Four LukS Subunits Alternating around a Central Axis[END_REF][START_REF] Lin | New Insights into the Prevention of Staphylococcal Infections and Toxic Shock Syndrome[END_REF].

Receptor-independent PFTs include in particular the family of Phenol-Soluble Modulins (PSMs) and delta-hemolysins (Hld). Hld is encoded by hemolysin δ gene, which is located in the coding sequence of RNAIII, a major riboregulator of virulence of S. aureus [START_REF] Janzon | The Role of the Delta-Lysin Gene (Hld) in the Regulation of Virulence Genes by the Accessory Gene Regulator (Agr) in Staphylococcus Aureus[END_REF]D. Oliveira, Borges, and Simões 2018;[START_REF] Tam | Staphylococcus Aureus Secreted Toxins and Extracellular Enzymes[END_REF]. As for PSMs, they represent a family of small protein toxins that are soluble in phenol. These are surface-active, amphipathic, and alpha-helical peptides, which are classified in different groups based on their length [START_REF] Peschel | Inactivation of the Dlt Operon in Staphylococcus Aureus Confers Sensitivity to Defensins, Protegrins, and Other Antimicrobial Peptides[END_REF]Michael Otto 2016;[START_REF] Donvito | Synergistic Hemolytic Activity of Staphylococcus Lugdunensis Is Mediated by Three Peptides Encoded by a Non-Agr Genetic Locus[END_REF][START_REF] Mehlin | An Inflammatory Polypeptide Complex from Staphylococcus Epidermidis: Isolation and Characterization[END_REF][START_REF] Watson | The Amino Acid Sequence of a Gonococcal Growth Inhibitor from Staphylococcus Haemolyticus[END_REF]): (I) PSMs type α and Hld, and (II) PSMs type β. Through their physicochemical properties, they have been shown to be essential for the structuring of biofilms formed by S. aureus, as well as for the detachment step allowing the dissemination of bacteria [START_REF] Periasamy | Phenol-Soluble Modulins in Staphylococci[END_REF]). All PSMαs have been shown to actively participate in the escape of S. aureus from the Table 3_ Virulence regulators (Adapted from [START_REF] Jenul | Regulation of Staphylococcus Aureus Virulence[END_REF] immune system by promoting its exit from the phagolysosome [START_REF] Grosz | Cytoplasmic Replication of Staphylococcus Aureus upon Phagosomal Escape Triggered by Phenol-Soluble Modulin α[END_REF][START_REF] Münzenmayer | Influence of Sae-Regulated and Agr-Regulated Factors 206 on the Escape of Staphylococcus Aureus from Human Macrophages[END_REF]. Furthermore, PSMα1-4, encoded by the psmα locus (especially PSMα4), are strongly cytolytic. Recent studies have shown their contribution in the lysis of neutrophils after phagocytosis of particularly virulent strains of S. aureus such as isolates of community origin and resistant to methicillin (D. [START_REF] Oliveira | Staphylococcus Aureus Toxins and Their Molecular Activity in Infectious Diseases[END_REF][START_REF] Surewaard | Staphylococcal Alpha-Phenol Soluble Modulins Contribute to Neutrophil Lysis after Phagocytosis[END_REF]; whereas hemolysin δ has moderate cytolytic activity and the PSMβ peptides are non-cytolytic (Andreas Peschel; Michael Otto 2016; Otto 2014). In addition, some hospital-acquired methicillin-resistant staphylococci (HA-MRSA) express PSM-mec, which is encoded by psm-mec in the SSCmec cassette, and also encodes PSMα [START_REF] Kaito | Mobile Genetic Element SCCmec-Encoded Psm-Mec RNA Suppresses Translation of AgrA and Attenuates MRSA Virulence[END_REF][START_REF] Tam | Staphylococcus Aureus Secreted Toxins and Extracellular Enzymes[END_REF].

Superantigens (SAgs) and superantigens-like proteins

Superantigens are a group of potent secreted immune-stimulatory proteins capable of inducing a variety of human toxic shock associated diseases. Staphylococcal superantigens are the only toxins that can cause disease by their very presence. They comprise T cell superantigens and B cell superantigens, which are able to trigger T cell and B cell activation and proliferation, respectively. T cell superantigens include such as staphylococcal enterotoxins (SEs), toxic shock syndrome toxin-1 (TSST-1) and exfoliating toxins [START_REF] Alouf | Mini-Review Staphylococcal and Streptococcal Superantigens : Molecular , Biological and Clinical Aspects[END_REF][START_REF] Baker | Superantigens: Structure-Function Relationships[END_REF][START_REF] Kreiswirth | The Toxic Shoch Syndrome Exotoxin Structural Gene Is Not Detectably Transmitted by a Prophage[END_REF]) and many other "superantigen-like" named SSI (SE-I H to SE-I Y; or SSL-5 and SSL-7); and B cell superantigens such as Spa. Among them, the best-known Sag TSST-1 is responsible for 95% of cases of toxic shock associated with menstruation (mTSS) and 50% of TSS cases caused by nonmenstrual infective foci (nmTSS) [START_REF] Bohach | Staphylococcal and Streptococcal Pyrogenic Toxins Involved in Toxic Shock Syndrome and Related Illnesses[END_REF][START_REF] Kreiswirth | The Toxic Shoch Syndrome Exotoxin Structural Gene Is Not Detectably Transmitted by a Prophage[END_REF][START_REF] Sharma | Clinical and Molecular Epidemiology of Staphylococcal Toxic Shock Syndrome in the United Kingdom[END_REF], and also involved in the apoptosis of host cells induced by S. aureus [START_REF] Hofer | Differential Effects of Staphylococcal Toxic Shock Syndrome Toxin-1 on B Cell Apoptosis[END_REF]. By creating a non-specific interaction between T cell receptors and complex receptors major class II histocompatibility (MHC II), TSST-1 enables immune response through T cell and this activation causes the production and massive release of pro-inflammatory cytokines responsible for the onset of toxic shock (D. [START_REF] Oliveira | Staphylococcus Aureus Toxins and Their Molecular Activity in Infectious Diseases[END_REF]. Another case of SAgs are exfoliating toxins, which belong to Serineprotease family and are responsible for a wide spectrum of symptoms, ranging from localized lesions to scalded skin syndrome (SSS) [START_REF] Bukowski | Exfoliative Toxins of Staphylococcus Aureus[END_REF][START_REF] Ladhani | Understanding the Mechanism of Action of the Exfoliative Toxins of Staphylococcus Aureus[END_REF][START_REF] Lee | Sequence Determination and Comparison of the Exfoliative Toxin A and Toxin B Genes from Staphylococcus Aureus[END_REF][START_REF] Tam | Staphylococcus Aureus Secreted Toxins and Extracellular Enzymes[END_REF].

D. Virulence regulators

Through different illustrations above, we can see that S. aureus possesses a large arsenal of cell wall and extracellular components involved in the virulence of the bacterium. The diverse range of these virulence factors implies that the pathogenicity of S. aureus is a complex process requiring an organized expression of these factors during different stages of infection (i.e. colonization, immune evasion, growth and cell division, bacterial dissemination). Indeed, S. aureus firstly upregulates the expression of genes coding for surface proteins involved in adhesion and defense against the host immune system; and only late in infection it starts to upregulate the production of toxins that facilitate tissue propagation. To control the production of the virulence determinants during infection, S. aureus employs several regulatory systems that respond to bacterial cell density (quorum sensing) and environmental signals (e.g. pH, osmolarity, and nutrient availability, temperature, and oxygen tension). These regulatory systems can be divided into five broad categories: two-component signal transduction systems, global transcriptional regulators, the SarA protein family, the sigma factors and the most recent group is regulatory RNAs (sRNAs). Here, we only describe several major regulator systems; however, all regulatory systems with their putative functions are listed concisely in Table 3.

1. Two-component signal transduction systems (TCS) About sixteen TCS have been identified in S. aureus (reviewed in (Jenul and Horswill 2019)). Extracellular signal receptors are a major source of information on external environmental conditions and allow S. aureus to adapt to changing environmental conditions. As the name suggests, TCS works with a "sensor" molecule from the external environment and a "response" element which, when is activated, allows the bacteria to control the expression of specific RNA and/or proteins in order to respond accordingly to exposure to environmental stresses. For instance, one of the classic TCS is the staphylococcal respiratory response AB (SrrAB) [START_REF] Haag | A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes[END_REF][START_REF] Tiwari | The SrrAB Two-Component System Regulates Staphylococcus Aureus Pathogenicity through Redox Sensitive Cysteines[END_REF][START_REF] Yarwood | Identification of a Novel Two-Component Regulatory System That Acts in Global Regulation of Virulence Factors of Staphylococcus Aureus[END_REF]. srrA encodes a 28-kDa, 241-amino-acid response regulator, and srrB a 66-kDa, 583-amino-acid histidine kinase. The response regulator, SrrA, localizes in the cytoplasmic and SrrB, the transmembrane histidine kinase, localizes on the membrane. SrrAB seems to respond to oxygen levels. Once SrrB has received the signal, it is auto phosphorylated and associated with SrrA, allowing the phosphorylation of SrrA. This modification in SrrA alters the DNA-binding activity of this response regulator to the promoter regions of the agr, spa and tst loci and consequently alters the transcription of these genes [START_REF] Pragman | Virulence Regulation in Staphylococcus Aureus: The Need for in Vivo Analysis of Virulence Factor Regulation[END_REF]) (Figure 13).

However, among all TCS studied, two major illustrations are the accessory gene regulator system (agr) and the staphylococcal accessory element system (sae).

First, the agr system has been extensively studied since its discovery several decades ago by [START_REF] Mallonee | Chromosomal Mapping of a Gene Affecting Enterotoxin A Production in Staphylococcus Aureus[END_REF]. This system is also called the quorum sensing system, due to its implication in the regulation of different virulence genes, according to the bacterial density during the infection (Richard P. Novick 2003). The agr locus occurs as two divergent transcription units (G. Y. C. [START_REF] Cheung | Role of the Accessory Gene Regulator Agr in Community-Associated Methicillin-Resistant Staphylococcus Aureus Pathogenesis[END_REF][START_REF] Peng | Cloning, Characterization and Sequencing of an Accessory Gene Regulator (Agr) in Staphylococcus Aureus[END_REF]. The first produces an RNA molecule called ARNII, which encodes four proteins, AgrA, B, C and D. The other a transcript is a sRNAs called RNAIII containing the hemolysin-delta gene (hld) [START_REF] Morfeldt | Activation of Alpha-Toxin Translation in Staphylococcus Aureus by the Trans-Encoded Antisense RNA, RNAIII[END_REF][START_REF] Novick | Synthesis of Staphylococcal Virulence Factors Is Controlled by a Regulatory RNA Molecule[END_REF]) (Figure 14A). The agrD transcript encodes a peptide precursor of the extracellular quorum signal of Agr, called autoinducing peptide (AIP) [START_REF] Lyon | Key Determinants of Receptor Activation in the Agr Autoinducing Peptides of Staphylococcus Aureus[END_REF]. The agrB gene product is a transmembrane endopeptidase responsible for exporting of the AIP (L. [START_REF] Zhang | Membrane Anchoring of the AgrD N-Terminal Amphipathic Region Is Required for Its Processing to Produce a Quorum-Sensing Pheromone in Staphylococcus Aureus[END_REF]. The agrC and agrA genes encode a two-component signal transduction system with AgrC as histidine kinase sensor and AgrA as its associated response regulator (R. P. Novick et al. 1995;[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]. When extracellular concentration of AIP passes the threshold, AIP binds to AgrC and leads to its own auto-phosphorylation, following by the phosphorylation of AgrA (R. P. Novick et al. 1995;[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] Roux | Cody-Mediated Regulation of the Staphylococcus Aureus Agr System Integrates Nutritional and Population Density Signals[END_REF]. It can up-regulate the expressions of several exoproteins (e.g., α-, β-, γ-hemolysin, and leukotoxins), lipases, phenol-soluble modulins, and toxic shock syndrome toxins (TSST), and represses the transcription of some cell wall-associated proteins (e.g., protein A, Figure 14. Two component systems. (A) The Agr system. The pro-AIP peptide is processed and secreted by AgrB, binds to an extracellular loop in the receptor-HPK (AgrC), activating autophosphorylation (or dephosphorylation), followed by phosphorylation or dephosphorylation of the response regulator, AgrA, which activates the two agr promoters, P2 and P3, leading to the production of RNA III [START_REF] Jenul | Regulation of Staphylococcus Aureus Virulence[END_REF]. (B) Organization of the sae operon. (C) The proposed mode of action of the Sae system. Upon exposure to signal(s), SaeS autophosphorylates and then transfers the phosphoryl group to SaeR. The phosphorylated SaeR binds to its binding sequence and activates transcription from target promoters including the saeP1 promoter. From saeP1, SaeP and SaeQ are produced and bind to SaeS in the membrane. As a lipoprotein, SaeP is expected to interact with the extracellular linker peptide of SaeS. On the other hand, SaeQ is thought to interact with the cytoplasmic domain of SaeS (Q. [START_REF] Liu | The SaeRS Two-Component System of Staphylococcus Aureus[END_REF].

coagulase, and fibronectin binding protein) [START_REF] Bronner | Regulation of Virulence Determinants in Staphylococcus Aureus: Complexity and Applications[END_REF][START_REF] Le | Quorum-Sensing Regulation in Staphylococcian Overview[END_REF]. Through the genes it regulates, the quorum sensing Agr system is causally linked to the pathogenesis of S. aureus in skin and tissue infections, via the expression of toxins, but also in endocarditis, pneumonia, and osteomyelitis via its involvement in the biofilm life cycle [START_REF] Boles | Agr-Mediated Dispersal of Staphylococcus Aureus Biofilms[END_REF]D. Oliveira, Borges, and Simões 2018;[START_REF] Vuong | A Crucial Role for Exopolysaccharide Modification in Bacterial Biofilm Formation, Immune Evasion, and Virulence[END_REF]). As for the regulation of agr locus expression, some members of the SarA Next, the sae system is a two-component system with four elements: SaeP, SaeG, SaeR and SaeS. However, only SaeR and SaeS are identified as the response regulator and histidine kinase sensor (A. T. [START_REF] Giraudo | Characterization of a Tn551-Mutant of Staphylococcus Aureus Defective in the Production of Several Exoproteins[END_REF][START_REF] Rogasch | Influence of the Two-Component System SaeRS on Global Gene Expression in Two Different Staphylococcus Aureus Strains[END_REF]) (Figure 14B andC).

Besides its implication in biofilm formation [START_REF] Haag | A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes[END_REF]Jenul and Horswill 2019;Q. Liu, Yeo, and Bae 2016), the system also regulate a wide range of virulence factors involved in bacterial adhesion, toxicity, and immune evasion. For instance, Sae system activates the expression of several hemolysins (hla, hlb, hld) and coagulase (Ana T. [START_REF] Giraudo | The Sae Locus of Staphylococcus Aureus Controls Exoprotein Synthesis at the Transcriptional Level[END_REF][START_REF] Goerke | Role of Staphylococcus Aureus Global Regulators Sae and ΣB in Virulence Gene Expression during Device-Related Infection[END_REF], and represses some exoenzymes (plc, sspA, aur) [START_REF] Rogasch | Influence of the Two-Component System SaeRS on Global Gene Expression in Two Different Staphylococcus Aureus Strains[END_REF].

From then, many more TCSs have been discovered: ArlRS system potentially involved in cell division and adhesion [START_REF] Fournier | The Two-Component System ArlS-ArlR Is a Regulator of Virulence Gene Expression in Staphylococcus Aureus[END_REF], LytRS and WalKS systems in biofilm formation [START_REF] Dubrac | New Insights into the WalK/WalR (YycG/YycF) Essential Signal Transduction Pathway Reveal a Major Role in Controlling Cell Wall Metabolism and Biofilm Formation in Staphylococcus Aureus[END_REF][START_REF] Sharma-Kuinkel | The Staphylococcus Aureus LytSR Two-Component Regulatory System Affects Biofilm Formation[END_REF]) etc. (Table 2).

Global transcriptional regulators

The mechanism by which the expression if virulence factors are regulates by S. aureus are complex and are not completely elucidated. The regulation of S. aureus pathogenicity is achieved by different pathways, by the quorum-sensing accessory gene regulator, agr system, or by the family of winged helix DNA-binding proteins of SarA family, or the nutritionally controlled regulators. These latter global regulators act a sensor of the nutritional status of the bacterium, and contribute to the decision by S. aureus of "when and in what amounts" to express toxins, adhesins involved in biofilm formation, etc.

CodY was first discovered in B. subtilis and L. lactis as a regulator to metabolic genes involved in nitrogen and nucleotide metabolism [START_REF] Molle | Additional Targets of the Bacillus Subtilis Global Regulator CodY Identified by Chromatin Immunoprecipitation and Genome-Wide Transcript Analysis[END_REF][START_REF] Sonenshein | CodY, a Global Regulator of Stationary Phase and Virulence in Gram-Positive Bacteria[END_REF], before being emerging as a highly conserved regulatory protein of virulence in S. aureus [START_REF] Pohl | CodY in Staphylococcus Aureus: A Regulatory Link between Metabolism and Virulence Gene Expression[END_REF]) (Figure 15) According to genome-wide analysis in [START_REF] Majerczyk | Direct Targets of CodY in Staphylococcus Aureus[END_REF], CodY regulates virulence genes through three different mechanisms: (I) agr expression-dependent regulation such as cap, spa, fnbA and coa; (II) direct regulation by CodY, (III) hla and capsule synthesis (like ica operon) [START_REF] Majerczyk | Direct Targets of CodY in Staphylococcus Aureus[END_REF]. One of the most well-known target of CodY is agrBCDA operon [START_REF] Roux | Cody-Mediated Regulation of the Staphylococcus Aureus Agr System Integrates Nutritional and Population Density Signals[END_REF]. At the exponential phase of bacterial growth, when the intracellular concentrations of branched-chain amino acids (BCAAs) and GTP are important, CodY binds to P2 and P3 promoters and represses the transcription of agr operon and rnaIII [START_REF] Roux | Cody-Mediated Regulation of the Staphylococcus Aureus Agr System Integrates Nutritional and Population Density Signals[END_REF].

Another global regulator is CcpA encoding for catabolite control protein A that belongs to global regulatory of the carbon catabolite repression (CCR), which is involved in carbon utilization and metabolization of the preferred carbon source (i.e. glucose). In the early 1990s, CcpA was first studied in B. subtilis for its specific binding to DNA sequence, cre (for catabolite-responsive element) [START_REF] Henkin | The Role of the CcpA Transcriptional Regulator in Carbon Metabolism in Bacillus Subtilis[END_REF], and later was demonstrated on S.

xylosus [START_REF] Jankovic | Carbon Catabolite Repression by the Catabolite Control Protein CcpA in Staphylococcus Xylosus[END_REF]. However, little was known for this repressor in S. aureus until the 2000s. CcpA was demonstrated to involve in the virulence determinants regulation such as RNAIII, hla and spa [START_REF] Seidl | Staphylococcus Aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance[END_REF](Seidl et al. , 2008(Seidl et al. , 2008(Seidl et al. , 2009;;[START_REF] Ueda | Sugar-Responsive Gene Expression and the Agr System Are Required for Colony Spreading in Staphylococcus Aureus[END_REF] and recently was showed to regulate an sRNA implicated in the bacterial response to glucose consumption, RsaI (D. [START_REF] Bronesky | A Dual SRNA in Staphylococcus Aureus Induces a Metabolic Switch Responding to Glucose Consumption[END_REF]). In addition, CcpA also represents an auxiliary factors reported to reduce methicillin resistance in S. aureus strain COL [START_REF] De Lencastre | Antibiotic Resistance as a Stress Response: Complete Sequencing of a Large Number of Chromosomal Loci in Staphylococcus Aureus Strain COL That Impact on the Expression of Resistance to Methicillin[END_REF] and is required for promoted colony spreading in S. aureus [START_REF] Ueda | Sugar-Responsive Gene Expression and the Agr System Are Required for Colony Spreading in Staphylococcus Aureus[END_REF]) (Figure 16).

Yet another global regulator, Fur (Ferric uptake regulator), is one of the four distinct families of metallo-regulatory proteins, beside DtxR (diphtheria toxin repressor), MerR, and ArsR [START_REF] Fuangthong | Recognition of DNA by Three Ferric Uptake Regulator (Fur) Homologs in Bacillus Subtilis[END_REF][START_REF] Troxell | Transcriptional Regulation by Ferric Uptake Regulator (Fur) in Pathogenic Bacteria[END_REF]. Firstly purified from E. coli in 1988, Fur was demonstrated to function as a dimer in the presence of iron, one of the essential elements required for the growth and the metabolism of prokaryotes [START_REF] Carpenter | This Is Not Your Mother's Repressor: The Complex Role of Fur in Pathogenesis[END_REF][START_REF] Chandrangsu | Metal Homeostasis and Resistance in Bacteria[END_REF][START_REF] Hantke | Iron and Metal Regulation in Bacteria[END_REF]). Fur utilizes Fe2+ as a cofactor and not only represses expression of iron acquisition genes in the presence of iron [START_REF] Horsburgh | In Staphylococcus Aureus, Fur Is an Interactive Regulator with PerR, Contributes to Virulence, and Is Necessary for Oxidative Stress Resistance through Positive Regulation of Catalase and Iron Homeostasis[END_REF], but also sRNA in E.coli [START_REF] Massé | A Small RNA Regulates the Expression of Genes Involved in Iron Metabolism in Escherichia Coli[END_REF] (Figure 17). In many bacteria, a paradigm being RyhB in E. coli, an iron-sparing sRNA, negatively regulating many mRNAs encoding dispensable iron-related proteins [START_REF] Chareyre | Bacterial Iron Homeostasis Regulation by SRNAs[END_REF] with functional homologs in many bacteria (Oglesby-Sherrouse and Murphy 2013). In S. aureus, however, no such iron responsive sRNA was identified until now. Furthermore, since iron and heme-iron overloads are toxic for the bacteria, most of them have developed additional regulatory mechanisms that fine-tune the acquisition and metabolism of iron and heme-iron in the absence of Fur repression [START_REF] Laakso | A Heme-Responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus Aureus[END_REF], as well as mechanisms to export iron excess, such as the Heme Responsible Transporter (HrtAB) [START_REF] Stauff | Staphylococcus Aureus HrtA Is an ATPase Required for Protection against Heme Toxicity and Prevention of a Transcriptional Heme Stress Response[END_REF].

The SarA protein family

The SarA protein family has the particular structure of a three-dimensional "wingedhelix", composed of 3 α-helices and 3 β-sheets, allowing the protein to bind to nucleic acids (Ambrose L. [START_REF] Cheung | The SarA Protein Family of Staphylococcus Aureus[END_REF]. SarA was the first transcriptional factor discovered by (A. L. [START_REF] Cheung | Cloning and Sequencing of SarA of Staphylococcus Aureus, a Gene Required for the Expression of Agr[END_REF]. The sarA gene is under the control of three different promotors (P1, P2 and P3), resulting in the transcription of three transcripts but the constitutive production of a single protein (Ambrose L. [START_REF] Cheung | The SarA Protein Family of Staphylococcus Aureus[END_REF]Jenul and Horswill 2019). SarA has been shown to regulate the expression of multiple virulence factors. For instance, SarA upregulates tst, hla, hlb and hld transcription and downregulates the transcription of sak and spa (Ambrose L. [START_REF] Cheung | The SarA Protein Family of Staphylococcus Aureus[END_REF][START_REF] Ziebandt | Extracellular Proteins of Staphylococcus Aureus and the Role of SarA and ΣB[END_REF]. In addition to SarA, there are 11 other transcriptional factors, identified as homologues of SarA, such as Rot, MgrA/Rat, SarS, SarR, SarZ, SarT, SarU, SarV, SarX, SarY and TcaR (reviewed in (Jenul and Horswill 2019)) and their regulatory networks intersect (Figure 18). For instance, MgrA plays a role in the activation of sarZ, sarX and sarS expression, apart from controlling the expression of several virulence factors like hla, coa, spa (Jenul and Horswill 2019;[START_REF] Luong | Mgr, a Novel Global Regulator in Staphylococcus Aureus[END_REF]. Another example is one of the most well-known transcriptional factors of SarA-like family, Rot (Repressor of toxins), which favors the expression of SarS. Rot also upregulates the expression of several surface proteins (Spa) and inhibits the expression of some secreted factors (ETB, Hla, several proteases and toxins, which is the opposite of the Agr system (Jenul and Horswill 2019;[START_REF] Saïd-Salim | Global Regulation of Staphylococcus Aureus Genes by Rot[END_REF].

Sigma factors

Sigma factors comprise major sigmaA (σA) and three alternative sigma factors: sigmaB (σB), sigmaH (σH) and sigmaS (σS) [START_REF] Gruber | Multiple Sigma Subunits and the Partitioning of Bacterial Transcription Space[END_REF][START_REF] Mittenhuber | A Phylogenomic Study of the General Stress Response Sigma Factor ΣB of Bacillus Subtilis and Its Regulatory Proteins[END_REF][START_REF] Morikawa | A New Staphylococcal Sigma Factor in the Conserved Gene Cassette: Functional Significance and Implication for the Evolutionary Processes[END_REF]L. N. Shaw et al. 2008;[START_REF] Tao | Alternative Sigma Factor ΣH Modulates Prophage Integration and Excision in Staphylococcus Aureus[END_REF][START_REF] Wu | Sigma-B, a Putative Operon Encoding Alternate Sigma Factor of Staphylococcus Aureus RNA Polymerase: Molecular Cloning and DNA Sequencing[END_REF]. They are involved in the imitation of transcription by binding to the core RNA polymerase to form the holoenzyme that binds to specific promoters and allow the bacteria to adapt quickly to stress and express specific virulence factors when required during infection. The primary sigma factor, σA, is responsible for the expression of housekeeping genes essential for growth. The alternative sigma factor H (σH) has been shown to modulate the transcription of phage integrase (int), to stabilize the lysogens in the host cell [START_REF] Tao | Alternative Sigma Factor ΣH Modulates Prophage Integration and Excision in Staphylococcus Aureus[END_REF];while σS activation enhances the severity of infection, the immune response, and the survival of S. aureus in mouse infection model [START_REF] Miller | The Extracytoplasmic Function Sigma Factor Σs Protects 205 against Both Intracellular and Extracytoplasmic Stresses in Staphylococcus Aureus[END_REF]L. N. Shaw et al. 2008). However, among alternative sigma factors, the most well-studied, σB, has been demonstrated to controls a large panel of genes involved in cellular functions (e.g. stress response). At least 30 virulence genes implicated in biofilm formation, cellular internalization, antibiotics resistance, etc. [START_REF] Bischoff | Microarray-Based Analysis of the Staphylococcus Aureus ΣB Regulon[END_REF]Jenul and Horswill 2019), were regulated by σB through recognizing and binding to a consensus sequence (GTTTAA-12-15-GGGTAT) located in the promoter region of its target genes [START_REF] Homerova | Optimization of a Two-Plasmid System for the Identification of Promoters Recognized by RNA Polymerase Containing Staphylococcus Aureus Alternative Sigma Factor ΣB[END_REF]).

Regulatory RNAs

In addition to proteins that regulate virulence factors, there are RNAs that are involved in the regulation of virulence factors and in the rapid adaptation of bacteria to different environmental conditions (Table 4). In Staphylococcus, RNAIII is the most well-known riboregulator of various virulence genes. It is the effector of the agr system that coordinates expression of S. aureus genes based on bacterial density. Although RNAIII remains the most well-known, there are more sRNAs recently studied in the bacterium.

Regulatory sRNAs of S. aureus will be described in more details in the next chapter.

V. Bacterial small regulatory RNAs

A. The discovery of small regulatory RNAs

To date, there are three main types of "classic" RNAs: messenger RNAs (mRNA), transfer RNAs (tRNA) and ribosomal RNAs (rRNA). The mRNAs are translated into proteins, while the tRNAs and the rRNAs have roles during the translation of the mRNA. tRNAs and rRNAs are non-coding RNAs, meaning that they do not have the information necessary for the synthesis of a protein. However, another type of non-coding RNA has been identified, namely regulatory RNAs (sRNAs). They allow the bacteria to regulate the expression of different factors involved in the adaptation to environmental changes.

In prokaryotes, 6S RNA was among the first regulatory RNAs identified in 1967 from E. coli [START_REF] Hindley | Fractionation of 32P-Labelled Ribonucleic Acids on Polyacrylamide Gels and Their Characterization by Fingerprinting[END_REF]) and rapidly sequenced [START_REF] Bronwlee | The Small RNA SprX Regulates the Autolysin Regulator WalR in Staphylococcus Aureus[END_REF]). However, it took about thirty years to identify its function in sequestering the sigma70 subunit of RNA polymerase (Karen Montzka [START_REF] Wassarman | 6S RNA Regulates E. Coli RNA Polymerase Activity[END_REF]. Not long after, another sRNA, RNAI, was identified and demonstrated to regulate the replication of the ColE1 plasmid of E. coli [START_REF] Stougaard | RNAs Involved in Copy-Number Control and Incompatibility of Plasmid R1[END_REF][START_REF] Tomizawa | Inhibition of ColE1 RNA Primer Formation by a Plasmid-Specified Small RNA[END_REF]) and of the plasmid pT181 in S. aureus (Richard P. [START_REF] Novick | PT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator[END_REF][START_REF] Pluta | Antisense and yet Sensitive: Copy Number Control of Rolling Circle-Replicating Plasmids by Small RNAs[END_REF]

(See Chapter V/ Section C/ 1.).
In the early 2000s, few regulatory RNAs had been identified in part due to available techniques like computational approaches, which allowed the identification of RNAs using different parameters to define sRNAs [START_REF] Bronsard | SRNA and Cis-Antisense SRNA Identification in Staphylococcus Aureus Highlights an Unusual SRNA Gene Cluster with One Encoding a Secreted Peptide[END_REF][START_REF] Livny | High-Throughput, Kingdom-Wide Prediction and Annotation of Bacterial Non-Coding RNAs[END_REF][START_REF] Mraheil | Comparative Genome-Wide Analysis of Small RNAs of Major Gram-Positive Pathogens: From Identification to Application[END_REF][START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF]. From then on, more bioinformatics predictions as well as the explosion of high throughput techniques (RNA-seq, DNA micro-arrays, clonage shotgun) have allowed the identification of many RNAs [START_REF] Altuvia | Regulatory Small RNAs: The Key to Coordinating Global Regulatory Circuits[END_REF][START_REF] Hüttenhofer | Experimental Approaches to Identify Non-Coding RNAs[END_REF][START_REF] Kazantsev | Bacterial RNase P: A New View of an Ancient Enzyme[END_REF][START_REF] Moore | The TmRNA System for Translational Surveillance and Ribosome Rescue[END_REF]. Indeed, advances in high-throughput sequencing analysis have revealed the existence of hundreds of potential regulatory RNAs, although only a fraction of which have been validated in vivo. However, the lack of a consensual and fully annotated S. aureus genome added to the problem of sRNAs nomenclature. To overcome this matter, [START_REF] Sassi | SRD: A Staphylococcus Regulatory RNA Database[END_REF] provided a Staphylococcus Regulatory RNA Database (SRD) that gathers a list of sRNAs identified and validated experimentally throughout the years. Not long after, Bouloc and his team performed in silico analysis to identify sRNAs likely to act in trans and to elucidate redundancies in the literature due to the use of different nomenclatures and concluded about 50 bona fide sRNAs in HG003 strain, a NCTC825 derivative strain commonly used for S. aureus genetic regulation studies (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF]. More recently, Carroll's group created an updated S. aureus genome annotation report, including annotations for 303 known sRNAs in USA300, associating with publicly available RNA-Seq data sets in order to recover lost information on sRNA expression, stability, and potential to encode peptides [START_REF] Sorensen | Reading between the Lines: Utilizing RNA-Seq Data for Global Analysis of SRNAs in Staphylococcus Aureus[END_REF]).

B. Classification and Mode of action

The discovery and progressive characterization of bacterial sRNAs have revealed various regulatory mechanisms. They can act on different targets: nucleic acids (RNA and DNA) or proteins. According to their mechanism of action, they have been separated into several classes: riboswitches, RNAs regulating proteins, DNAs or RNAs by base-pairings.

They can also act at different stages: during DNA replication or repair or gene expression, or at different levels of expression of the target: the transcriptional level, the translational level and/or the stability of the target RNA.

Riboswitches

Riboswitches are bacterial-specific, structured noncoding RNA commonly used by bacteria to detect a wide range of metabolites, amino acids, ions and even temperature changing (thermosensors) to regulate genes expression. The majority of the known riboswitches reside in the 5'or 3' UTR of the mRNAs targets and are identified as cis-acting RNAs where they control transcription or translation of the targets. They are consisted of two domains: a detection domain (aptamer) which binds to a ligand molecule causing a modification of the conformation of an adjacent expression platform; and a regulatory domain (expression platform) that converts ligand binding into a change in gene expression by adopting an alternative RNA structure [START_REF] Tucker | Riboswitches as Versatile Gene Control Elements[END_REF].

So far, there are five established or predicted mechanisms of riboswitch-mediated gene regulation in prokaryotes (reviewed in [START_REF] Breaker | Riboswitches and the RNA World[END_REF])) (Figure 19). The most common mechanism is the modulation of transcription termination; ligand binding to the aptamer leads to secondary structural modification of mRNA and forms a competitive secondary structure or anti-terminator, which obstructs the transcription and release a nascent RNA; for example, the bacterial response to intracellular Mg 2+ in Salmonella enterica. In low Mg 2+ concentration condition, the sensor PhoQ activates the response PhoP by phosphorylation; PhoP~P then binds to the promoter of mtgA gene and promotes the transcription of this gene, which encodes for protein involved in internalization of Mg 2+ .

In high concentration of Mg 2+ , Mg 2+ fixes to the 5' UTR of mtgA and blocks the transcription. Another well-known mechanism is the modulation of translation initiation; ligand binding to the riboswitch aptameric provokes in conformation changing and allows the recruitment of the ribosome to the RBS of target mRNA and initiates the translation.

For instance, in lactic acid bacteria, S-adenosylmethionine (SAM) has been demonstrated to bind the SMK box within the 5' UTR of metK gene and cause a rearrangement of the RNA structure resulting in sequestration of SD and preventing the translation of MetK (SAM synthetase) [START_REF] Fuchs | The SMK Box Is a New SAM-Binding RNA for Translational Regulation of SAM Synthetase[END_REF].

In addition, several other interesting mechanisms for riboswitch-mediated gene regulation are ribozymes, which possess a double function: they regulates gene expression in response to a metabolite and catalyze their own cleavage [START_REF] Winkler | Regulation of Bacterial Gene Expression by Riboswitches[END_REF], and thermosensors that modulate gene translation dependently on the temperature such as the case of prfA gene in L. monocytogenes [START_REF] Johansson | An RNA Thermosensor Controls Expression of Virulence Genes in Listeria Monocytogenes[END_REF].

At low temperature (under 30°C), the 5' UTR of prfA forms a secondary structure that masks the SD resulting in preventing translation, whereas at high temperature, this secondary structure enables the RBS and allows translation initiation [START_REF] Johansson | An RNA Thermosensor Controls Expression of Virulence Genes in Listeria Monocytogenes[END_REF]. Interestingly, in the same bacterial model, two other riboswitches (SreA and SreB) have been demonstrated to act as noncoding RNAs and to regulate in trans the expression of prfA in L. monocytogenes in response to the presence or the absence of Sadenosylmethionine (SAM) [START_REF] Loh | A Trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria Monocytogenes[END_REF]. Riboswitches of the SAM, SreA and SreB family have been shown to control the expression of the PrfA protein by binding to the 5' UTR of the prfA mRNA [START_REF] Loh | A Trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria Monocytogenes[END_REF]). This binding inhibits the translation of prfA mRNA which encodes a virulence regulator of Listeria monocytogenes.

To date, there are approximatively 40 different classes of riboswitches discovered, validated, and modeled with their respective ligands [START_REF] Mccown | Riboswitch Diversity and Distribution[END_REF]). Among them, some of the riboswitches, such as AdoBcl (Adenosyl cobalamin) or SAM or FMN (Flavin mononucleotide) riboswitches, occur the widest number of pathogens like C. difficile, L. monocytogenes, S. pneumonia, S. aureus, E. faecalis and many more (reviewed in [START_REF] Bédard | Riboswitch Regulation Mechanisms: RNA, Metabolites and Regulatory Proteins[END_REF]); therefore, they could be excellent potential targets for antibacterial drug treatments (reviewed in [START_REF] Panchal | Riboswitches as Drug Targets for Antibiotics[END_REF]).

sRNAs interacting with proteins

More than a decade of intensive study of small, noncoding RNAs (sRNAs) in bacteria has identified hundreds of sRNAs. A growing and divergent group of sRNAs interact with and modify proteins. By mimicking the structures of RNA or DNA targets, these RNAs bind proteins to inhibit their activity. This is the least well-known class because only a small number of sRNAs have been identified as modulating the activity of proteins.

6S RNA, first discovered in E. coli in 1967 by Hindley, is found in most bacteria and its expression/ accumulation is species-specific; some species have multiple 6S RNAs such as B. subtilis with two copies whereas E. coli only has one copy [START_REF] Trotochaud | A Highly Conserved 6S RNA Structure Is Required for Regulation of Transcription[END_REF]. This RNA is a global regulator that downregulates transcription of multiple genes due to its interaction with the primary holoenzyme form of RNA polymerase (RNAP). Indeed, 6S has a highly conserved structure mimicking the conformation of a DNA during transcription initiation, which allows 6S to interact with the sigma 70 subunit of RNAP [START_REF] Cavanagh | 6S RNA, a Global Regulator of Transcription in Escherichia Coli, Bacillus Subtilis, and Beyond[END_REF][START_REF] Trotochaud | A Highly Conserved 6S RNA Structure Is Required for Regulation of Transcription[END_REF]) (Figure 20). Moreover, it is suggested that 6S RNA alters the competition between different sigma factors by binding to sigma 70, which might lead to an increase in activity for sigma S-dependent transcription in vivo (reviewed in [START_REF] Cavanagh | 6S RNA, a Global Regulator of Transcription in Escherichia Coli, Bacillus Subtilis, and Beyond[END_REF]). Albeit E. coli 6S RNA is important for modulating stress and optimizing survival during nutrient limitation; however, its functions are also speciesspecific, just like its expression. For instance, 6S RNA has been demonstrated to be associated with bacterial extracellular vesicles (Evs) in S. aureus [START_REF] Joshi | Transcriptome Profiling of Staphylococcus Aureus Associated Extracellular Vesicles Reveals Presence of Small RNA-Cargo[END_REF]).

Furthermore, it has been proven in vitro and in vivo, a synthesis of a short transcript of 14 to 20 nucleotides (RNAP) encoded from 6S RNA by RNA polymerase, although no information on the specific function of this transcript is known [START_REF] Svensson | Small RNAs in Bacterial Virulence and Communication[END_REF]; Karen M. [START_REF] Wassarman | Synthesis-Mediated Release of a Small RNA Inhibitor of RNA Polymerase[END_REF].

CsrB and CrsB RNAs belong to the carbon storage regulator (Csr) system in E. coli and the homologous repressor of secondary metabolites (Rsm), found in other bacteria. CrsB and CrsC are identified as regulatory RNAs that modulate the activity of CsrA (or RsmA in other bacteria), an RNA-binding protein (RDP). This RBP is a pleiotropic regulator of carbon metabolisms and regulates gene expression post-transcriptionally at stationary growth phase. CsrA interacts with GGA sequence motif in the 5' UTR of its mRNA targets and affects ribosome binding and/or mRNA stability (M. Y. [START_REF] Liu | The RNA Molecule CsrB Binds to the Global Regulatory Protein CsrA and Antagonizes Its Activity in Escherichia Coli[END_REF][START_REF] Weilbacher | A Novel SRNA Component of the Carbon Storage Regulatory System of Escherichia Coli[END_REF]) (Figure 21). As for CsrB and CrsC, their structure contain multiple GGA motifs; when they are sufficiently expressed, they could isolate and antagonize CsrA protein. CRISPR loci are composed of a leader RNA, "direct repeat" and spacers. The leader sequence is located at 5' end of each CRISPR, directly adjacent to the first direct repeat, and is often rich in A+T residues. These repeated sequences are sometimes partially palindromic, which allows them to form very stable secondary structures [START_REF] Kunin | Evolutionary Conservation of Sequence and Secondary Structures in CRISPR Repeats[END_REF] and are separated by portions of variable DNA sequence called "spacers". The spacers correspond to captured viral or plasmid segments that are acquired from foreign DNA od plasmids or viruses during adaptation or immunization.

Their number can reach several hundred units but most loci contain less than 50 [START_REF] Horvath | Comparative Analysis of CRISPR Loci in Lactic Acid Bacteria Genomes[END_REF].

CRISPR/Cas mechanism is based on the acquisition of foreign genes through the recognition and specific degradation of foreign genetic elements [START_REF] Makarova | A Putative RNA-Interference-Based Immune System in Prokaryotes: Computational Analysis of the Predicted Enzymatic Machinery, Functional Analogies with Eukaryotic RNAi, and Hypothetical Mechanisms of Action[END_REF] (Figure 22). CRISPR systems work in association with genes encoding Cas proteins, which are adjacent to each CRISPR loci [START_REF] Jansen | Identification of Genes That Are Associated with DNA Repeats in Prokaryotes[END_REF]. The cas genes code for a large family of proteins heterogeneous which contain functional domains typical of nucleases, helicases, and polynucleotide binding proteins [START_REF] Haag | A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes[END_REF]. When phage invades a bacterium, phage nucleic acids proliferate in the cell and new phage particles are produced, leading to the death of the majority of susceptible bacteria. This immune function is executed in a three-stage process: (I) Adaptation, (II) Processing and (III) Interference. First, a small number of bacteria acquire spacers derived from these invading foreign nucleic acids giving them immunity (Adaptation). This immunity is linked to the recognition and degradation by the CRIPSR-Cas system of foreign nucleic acids.

Then, the CRISPR locus is transcribed into a single pre-crRNA (CRISPR RNA) which will be matured to produce a collection of small crRNA (CrRNA Biogenesis) (Processing). Each crRNA, in association with Cas proteins forms the CRISPR-Cas system. Small crRNAs recognize and interact by sequence complementarity with phage nucleic acids causing their degradation (Interference) (reviewed in [START_REF] Faure | CRISPR-Cas in Mobile Genetic Elements: Counter-Defence and Beyond[END_REF][START_REF] Samson | The CRISPR-Cas Immune System and Genetic Transfers: Reaching an Equilibrium[END_REF]). NanoRNAs are identified as another class of regulatory RNAs. The frequent transcription and rapid degradation of mRNAs are needed for bacteria to adapt to a wide range of nutritional and environmental changes. mRNA decay is initiated by a series of endo-/ exoribonucleases and leaves behind 2-to 5-nts long oligoribonucleotides called "nanoRNAs". They could also be produced from products of RNA cleavage during transcription elongation by RNAP backtracking or products of abortive transcription initiation (reviewed in (Nickels and Dove 2011)) (Figure 23). They have been demonstrated to prime transcription initiation in bacteria by being incorporated directly into a target transcript. In addition, it is believed that nanoRNAs act as activators or repressors of the transcription of target genes depending on the state of phosphorylation of their 3 'end. Addition of nucleotides to the 5 ′ end of RNAs transcribed by nanoRNAs could alter the secondary structure and thus modulate translation and / or stability of transcripts. The degradation and therefore the regulation of these nanoRNAs is effected by a 3'-5' exonuclease, oligoribonuclease (Orn). In the absence of Orn, nanoRNAs can be used as an RNA polymerase primer to initiate RNA transcription that might be harmful for cell with unknown mechanism. Goldman et al. has shown that in E. coli, when Orn is inactivated, the amount of nanoRNAs increases significantly, followed by the loss of viability of the bacteria [START_REF] Nickels | NanoRNAs: A Class of Small RNAs That Can Prime Transcription Initiation in Bacteria[END_REF]. Since the use of nanoRNAs to initiate transcription could have an overall influence on gene expression, they can therefore be considered as regulators of gene expression by modulating the initiation of gene transcription and / or transcript stability [START_REF] Liao | The Special Existences: NanoRNA and NanoRNase[END_REF][START_REF] Nickels | NanoRNAs: A Class of Small RNAs That Can Prime Transcription Initiation in Bacteria[END_REF].

sRNAs interacting with RNAs

The vast majority of sRNAs interact with RNAs, they can be classified into two categories: sRNAs encoding in cis, which is located on the opposite strand of the gene encoding their target mRNAs, and sRNAs encoding in trans that is located in a different chromosomal region than the gene for their target mRNA [START_REF] Waters | Regulatory RNAs in Bacteria[END_REF].

Cis-encoded sRNAs

Cis-encoded sRNAs are expressed from the same loci as mRNA targets on the opposite strand of DNA. This results in perfect and usually extended complementarity between sRNAs and their target mRNAs. They can inhibit the expression of their targets by several mechanisms: transcriptional interference, early termination of transcription, alteration of stability or modulation of the translation of the target mRNA [START_REF] Georg | Cis-Antisense RNA, Another Level of Gene Regulation in Bacteria[END_REF] (reviewed in [START_REF] Stork | Transcription Termination within the Iron Transport-Biosynthesis Operon of Vibrio Anguillarum Requires an Antisense RNA[END_REF])).

The first type of cis-encoded sRNAs are RNAs antisense (asRNAs). They are generally found on plasmids or other mobile genetic elements, such as transposons or bacteriophages, but also on the chromosome [START_REF] Gerhart | Antisense RNA Control in Bacteria, Phages, and Plasmids[END_REF][START_REF] Svensson | Small RNAs in Bacterial Virulence and Communication[END_REF][START_REF] Thomason | Bacterial Antisense RNAs: How Many Are There, and What Are They Doing?[END_REF][START_REF] Waters | Regulatory RNAs in Bacteria[END_REF][START_REF] Westermann | Regulatory RNAs in Virulence and Host-Microbe Interactions[END_REF]). Thus, they control the maintenance and the stability of the mobile genetic elements by acting, for example, on the inhibition of the maturation of the primers. For instance, RNAI in E. coli, a 108 nucleotide RNA which is encoded on the opposite strand of the gene encoding RNAII, has been demonstrated to inhibit primer formation and consequently prevent RNA-DNA hybrid for plasmid ColE1 [START_REF] Stougaard | RNAs Involved in Copy-Number Control and Incompatibility of Plasmid R1[END_REF][START_REF] Tomizawa | Inhibition of ColE1 RNA Primer Formation by a Plasmid-Specified Small RNA[END_REF]) (Figure 24). RNAI is also involved on the transcriptional reduction of rep mRNA [START_REF] Brantl | Regulatory Mechanisms Employed by Cis-Encoded Antisense RNAs[END_REF][START_REF] Svensson | Small RNAs in Bacterial Virulence and Communication[END_REF][START_REF] Westermann | Regulatory RNAs in Virulence and Host-Microbe Interactions[END_REF]) (See Chapter V/ Section C/1.). Another sRNA discovered in bacteria that acts as asRNA and is involved in plasmid and transposon copy-number control is RNA-OUT in the case of IS10. Translation of the transposase mRNA (RNA-IN) is inhibited by the cis-encoded asRNA, RNA-OUT via base pairing between the 5' end of RNA-IN and the terminal loop domain of RNA-OUT [START_REF] Simons | Translational Control of IS10 Transposition[END_REF].

The next category of cis-encoded sRNAs is the toxin-antitoxin system (TAS). They are twogene elements composed of a gene encoded for a stable protein toxin (less than 60 amino acids), whose expression leads to growth cessation or cell death, and a corresponding antitoxin that neutralizes the toxicity during bacterial growth. TAS are classified into six groups based on the nature of the antitoxin and mode of action since all toxins are proteins. For instance, antitoxins that are non-coding RNAs belong to type I and type III systems, and antitoxins in other types are low-molecular-weight proteins (reviewed in (Camille Riffaud, Pinel-Marie, and Felden 2020)). Here, we only show TAS type I where antitoxin is a cis-encoded RNA acting in trans (Figure 25). Binding between RNA antitoxin to the toxin gene inhibits its translation and causes the degradation of toxin gene [START_REF] Svensson | Small RNAs in Bacterial Virulence and Communication[END_REF]. For example, in E. coli, ibs gene encodes for a peptide whose overexpression provokes cell membrane disruption and Sib RNA antitoxin can repress this toxicity through base pairing in ibs mRNA ORF [START_REF] Fozo | Small Toxic Proteins and the Antisense RNAs That Repress Them[END_REF][START_REF] Fozo | Small Toxic Proteins and the Antisense RNAs That Repress Them[END_REF]. The function of these chromosome-encoded TA systems remains unclear; however, they might mediate stress metabolism adaptation, persistent cell formation, or antibiotic resistance [START_REF] Svensson | Small RNAs in Bacterial Virulence and Communication[END_REF][START_REF] Westermann | Regulatory RNAs in Virulence and Host-Microbe Interactions[END_REF]. Indeed, type I TA systems have been demonstrated to involve in the bacterial pathogens like TisB/IstR-A in E. coli [START_REF] Edelmann | Post-Transcriptional Deregulation of the TisB/IstR-1 Toxin-Antitoxin System Promotes SOS-Independent Persister Formation in Escherichia Coli[END_REF]Vogel, Jörg;[START_REF] Vogel | The Small RNA IstR Inhibits Synthesis of an SOS-Induced Toxic Peptide[END_REF]Wagner, E. Gerhart H;[START_REF] Altuvia | Regulatory Small RNAs: The Key to Coordinating Global Regulatory Circuits[END_REF] or SprA1/ SprA1-as in S. aureus [START_REF] Beaume | Cartography of Methicillin-Resistant S. Aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions[END_REF][START_REF] Sayed | A Cis-Antisense RNA Acts in Trans in Staphylococcus Aureus to Control Translation of a Human Cytolytic Peptide[END_REF].

Recently, according to transcriptomic studies of Listeria spp., a new class of asRNAs has been categorized, called excludons. They are made up of long antisense RNAs (lasRNAs) that overlap multiple coding phases. LasRNA transcription is initiated from a promoter located on the complementary strand of an mRNA, generating asRNA that overlaps the entire coding phase of the opposite strand. However, transcription does not end at the end of this overlap but extends to the neighboring gene encoded on the same strand.

LasRNAs inhibit the expression of one group of genes while activating the expression of a second group of genes with related physiological functions [START_REF] Sesto | The Excludon: A New Concept in Bacterial Antisense RNA-Mediated Gene Regulation[END_REF].

Trans-encoded sRNAs

This is another class of sRNAs acting by base pairing, which, unlike asRNAs, are not encoded at a same genetic locus at their target mRNAs and share only limited complementarity with their target and on many occasions, requires the assistance of a chaperone protein Hfq to facilitate sRNA-mRNA interaction. The interaction between sRNAs and its target mRNA regulates gene expression through various mechanisms: this may be due to a change in translation, mRNA stability and/or transcription efficiency. Trans-encoded sRNAs regulate its target expression through three major mechanisms: repression of translation followed by degradation by RNases or activation of translation, or repression of transcription.

The best represented class corresponds to the sRNAs inhibiting translation of mRNA targets. They are able to bind to the translation initiation site of the mRNA and prevent the fixation of Ribosome; therefore leads to inhibition of translation, whether or not coupled with the degradation of the target mRNA. This is the case in E. coli with RyhB sRNAs which, with the help of the Hfq chaperone, represses translation of sodB mRNA that encodes for superoxide dismutase, by recruiting two RNAs by RNase E and RNase III [START_REF] Massé | Coupled Degradation of a Small Regulatory RNA and Its MRNA Targets in Escherichia Coli[END_REF][START_REF] Morita | Translational Repression Is Sufficient for Gene Silencing by Bacterial Small Noncoding RNAs in the Absence of MRNA Destruction[END_REF] (Figure 26A). However, there are some cases where this interaction leads to translation activation by a conformational change in the secondary structure of the mRNA target resulting in ribosome recruitment. For instance, in E. coli, the same RyhB sRNA also activates the translation of shiA mRNA expressing a permease of shikimate, an aromatic compound which participates in the synthesis of siderophores [START_REF] Prévost | The Small RNA RyhB Activates the Translation of ShiA MRNA Encoding a Permease of Shikimate, a Compound Involved in Siderophore Synthesis[END_REF]) (Figure 26B). The RyhB sRNA illustrates a situation where an sRNA can regulate several targets and in different ways. In addition, some mRNAs expression could also be regulated by multiple sRNAs; for example, in enterica bacteria, glmS encoding for glutamine-fructose-6phosphate (GlcN6P) is regulated by two homologous sRNAs, GlmY and GlmZ. In depletion of GlcN6P, the two sRNAs bind to glmS mRNA and induce the translation to replenish GlcN6P (M. A. [START_REF] Khan | Two Small RNAs Conserved in Enterobacteriaceae Provide Intrinsic Resistance to Antibiotics Targeting the Cell Wall Biosynthesis Enzyme Glucosamine-6-Phosphate Synthase[END_REF].

It has been shown recently that, the interaction between a sRNAs and its target can also repress the transcription of the target mRNA. For example, in Salmonella, the formation of a duplex between ChiX sRNAs and chiPQ mRNA results in inhibition transcription [START_REF] Bossi | A Role for Rho-Dependent Polarity in Gene Regulation by a Noncoding Small RNA[END_REF]. sRNA allows exposure of rut sites normally hidden by the ribosome. These sites are used to bind the termination factor Rho which will lead to the detachment of the complex of transcription and induce the formation of a truncated mRNA [START_REF] Bossi | A Role for Rho-Dependent Polarity in Gene Regulation by a Noncoding Small RNA[END_REF]. Conversely, sRNAs can also suppress the Rho-dependent transcription terminator [START_REF] Sedlyarova | SRNA-Mediated Control of Transcription Termination in E. Coli[END_REF]. This type of regulation of sRNAs at the transcriptional level remains little described and does not represent the most common sRNA regulations on their targets.

Other sRNAs regulation mechanisms

RNAs have been proved to possess multiple functions. They could act as a sRNAs and also possessing an open reading frame (ORF) coding for a peptide. These are sRNAs that interact with mRNAs and either express a protein or interact with proteins to regulate their activity such as RNAIII and PSM-mec in S. aureus in virulence regulation (Delphine [START_REF] Bronesky | Staphylococcus Aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression[END_REF][START_REF] Qin | PSM-Mec-A Virulence Determinant That Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci[END_REF], SR1 in B. subtilis in arginine catabolism activation [START_REF] Heidrich | The Small Untranslated RNA SR1 from the Bacillus Subtilis Genome Is Involved in the Regulation of Arginine Catabolism[END_REF][START_REF] Licht | Implication of CcpN in the Regulation of a Novel Untranslated RNA (SR1) in Bacillus Subtilis[END_REF] and SgrS in E. coli in glucose/mannose transporter and virulence [START_REF] Wadler | A Dual Function for a Bacterial Small RNA: SgrS Performs Base Pairing-Dependent Regulation and Encodes a Functional Polypeptide[END_REF]. It has been shown that there are currently 10 sRNAs encode short proteins, of which five have an associated function (reviewed in [START_REF] Gimpel | Dual-Function Small Regulatory RNAs in Bacteria[END_REF]). Another case of sRNA with multiple functions is transfer-messenger RNA (tmRNA or SsrA) that displays both tRNA and mRNA assets.

TmRNA controls trans-translation, which is a process of rescuing the ribosomes stalled during translation of defective mRNAs, such as those lacking in-frame termination codons in all bacteria [START_REF] Dulebohn | Trans-Translation: The TmRNA-Mediated Surveillance Mechanism for Ribosome Rescue, Directed Protein Degradation, and Nonstop MRNA Decay[END_REF]), but also acts as an asRNA to regulate ctrMN operon in S. aureus (Y. [START_REF] Liu | SsrA (TmRNA) Acts as an Antisense RNA to Regulate Staphylococcus Aureus Pigment Synthesis by Base Pairing with CrtMN MRNA[END_REF].

Recent discoveries suggested the existence of new sRNAs acting as RNAs sponges in gene expression regulation. The term "sponge" RNA came from eukaryotic regulatory RNA studies demonstrating that engineered RNA can compete with mRNA for miRNA interaction [START_REF] Ebert | MicroRNA Sponges: Competitive Inhibitors of Small RNAs in Mammalian Cells[END_REF]. However, more and more RNA sponge cases have been recently studied in prokaryotes. In Salmonella enterica, mRNA-derived SroC sRNA acts as a sponge to sequester and trigger degradation of GcvB [START_REF] Miyakoshi | Cross Talk between ABC Transporter m RNA s via a Target m RNA -derived Sponge of the G Cv B Small RNA[END_REF] or the 3′ external transcribed spacers (3' ETS) of tRNA gene can also act as sRNA sponge to absorb transcriptional noise from repressed RNAs [START_REF] Lalaouna | Identification of SRNA Interacting with a Transcript of Interest Using MS2-Affinity Purification Coupled with RNA Sequencing (MAPS) Technology[END_REF]. In addition, the most recent case of RNA sponge in Gram-positive bacteria has been identified and validated in (Durand et al. 2021). RoxS is an sRNA implicated in oxidative stress response of B. subtilis and its expression and activity has been demonstrated to be affected by RoxA acting as a sponge for RoxS (Durand et al. 2021).

Proteins helpers of RNAs

Many proteins are associated with sRNAs and perform a multitude of functions such as the modification of the secondary structure of their RNA targets, the maturation of the sRNA, the promotion of the sRNA-mRNA interaction, and the degradation of the duplex formed. To date, new RNA-binding proteins were discovered such as the sequester CsrA/Rsm, the FinO/ProQ family and the CspD/E family, besides RNases and protein chaperone Hfq (reviewed in [START_REF] Quendera | RNA-Binding Proteins Driving the Regulatory Activity of Small Non-Coding RNAs in Bacteria[END_REF][START_REF] Romeo | Post-Transcriptional Regulation on a Global Scale: Form and Function of Csr/Rsm Systems[END_REF]). Hfq (Host factor Qβ) is widely identified as a global regulator and key element in sRNA-based regulation networks with ~30 % of sRNAs relying on this protein to carry out their functions in E. coli [START_REF] Vogel | Hfq and Its Constellation of RNA[END_REF]. In Salmonella, Hfq associates with almost half of the co-immunoprecipitated sRNAs [START_REF] Sittka | Deep Sequencing Analysis of Small Noncoding RNA and MRNA Targets of the Global Post-Transcriptional Regulator, Hfq[END_REF]. Beside involving in proteinprotein interactions [START_REF] Caillet | Identification of Protein-Protein and Ribonucleoprotein Complexes Containing Hfq[END_REF]) and in RNA degradation (reviewed in [START_REF] Quendera | RNA-Binding Proteins Driving the Regulatory Activity of Small Non-Coding RNAs in Bacteria[END_REF])), Hfq is also implicated in remodeling sRNA secondary structure. For instance, through binding to MicA sRNA, Hfq is demonstrated to change this sRNA secondary structure, to alter its stability and to allow exposure of the ompA binding site for pairing that leads to translational repression [START_REF] Henderson | Characterization of MicA Interactions Suggests a Potential Novel Means of Gene Regulation by Small Non-Coding RNAs[END_REF]). Although Hfq is well documented in other bacterial species to play an important role in gene expression regulation but whose role in S. aureus is unclear. Recent studies suggested that Hfq might play a role in the oxidative stress response in S. aureus, decreasing the bacteria's ability to survive in macrophages (Bouloc and Repoila 2016; W. [START_REF] Liu | Staphylococcus Aureus Pigmentation Is Not Controlled by Hfq[END_REF]. However, Hfq is not as an important regulator of gene expression as it is the case in Gram-negative bacteria, nor globally influences RNA stability.

Ribonucleases (RNases) are also classified as RBPS and play an important role in regulating the expression of target genes by sRNAs. For a very large number of sRNAs in bacteria, the interaction between sRNA and its mRNA prevents the binding of ribosomes and promotes the degradation of the mRNA by an RNase recruited during the formation of the duplex leading to an irreversible inhibition of translation of the target gene. RNases can recognize the single-stranded or double-stranded structures of RNAs (Figure 27). For instance, RNase E has been shown to act as a single-strand-specific endonuclease to initiate AmgR sRNA-mgt operon target degradation in Salmonella (reviewed in (Ul Haq, Müller, and Brantl 2020)); or RNase III as the double-stranded ribonuclease in Rat sRNA-3' UTR of txpA mRNA in B. subtilis [START_REF] Durand | The Essential Function of B. Subtilis RNase III Is to Silence Foreign Toxin Genes[END_REF]. Moreover, certain RNases are also involved in the degradation and processing of RNAs within a multi-protein complex called a degradosome whose composition differ between Gram-positive or negative bacteria. For instance, most of the genomes of Gram-positive bacteria produce two orthologous ribonucleases, RNase J1 and RNase J2. These enzymes are part of the degradosome complex of Gram-positive bacteria consisting of a PNPase, an enolase, a DEAD box helicase (CshA), a phosphofructokinase and RNase Y. In E. coli, the degradosome is made up of four enzymes: RNaseE, exonuclease 3′-5' PNPase, RhlB helicase and an enolase (reviewed in [START_REF] Carpousis | The RNA Degradosome of Escherichia Coli: An MRNA-Degrading Machine Assembled on RNase E[END_REF][START_REF] Haq | Intermolecular Communication in Bacillus Subtilis: RNA-RNA, RNA-Protein and Small Protein-Protein Interactions[END_REF]).

These RNases play an important role in post-transcriptional regulations and are, moreover, involved in the regulation of virulence factors.

Another case is ProQ of the FinO family that is commonly found in Proteobacteria [START_REF] Olejniczak | ProQ/FinO-Domain Proteins: Another Ubiquitous Family of RNA Matchmakers?[END_REF]. The majority of ProQ studies are performed in E. coli, Salmonella enterica and Legionella pneumonia (reviewd in [START_REF] Quendera | RNA-Binding Proteins Driving the Regulatory Activity of Small Non-Coding RNAs in Bacteria[END_REF]).

However, ProQ binding specificity is different from Hfq, based on RIL-seq performed by [START_REF] Melamed | RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles[END_REF], ProQ shows affinity for encoding sequences while Hfq is enriched in both sRNAs and mRNAs complex formation. One of the well-known example for ProQ role is the RaiZ sRNA -hupA mRNA [START_REF] Smirnov | Molecular Mechanism of MRNA Repression in Trans by a ProQ-dependent Small RNA[END_REF]. This complex formation ProQ/ RaiZ/ hupA is demonstrated to prevent the translation of the mRNA by masking the binding site of the 30S ribosome subunit [START_REF] Smirnov | Molecular Mechanism of MRNA Repression in Trans by a ProQ-dependent Small RNA[END_REF]) (Figure 28).

In addition, Cold-shock proteins (CSPs) constitute the largest nonribosomal RBP family in Gram-negative bacteria, including E. coli and S. enterica. Out of the nine and six CSP paralogs present in E. coli and S. enterica, CspA is an RNA chaperone that accumulates during growth at low temperatures and modulates both the transcription and translation of target genes required for bacterial survival in these conditions [START_REF] Bae | Escherichia Coli CspA-Family RNA Chaperones Are Transcription Antiterminators[END_REF][START_REF] Giuliodori | The CspA MRNA Is a Thermosensor That Modulates Translation of the Cold-Shock Protein CspA[END_REF][START_REF] Jiang | CspA, the Major Cold-Shock Protein of Escherichia Coli, Is an RNA Chaperone[END_REF]. Intriguingly, other family members, such as CspC and CspE, are not induced in response to cold shock but are highly expressed at higher temperatures and suggested to be implicated in stress response and virulence of Salmonella pathogenicity [START_REF] Michaux | New Insight into Cold Shock Proteins: RNA-Binding Proteins Involved in Stress Response and Virulence[END_REF].

C. Regulatory sRNAs in S. aureus

Since early 2000s, more sRNAs have been discovered in various S. aureus strains from computational analysis to experimental approaches like microarrays, clonage shotgun, and more recently via high-throughput RNA sequencing (RNA seq) (See Chapter V/ Section A). They are defined in general as stable molecules between 50 and 500 nts in size and their annotation depends on the approached technics and/or the localization of studied sRNAs. For example, sRNAs, whose the genes are located in pathogenicity islands of S. aureus, are called Spr (Small pathogenicity island RNA) [START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF] (Figure 29). Other are named RsaO sRNAs (for RNA of S. aureus Orsay) [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Marchais | Single-Pass Classification of All Noncoding Sequences in a Bacterial Genome Using Phylogenetic Profiles[END_REF]; or Rsa sRNAs (Staphylococcus aureus RNA) [START_REF] Geissmann | The Role of MRNA Structure in Translational Control in Bacteria[END_REF]. Regulatory RNAs discovered by experimental method from Geneva are named Teg sRNAs (Transcript from Experimental method from Geneva) [START_REF] Beaume | Cartography of Methicillin-Resistant S. Aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions[END_REF](Beaume et al. , 2011) ) and some other small groups of sRNAs that are still understudied such as the SSR (small stable RNAs) [START_REF] Anderson | Characterization of the Staphylococcus Aureus Heat Shock, Cold Shock, Stringent, and SOS Responses and Their Effects on Log-Phase MRNA Turnover[END_REF][START_REF] Retallack | A Role for a Small Stable RNA in Modulating the Activity of DNA-Binding Proteins[END_REF]. Moreover, there is situation where an sRNA studied in one work was named differently in another publish, for instance, RsaOW is named Teg17 [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Guillet | Emerging Functions for the Staphylococcus Aureus RNome[END_REF] or SprX alias RsaOR and Teg15 (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF]. With an overflow of regulatory RNAs identified during this last decade, our lab designed a platform, called "Staphylococcal Regulatory RNA Database" with an objective to compile all published regulatory RNAs and to simplify the identification by naming them Staphylococci regulatory RNA (srn) [START_REF] Sassi | SRD: A Staphylococcus Regulatory RNA Database[END_REF]. However, many more sRNAs have been identified and it becomes a challenge to carry on the update. Indeed, Madër and his collaborators recently provided a global analysis of transcriptional regulation and non-coding RNAs in S. aureus [START_REF] Mäder | Staphylococcus Aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions[END_REF]) and Bouloc's group provided a new reassessment of approximately 50 bona fide sRNAs in S. aureus by in silico analysis (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF].

Here, the sRNAs will be presented based on their original discovered name and their shared physiological function in S. aureus. At low cell density (during the exponential phase), the agr system is not induced: the level of expression of RNAIII is low and the mRNAs allowing tissue colonization (sa1000, spa, sa2353 and rot) are expressed. At high cell density (during the stationary phase), the agr system self-induces via quorum-sensing (QS). The expression of RNAIII allows the inhibition of translation of the above-mentioned target RNAs and the activation of the expression of hla. In addition, the coding phase of RNAIII is translated to produce hemolysin delta. Induction of the agr system by QS enables the coordinated response of the entire S. aureus population. This response allows the destruction of host tissue and promotes colonization by the bacteria (Adapted from [START_REF] Repoila | Small Regulatory Non-Coding RNAs in Bacteria: Physiology and Mechanistic Aspects[END_REF].

RNAI

RNAI was first studied in E. coli in the early 1980s for its contribution in the control of plasmid ColE1 replication [START_REF] Stougaard | RNAs Involved in Copy-Number Control and Incompatibility of Plasmid R1[END_REF][START_REF] Tomizawa | Inhibition of ColE1 RNA Primer Formation by a Plasmid-Specified Small RNA[END_REF] (See Chapter V/ Section B/3/3.1.). RNAI was later identified in S. aureus as a first cisencoded sRNA that controls the rolling-circle replication (RCR) of plasmid pT181 by transcriptional attenuation (Richard P. [START_REF] Novick | PT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator[END_REF]). The replication of small multiple copy plasmids carrying antibiotic resistance genes in Gram-positive bacteria generally occurs by RCR mechanism. And the most distinct trait of RCR is its initiation step by a replication initiator protein (Rep) encoded by the plasmid. For instance, pT181, a tetracycline resistant plasmid of S. aureus, regulates its own replication by expressing RNAI that blocks the expression of RepC. This regulation involves pairings between complementary loops in the mRNA leader (repC) and the asRNA (RNAI), which results in the formation of an anti-terminator at the 5′ to the repC initiation codon that blocks the access of the Ribosome. By masking the RBS, RNAI leads to a premature termination of transcription of repC [START_REF] Guillet | Emerging Functions for the Staphylococcus Aureus RNome[END_REF][START_REF] Novick | PT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator[END_REF] reviewed in Pluta and Espinosa 2018) (Figure 30).

RNAIII: A Paradigm

Discovered almost three decades ago, RNAIII is a 514 nucleotide RNA, that acts as the effector of the Agr "quorum sensing" system (R. P. [START_REF] Novick | Synthesis of Staphylococcal Virulence Factors Is Controlled by a Regulatory RNA Molecule[END_REF]. RNAIII accumulates during growth and peaks in the post-exponential phase of growth (R. P. [START_REF] Novick | Synthesis of Staphylococcal Virulence Factors Is Controlled by a Regulatory RNA Molecule[END_REF]. It allows the temporal regulation of the expression of virulence factors, that is to say, it controls the switch between colonization and bacterial infection of the host (Figure 31). RNAIII is a bifunctional RNA, encoding a PSM called δ-hemolysin (hld gene) [START_REF] Janzon | The Role of the Delta-Lysin Gene (Hld) in the Regulation of Virulence Genes by the Accessory Gene Regulator (Agr) in Staphylococcus Aureus[END_REF] and acts as a trans-acting sRNA on multiple target mRNAs [START_REF] Morfeldt | Activation of Alpha-Toxin Translation in Staphylococcus Aureus by the Trans-Encoded Antisense RNA, RNAIII[END_REF]) (Figure 32). This sRNA possesses an extraordinarily complex structure with 14 stem-loops [START_REF] Benito | Probing the Structure of RNAIII, the Staphylococcus Aureus Agr Regulatory RNA, and Identification of the RNA Domain Involved in Repression of Protein A Expression[END_REF], and three of them contribute highly to the regulation several virulence factors. Indeed, stem-loops 7, 13 and 14 with a consensus motif rich in cytosines, facilitate interactions with the guanine-rich RBS of its target mRNAs [START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF]. RNAIII, binding with its target mRNA, will block Ribosome to be recruited to the ribosome binding site (RBS) on the mRNA, thereby prevent translation initiation and, in some cases, facilitating the degradation of mRNA by RNase III [START_REF] Chevalier | Staphylococcus Aureus RNAIII Binds to Two Distant Regions of Coa MRNA to Arrest Translation and Promote MRNA Degradation[END_REF][START_REF] Romilly | Loop-Loop Interactions Involved in Antisense Regulation Are Processed by the Endoribonuclease III in Staphylococcus Aureus[END_REF]. In fact, the formation of a duplex between RNAIII and the target mRNAs by loop-loop interactions creates a binding site for RNase III, which will lead to specific cleavage in the regions of the interaction [START_REF] Romilly | Loop-Loop Interactions Involved in Antisense Regulation Are Processed by the Endoribonuclease III in Staphylococcus Aureus[END_REF]). These targets are particularly involved in S. aureus virulence such as the surface protein A (Spa), coagulase (Coa), fibrinogen binding protein (SA1000), homologs of the staphylococcal secretory antigen SsaA (SA2353 and SA2093), immunoglobulin binding protein (Sbi), lipoteichoic acid synthase (LtaS) and cell wall autolysin (LytM) (Amdahl et al. 2017;[START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF][START_REF] Bronesky | [END_REF]Chabelskaya, Gaillot, and Felden 2010a;[START_REF] Geisinger | Inhibition of Rot Translation by RNAIII, a Key Feature of Agr Function[END_REF]Huntzinger et al. 2005;[START_REF] Huntzinger | De plus, ARNIII inhibe l'initiation de la traduction de l'ARNm de rot codant pour le répresseur des toxines[END_REF]). In addition, RNAIII inhibits initiation of translation of rot mRNA, encoding the repressor of toxins Rot [START_REF] Mcnamara | Identification, Cloning, and Initial Characterization of Rot, a Locus Encoding a Regulator of Virulence Factor Expression in Staphylococcus Aureus[END_REF]Oscarsson, Tegmark-Wisell, and Arvidson 2006), which blocks the transcription of many exoproteins and toxins [START_REF] Saïd-Salim | Global Regulation of Staphylococcus Aureus Genes by Rot[END_REF]. Therefore, by inhibiting Rot production, RNAIII indirectly activates transcription of many exotoxins and indirectly inhibits synthesis of protein A at the transcriptional level.

In addition to targets downregulation, RNAIII is also capable of activating expression of few targets: alpha hemolysin (Hla), the extracellular adhesion protein (Map) and the transcriptional regulator MgrA. The interaction of RNAIII with the 5'UTR region of hla and map mRNAs releases the SD sequence thus promoting ribosome recruitment and translation initiation (Delphine [START_REF] Bronesky | Staphylococcus Aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression[END_REF]Y. Liu et al. 2011;[START_REF] Raina | Dual-Function RNAs[END_REF].

Moreover, by inducing Hla production, RNAIII favors cell lysis, which is part of S. aureus infection process [START_REF] Bramley | Roles of Alpha-Toxin and Beta-Toxin in Virulence of Staphylococcus Aureus for the Mouse Mammary Gland[END_REF][START_REF] Dinges | Exotoxins of Staphylococcus Aureus[END_REF][START_REF] Gray | Primary Sequence of the α-Toxin Gene from Staphylococcus Aureus Wood 46[END_REF]. More recently, the 3 'and 5' ends of RNAIII have both been shown to interact with the 5 'UTR of mgrA RNA, thus preventing mRNA from degradation by an unknown ribonuclease (Delphine [START_REF] Bronesky | Staphylococcus Aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression[END_REF][START_REF] Gupta | Erratum: RNAIII of the Staphylococcus Aureus Agr System Activates Global Regulator MgrA by Stabilizing MRNA[END_REF][START_REF] Raina | Dual-Function RNAs[END_REF].

Altogether, due to RNAIII regulatory action on multiple targets directly or indirectly via Rot and MgrA, two other global regulators of gene expression, shows the importance in the pathogenicity of S. aureus. Furthermore, it also shows that RNAIII is implicated in the switch between expression of various surface proteins and synthesis of secreted toxins, which displays the level of complexity in the regulation of virulence genes.

sRNAs implicated in S. aureus Metabolism

Many sRNAs have been demonstrated to be engaged in the metabolism of S. aureus, however, RsaI and RsaE are the most studied so far. First, RsaI (alias RsaOG) [START_REF] Geissmann | The Role of MRNA Structure in Translational Control in Bacteria[END_REF]) is involved in a signaling pathway responding to glucose uptake. Indeed, several direct targets of RsaI were found through MAPS approaches. Some are sRNAs, and most are mRNAs involved in glycolysis and pentose phosphate pathway (negatively regulated) and in fermentation processes (positively regulated) (D. [START_REF] Bronesky | A Dual SRNA in Staphylococcus Aureus Induces a Metabolic Switch Responding to Glucose Consumption[END_REF].

It has been shown that RsaI regulates its mRNA targets by binding to the SD sequence and masking the RBS and consequently inhibits the translation. For instance, RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations.

When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake (glcU_2) and the FN3K enzyme (fn3K) that protects proteins against damage caused by high glucose concentrations. This multifunctional RNA provides a signature for a metabolic switch when glucose is scarce and growth is arrested.

The second sRNA is RsaE, a sRNA conserved in S. aureus and B. subtilis, that controls enzymes involved in amino acid and peptide transport, cofactor synthesis, lipid metabolism, carbohydrate metabolism, the TCA cycle [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Geissmann | The Role of MRNA Structure in Translational Control in Bacteria[END_REF]) and in arginine degradation pathway [START_REF] Rochat | The Conserved Regulatory RNA RsaE Down-Regulates the Arginine Degradation Pathway in Staphylococcus Aureus[END_REF]). Hence, it was suggested that RsaE repressed several enzymes of the central metabolism under non-favorable conditions and it would contribute to readjust the cellular NAD+/NADH balance under stress conditions [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF]Durand et al. 2017). In addition, RsaE is showed to interact with the RBS of opp3A mRNA, encoding an ABC transporter component, to prevent formation of the ribosomal initiation complex and to restrain its translation.

Albeit RsaE functions is suggested to ensure a coordinate downregulation of the central metabolism when carbon sources become insufficient (Delphine Bronesky et al. 2019) 4. sRNAs implicated in Persistence SprF/SprG belong to the last two regulatory RNAs of the Spr "family", here also representing a type I toxin antitoxin system (TAS) discovered in 2014 (Pinel-Marie, Brielle, and Felden 2014). sprG1 encodes two toxic peptides, whose translation is repressed by the antitoxin SprF1. Recently, two novel type I toxin-antitoxin systems (TAS) were found to be located in the core genome and expressed in S. aureus, named sprG2/SprF2 and sprG3/SprF3. Identically to sprG1/ SprF1, sprG2 and sprG3 encode toxins and are neutralized respectively by their antitoxin SprF2 and SprF3. In the absence of antitoxins, toxins are translated and will accumulate in the membrane, causing the death of bacteria (Pinel-Marie, Brielle, and Felden 2014). Moreover, toxins are also secreted into the extracellular environment to act against other bacteria as well as against human erythrocytes. Altogether, it has been suggested that these systems and an involvement of the sprG/SprF TAS in S. aureus adaptation to antibiotic stress or in the escape of the immune system and may have a role in the entry into persistence of bacteria (C. [START_REF] Riffaud | Functionality and Cross-Regulation of the Four SprG/SprF Type I Toxin-Antitoxin Systems in Staphylococcus Aureus[END_REF].

sRNAs implicated in Antibiotic resistance

SprX was initially identified by Bohn and the collaborators in 2010 under the name of RsaOR [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF]. It is a sRNA with a variable copy number depending on the strain since it is found in a single copy in the N315 strain, two copies in the HG001 strain and three copies in the Newman strain. The first data on this sRNAs showed an implication in antibiotic resistance [START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF]). To better understand the mechanism by which SprX modulates antibiotic resistance, a proteomics study was performed to identify the SpoVG protein as a target of SprX. SpoVG is a protein involved in capsule formation and resistance to methicillin and glycopeptides [START_REF] Schulthess | Functional Characterization of the ΣB-Dependent YabJ-SpoVG Operon in Staphylococcus Aureus: Role in Methicillin and Glycopeptide Resistance[END_REF]). Thus, by inhibiting the spoVG translation of the yabJ-spoVG operon via binding at the spoVG translation initiation site, SprX inhibits the expression of SpoVG and thus decreases resistance to glycopeptides [START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF]). In addition, SprX has been demonstrated to be involved in the virulence of S. aureus by regulating toxins and autolysin, which will be described in the next section (Buchad and Nair 2021; Kathirvel, Buchad, and Nair 2016).

sRNAs implicated in Virulence

In S. aureus, many sRNAs have been identified in the last decade and many scientific reports have shown a plethora of sRNAs is implicated in the virulence of the bacteria. Like mentioned before, sRNAs identified in S. aureus are called by different annotations depending on the original discovery. Here, they will be divided into four main groups based on their original discovered annotations: Rsa "family", Spr "family", Teg "family" and other sRNAs.

Rsa sRNAs "family"

The study of RsaA made it possible to identify a known target, namely mgrA mRNA, encoding for a transcriptional factor. RsaA represses the translation of MgrA, involved in biofilm formation and capsule synthesis (Romilly et al. 2014). In addition, RsaA also represses other targets, in particular four mRNAs belonging to the family of enzymes SsaA as well as FlipR (Tomasini et al. 2017a), which interferes in opsonization via complements pathway. Moreover, RsaA has been shown in animal models to suppress staphylococcal virulence. These data show us a rare anti-virulent effect in bacteria and which, as suggested for RsaA (Romilly et al. 2014), could promote commensal behavior of the bacteria in order to avoid the immune system of the host.

Beside the contribution in the metabolism of S. aureus previously mentioned, through masking the RBS at the 3' UTR of the target, RsaI also represses the expression of icaR mRNA, the repressor of the icaADBC operon, which encodes for proteins involved in the synthesis of the main exopolysaccharide constituting biofilms (Delphine Bronesky et al. 2019). Hence, RsaI would contribute to PIA-PNAG synthesis by at least reducing the IcaR repressor protein levels. In addition, the authors also identified other Rsa sRNAs (RsaD, RsaE and RsaG) enriched in complexes purified through their interaction with RsaI (Delphine Bronesky et al. 2019).

RsaE is not only involved in the metabolism described earlier, but also is implicated in the synthesis of PIA in the biofilm through its negative regulation of IcaR, causing the depletion of the icaADBC operon [START_REF] Schoenfelder | 15 PLoS Pathogens The Small Non-Coding RNA Rsae Influences Extracellular Matrix Composition in Staphylococcus Epidermidis Biofilm Communities[END_REF]. Moreover, a homologous of RsaE sRNA in Bacillus subtilis, RoxS, that has shown that RsaE expression is induced by the presence of in response to reactive oxygen species (nitric oxide (NO)) through SrrAB system. Thus, S. aureus RsaE may also intervene in the survival of cells against host immune reactions [START_REF] Durand | SRNA and MRNA Turnover in Gram-Positive Bacteria[END_REF](Durand et al. , 2017)).

Recently, through MAPS approach, sodA mRNA was identified as one of the targets of RsaC. This gene encodes for a superoxide dismutase implicated in oxidative stress response (Lalaouna et al. 2019). Through binding to the RBS of sodA mRNA, RsaC inhibits the translation of sodA mRNA. In addition, RsaC also binds to sarA mRNA in vitro and therefore inhibits its translation by binding at the level of the SD sequence. SarA being a transcriptional repressor of SodM, this would accentuate the effects of RsaC on the activation of the expression of the latter (Lalaouna et al. 2019). All these data show the role of RsaC, interconnecting manganese bioavailability and ROS detoxification and suggesting a role in the resistance to oxidative burst of immune cells via SodM.

Spr sRNAs "family"

SprD is a sRNA encoded in SaPI of S. aureus and is involved in the virulence of the bacteria since it could be demonstrated that the expression of SprD allowed an efficient infection in a mouse model [START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF]. Indeed, unlike the parental strain N315 which caused renal abscesses and death in mice, the deletion mutant resulted in an absence of mortality in mice [START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF].

According to proteomic study by of extracellular proteins between the parental strain and the deletion mutant, the protein Sbi, a protein involved in immune evasion mechanisms, was identified as a target of SprD. Subsequently, SprD inhibits translation of sbi mRNA in vitro by binding to its RBS. However, this inhibition of Sbi alone does not explain the effect of SprD on the virulence of S. aureus. This is why studies were continued and in 2014 showed that Sbi was also repressed by RNAIII [START_REF] Chabelskaya | Dual RNA Regulatory Control of a Staphylococcus Aureus Virulence Factor[END_REF]. Thus, these two regulatory RNAs appear to act synergistically to regulate Sbi expression efficiently and is the first example of its kind (Figure 33). SprC was studied for its targets through a comparative proteomic study [START_REF] Pabic | A Bacterial Regulatory RNA Attenuates Virulence, Spread and Human Host Cell Phagocytosis[END_REF]). Thus, it has been demonstrated that SprC is involved in virulence and bacterial propagation in an animal model of sepsis. In addition, a role in the internalization of bacteria by host immune cells has also been shown. The overexpression of SprC reduces the internalization of S. aureus by THP1 monocytes, as well as by THP1 differentiated into associated with a decrease in bacterial resistance to oxidative stress [START_REF] Pabic | A Bacterial Regulatory RNA Attenuates Virulence, Spread and Human Host Cell Phagocytosis[END_REF].

SprC and also interferes the internalization via epithelial cells by negatively regulates the production of autolysin (atl) by direct inhibition [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][START_REF] Pabic | A Bacterial Regulatory RNA Attenuates Virulence, Spread and Human Host Cell Phagocytosis[END_REF]) (Figure 33).

Although SprX (alias RsaOG) has been demonstrated to be involved in glycopeptides resistance, in 2014, a second study, this time on one copy of the Newman strain (SprX1), identified several other targets of SprX [START_REF] Kathirvel | Enhancement of the Pathogenicity of Staphylococcus Aureus Strain Newman by a Small Noncoding RNA SprX1[END_REF]. The overexpression of SprX1 induced an increase in the expression of several virulence factors such as hemolysin δ (Hld) and clumping factor ClfB, which involve in the adhesion of bacteria and in the formation of biofilms as well as in virulence in mouse models, thus showing the importance of this sRNAs in the pathogenesis of S. aureus. In addition, in 2017, a study using an innovative double plasmid reporter system identified another SprX target, the extracellular complement-binding protein Ecb [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF]. It is a secreted protein allowing the protection of bacteria against the host immune system by binding to CFH. Thus, by inhibiting the translation of ecb by binding to RBS, SprX decreases the expression of Ecb and therefore the protection of bacteria against the immune system [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF]. Another new study of SprX contribution to S. aureus pathogenicity has been released by Buchad and Nair 2021. The authors have demonstrated that, through binding to the 5' region of walR mRNA, SprX enhances the transcription of this mRNA and consequently modulates positively the expression of several autolysins such as AtlA, IsaA and LytM (Buchad and Nair 2021), since WalR is known as a positive regulator of autolysin (Figure 33).

The study of SprA revealed that it is in fact a TAS type I. It is composed of an unstable SprA1AS antitoxin localized on the opposite strand of the toxin PepA1 (encoded by SprA1), which has hemolytic and antibacterial activities [START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF][START_REF] Sayed | A Cis-Antisense RNA Acts in Trans in Staphylococcus Aureus to Control Translation of a Human Cytolytic Peptide[END_REF]. In addition, a second copy of this system has been identified, the SprA2/SprA2AS system, with a similar mechanism of action and predominantly cytolytic activity [START_REF] Germain-Amiot | A Novel Staphylococcus Aureus Cis-Trans Type I Toxin-Antitoxin Module with Dual Effects on Bacteria and Host Cells[END_REF]. Moreover, comparing to PepA1, PepA2 showed a greater toxic effect on human erythrocytes suggesting an effect of this system in the virulence of S. aureus (Germain-Amiot et al. 2019) (Figure 33).

Teg sRNA "family"

Recent transcriptome-sequencing (RNA-Seq) analysis indicated that one of the sRNA, Teg49, resides within the sarA P3 and P1 promoter region in SH100 S. aureus strain [START_REF] Manna | Small RNA Teg49 Is Derived from a SarA Transcript and Regulates Virulence Genes Independent of SarA in Staphylococcus Aureus[END_REF] and is showed to be involved in virulence regulation in sarAdependent or sarA-independent manner. In fact, through RNA-seq analysis, Teg49 seems to affect the expression of a wide range of genes possessing different functions such as metabolism-related transcripts, cell wall-related genes production, cellular processes, and virulence factors. To further validate RNA-seq data, quantitative RT-PCR was performed and showed that Teg49 downregulates two regulatory genes, saeR and lytS among other targets, and upregulates some known virulence factors like lukF and splA [START_REF] Manna | Small RNA Teg49 Is Derived from a SarA Transcript and Regulates Virulence Genes Independent of SarA in Staphylococcus Aureus[END_REF]). In addition, a mouse skin abscess model of infection revealed a modest but significant reduction in the bacterial load in the infected skin tissue, hence demonstrating that Teg49 plays a perceptible role in virulence gene regulation in S. aureus, although the exact mechanism has yet to be elucidated.

Another interesting Teg sRNA recently studied is called Teg41 (alias RsaX05/srn_1080) [START_REF] Beaume | Cartography of Methicillin-Resistant S. Aureus Transcripts: Detection, Orientation and Temporal Expression during Growth Phase and Stress Conditions[END_REF][START_REF] Geissmann | The Role of MRNA Structure in Translational Control in Bacteria[END_REF][START_REF] Subramanian | Genomic Analysis of RNA-Seq and SRNA-Seq Data Identifies Potential Regulatory SRNAs and Their Functional Roles in Staphylococcus Aureus[END_REF]. In 2019, Teg41 was remarked to be located immediately downstream of the αPSM transcript [START_REF] Zapf | The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus Aureus[END_REF], suggesting that Teg41 might affect αPSMs production. Indeed, their study showed that overexpression of Teg41 leads to an increase of αPSM production and consequently, an increase in hemolytic activity of S. aureus. stability, proposing that binding of Teg41 to the αPSM transcript stabilizes the transcript and facilitates the translation of the αPSM peptides. In conclusion, Teg41 activity definitely play a favorable role in the virulence of S. aureus [START_REF] Zapf | The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus Aureus[END_REF]).

sRNA-mediated, positive gene regulation frequently occurs at the level of transcript

Other sRNAs

Other sRNAs have been identified in S. aureus, by different approaches at the end of the 2000s. This was made possible by the democratization of bioinformatic tools and by highthroughput RNA sequencing technologies. For instance, ArtR [START_REF] Xue | ArtR, a Novel SRNA of Staphylococcus Aureus, Regulates α-Toxin Expression by Targeting the 5′ UTR of SarT MRNA[END_REF] upregulating hla expression indirectly through the degradation of sarT; or SbrA-C as σBdependent transcripts with unknown yet functions [START_REF] Nielsen | Searching for Small Σb-Regulated Genes in Staphylococcus Aureus[END_REF]). Another sRNAs is identified during a study aiming to analyze by microarray the modifications of the transcriptome in response to different stresses (hot shock, cold stringent condition, and SOS responses) [START_REF] Morrison | Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus Aureus USA300 Representative[END_REF]), called Ssr42. Ssr42 is expressed during the stationary phase of growth and controls a multitude of virulence factors such as Hla. It is involved in the lysis of host erythrocytes, resistance to lysis by leukocytes and especially in the virulence of staphylococcus in animal models [START_REF] Morrison | Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus Aureus USA300 Representative[END_REF]).

In addition, interestingly, a new sRNAs was found in 2016, PSM-mec [START_REF] Qin | PSM-Mec-A Virulence Determinant That Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci[END_REF] (Figure 34). Among all known PSMs encoded from core genome, PSM-mec, is an exception, whose gene is found in specific sub-types of SCCmec methicillin resistance mobile genetic elements present in methicillin-resistant Staphylococcus aureus. While PSM-mec peptide possesses a pro-inflammatory activity (inducing calcium flux and chemotaxis) and a cytolytic activity on erythrocytes and neutrophils, the psm-mec RNA exerts it gene regulatory activity through its interaction with the agrA transcript resulting in an overall repression of agrA activity [START_REF] Kaito | Mobile Genetic Element SCCmec-Encoded Psm-Mec RNA Suppresses Translation of AgrA and Attenuates MRSA Virulence[END_REF][START_REF] Qin | PSM-Mec-A Virulence Determinant That Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci[END_REF]). However, these effects are highly strain-dependent, which is possibly due to differences in PSM-mec peptide vs. psm-mec RNA-controlled effects. Albeit the psm-mec locus influences cytolytic capacity, methicillin resistance, biofilm formation, cell spreading, and the expression of other virulence factors, such as other PSMs, which results in a significant impact on immune evasion and diseases.

Recently, we have identified within phage Φ12 a gene encoding for a new sRNA that is expressed in proximity of SprX2 sRNA and called it SprY. Its localization in a region of the genome containing many virulence factors suggests that this sRNA could be involved in the virulence of S. aureus. The objective of my thesis is to characterize SprY expression profile, to identify its targets, its mechanisms of action and the physiological roles of SprY in HG003 S. aureus strain.

1. Firstly, we studied SprY expression profile. For this experiment, we verified SprY expression different S. aureus strains such as N315, HG003, USA300 and Newman.

However, for my thesis project, we mostly focused on SprY expression in HG003 wildtype strain (WT) and strains deleted for sprX2 or sprY (HG003 ΔsprX2 or HG003 ΔsprY). In addition, since sRNAs are known to allow bacterial adaptation to environmental changes, we also studied SprY expression in response to different stresses. Madër et al. 2016), is considered bona fide sRNA (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF]) and sRNAs are demonstrated to be implicated in expression regulation of targeted genes, we were looking for potential direct targets of SprY. To obtain this objective, we studied SprY targets using two different approaches: one is in silico predictions via CopraRNA, and the other approach is a high-speed method "MS2", based on the purification of the complexes sRNAs with its target RNA in vivo. Molecular biology methods and structural prediction will be used for the study of the mechanism of action of SprY sRNA.

Secondly, since SprY (alias S629 in

3. Next, we studied the involvement of SprY in the virulence and the hemolytic activity of S. aureus. Thus, SprY function was evaluated in mouse model staphylococcal infections by the method already used to show the importance of SprD in [START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF].

I. Characterization of SprY

A. Localization of sprY gene in HG003 S. aureus strain During studies of sRNA SprX2 and neighboring sequences, we identified by serendipity another sRNA that we called SprY. Located on phage Φ12 between sprX2 gene and the SAOUHSC_01515 gene encoding for putative peptidoglycan hydrolase, spry gene was also identified as a transcript of an sRNA by Madër and his collaborators that they designated S629 [START_REF] Mäder | Staphylococcus Aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions[END_REF]) (Figure 35). Not long after, Bouloc's lab performed a study in which this sRNA was validated as a bona fide sRNA: likely a trans-acting sRNA, not expressed from the opposite strand of a coding gene (W Liu et al. 2018). We determined the transcription start of sprY gene and the size of SprY sRNA of about 125 nucleotides (Le Huyen et al. 2021). We also showed a predicted secondary structure of SprY consisting of three stem loops and the last one being a Rho-independent transcriptional terminal site (Figure 3S in Le Huyen et al. 2021). Furthermore, during our work on sprY gene, we noticed two similar sequences to sprY located in two other phages (Φ11 and Φ13) of S. aureus HG003 strain. We named sprY1 the first sequence, located on phage Φ13 between sprX1 gene and the sak gene (Figure 35), with 55.47% sequence identity to sprY according to sequence alignment analysis (Figure 36A). However, sprY1 expression was not detected in HG003 strain by Northern Blot (Figure 36B). The second sequence with 73.4 % of identity with sprY gene, sprY3, corresponds to srn_9342 in Newman strain [START_REF] Bronsard | SRNA and Cis-Antisense SRNA Identification in Staphylococcus Aureus Highlights an Unusual SRNA Gene Cluster with One Encoding a Secreted Peptide[END_REF]. sprY3/ srn_9342 gene is located on phage Φ11 between SAOUHSC_02018 and SAOUHSC_02019 genes encoding respectively for a hypothetical protein and an autolysin (Figure 35 and Figure 36C). In addition, Northern Blot showed the expression of two transcripts of SprY3 with different sizes depending on the growing phase, which is consistent with the study of Bronsard et al. 2018 (Figure 36D). In general, the three sequences seem to share a highly conserved 3' end that corresponds to a terminal transcription sequence (TTS) (Figure 36) and the rest of the sequences vary greatly from one sequence to another one. My thesis project focused only on characterizing the sprY gene and studying its biological contribution to S. aureus pathogenicity.

B. Analysis of sprY expression

We showed previously that sprY is encoded on phage Φ12 (or Sa2) that is conserved in several S. aureus strains other than NCTC8325 such as Newman [START_REF] Herron-Olson | Molecular Correlates of Host Specialization in Staphylococcus Aureus[END_REF]) and USA300 [START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF]) among others. However, not all Sa2 phages encode the sprY gene like in the case of USA300 strain; according to genomic alignment analysis by Basic Local Alignment Search Tool (BLAST) and to the conservation analysis of sRNAs in the Firmicutes phylum in (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF]). To verify this observation, we performed Northern Blot assay using total RNA extraction of different S. aureus strains (HG003, USA300 and Newman) at exponential phase and stationary phase of bacterial growth. In addition, total RNA extraction of N315 strain was used as control negative since it does not contain bacteriophage Sa2 [START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF]. As expected, we only observed SprY expression in HG003 and Newman, and not in USA300 and N315 strains (Figure 37A). As for the contribution of SprY in staphylococcal infection, we tested of sprY expression in different clinical isolates. Our preliminary data showed the presence of SprY in septic shock and sepsis but none in colonization isolates (Figure 37B), which led us to wonder if in some infectious conditions, these features could confer advantages and might affect bacterial pathogenesis. Next, to analyze the promoter sequence of sprY and to test if sprY and sprX2 are expressed independently, we constructed different vectors including sprX2 gene with different portions of sprY gene (Figure 38A). The S. aureus RN4220 strain that lacks sprY and sprX2 genes was transformed with these vectors and then the expression of sprY and sprX2 was analyzed. RN4220 strain harboring the empty vector pICS3 was used as a negative control. Our Northern Blot results showed similar SprX2 sRNA expression levels from three first plasmids constructions (1-3) (Figure 38B). In addition, sprY expression was observed when the whole sequence of sprY is included in the plasmid designs whether the sprX2 sequence was included or not (Figure 38C) and an approximative estimation of 35 nts upstream of sprY is sufficient to express sprY. Thus, this analysis allowed us to estimate the promoter of the sprY gene and to show that the two sRNAs express independently from plasmids in RN4220 strain. Next, we verified if the endogenous expression of sprY and sprX2 is also independent in HG003 strain, which contains sprX2 and sprY genes. For that, we tested their expressions in HG003 deleted for sprX2 or sprY (HG003 ΔsprX2 or HG003 ΔsprY) as well as in HG003 overexpressing sprX2 or sprY under the control of its own promoter (HG003 pICS3-sprX2 and HG003 pICS3-sprY) (Le Huyen et al. 2021). Our results showed that the absence of one sRNA or the overproduction of one does not affect the expression of the other sRNA (Figure 2C in Le Huyen et al. 2021). Taken together, regardless of the proximity of sprX2 and sprY genes, their expressions are independent of each other.

II. Identification of SprY direct targets by different approaches

A. In silico prediction: SAOUHSC_03046 mRNA In general, most sRNAs with known functions act by base pairing to mRNA targets. To identify SprY potential direct targets, we performed predictions in silico using TargetRNA2 [START_REF] Kery | TargetRNA2: Identifying Targets of Small Regulatory RNAs in Bacteria[END_REF] and CopraRNA [START_REF] Tjaden | TargetRNA: A Tool for Predicting Targets of Small RNA Action in Bacteria[END_REF]Wright et al. 2014). Since SAOUHSC_03046 mRNA came up as the only common result from both predictions analysis (Annex 1 and Annex 2), we decided to study the impact of SprY on its expression. In addition, the analysis with IntaRNA [START_REF] Busch | IntaRNA: Efficient Prediction of Bacterial SRNA Targets Incorporating Target Site Accessibility and Seed Regions[END_REF]Mann, Wright, and Backofen 2017) confirmed a potential base pairing between SAOUHSC_03046 mRNA and SprY involving the 2 nd hairpin of SprY (49 th to 95 th nucleotide) and the 5'UTR and 36 nts of SAOUHSC_03046 mRNA (Figure 39A andB). Then, we verified the interaction between SprY and SAOUHSC_03046 mRNA by EMSA using synthetic RNAs. A complex between SprY and SAOUHSC_03046 mRNA was observed (Figure 39C). The predicted zone was tested furtherly by using SprYmB, an SprY allele bearing point mutations corresponding to the predicted SAOUHSC_03046 binding sequence (Figure 39A). Expectedly, SprYmB lost the ability to bind to the mRNA target, implying that the predicted zone of SprY is required for the interaction (Figure 39C). Altogether, our results confirmed the interaction between SprY and SAOUHSC_03046 mRNA in vitro.

Furthermore, the fact that SprY binds to the 5' UTR of SAOUHSC_03046 mRNA including the ribosome binding site (RBS), suggests the obstruction of its translation initiation. To test this hypothesis, we performed a toeprint assays. First, a ternary initiation complex made of purified 70S ribosomes, initiator tRNAfMet, and SAOUHSC_03046 mRNA was formed. A toeprint was then detected ~15 nts downstream from the AUG initiation codon of the SAOUHSC_03046 mRNA, indicating that the ribosome fixation blocked the elongation of reverse transcription (Figure 39D). SprY significantly reduced the toeprint in a concentration-dependent manner, indicating that SprY inhibits binding of the Ribosome onto the SAOUHSC_03046 mRNA in vitro (Figure 39D).

Next, we wanted to test if SprY affects SAOUHSC_03046 expression at mRNA and/or translational level in vivo since SprY binds to the mRNA and masks the RBS of the mRNA.

We first studied SAOUHSC_03046 mRNA expression level by qPCR, using HG003 strain harboring empty vector pICS3 or overexpressing sprY or sprYmB (pICS3-sprY or pICS3-sprYmB). The expression of sprY and sprYmB in these strains was verified by Northern Blot (Figure 40B). Surprisingly, SprY overexpression did not affect SAOUHSC_03046 mRNA level, whereas the overexpression of SprYmB showed a subtle increase of SAOUHSC_03046 transcript at exponential phase and a significant increase in the target mRNA level at stationary phase (Figure 40A). We then hypothesized that SprY could affect SAOUHSC_03046 at translational level rather than its mRNA quantity. For this, we constructed a SAOUHSC_03046-gfp translational gene fusion under the control of the constitutive PtufA promoter in pCN33 vector resulting in pCN33-PtufA-3046-gfp. HG003 containing empty pICS3 or pICS3-sprY or pICS3-sprYmB were co-transformed with pCN33-PtufA-3046-gfp in HG003 strain. Bacterial growth of all strains used for this experiment was essentially equivalent (Figure 40E). Overproduction of SprY significantly reduced SAOUHSC_03046 translation (Figure 40C andD). Moreover, the overexpression of SprYmB increased the fluorescence intensity in strain containing construction pCN33-PtufA-3046-gfp (Figure 40C andD), which correlated with the increment at mRNA level.

Taken together, our results showed that SprY downregulates the post-transcriptional level of SAOUHSC_03046 but also could affects its mRNA level by an unknow mechanism not through binding to its mRNA but maybe the presence of an intermediate factor.

To further understand the physiological role of this regulation in S. aureus, we noticed that SAOUHCS_03046 encodes for a protein of XRE family proteins (Xenope response element) [START_REF] Ibarra | Global Analysis of Transcriptional Regulators in Staphylococcus Aureus[END_REF]) by genomic comparison. It also showed that SAOUHCS_03046 gene in HG003 strain represents a homologue of 100% identity of and SAOUHSC_1342a mRNA was observed with an increasing concentration of the mRNA (Figure 43C). We also challenged the interaction region by SprYmB (described previously in Figure 39A). As we expected, SprYmB lost its ability to bind SAOUHSC_1342a mRNA, which confirmed the predicted interaction zone of SprY (Figure 43C). Next, we tested the effect of SprY and SprYmB overexpression on SAOUHSC_1342a at post-translational level.

For this, we co-transduced RN4220 S. aureus strain with pCN33-PtufA-1342a-gfp and pICS3 or pICS3-sprY or pICS3-sprYmB. SprY overproduction significantly reduced SAOUHSC_1342a-GFP expression, whereas the overexpression of SprYmB resulted in a similar fluorescence intensity as WT strains harboring empty plasmid (Figure 44A). In addition, bacterial growth of all strains used for this experiment was essentially comparable (Figure 44B). Taken together, we hypothesize that through binding to the 5' end of SAOUHSC_1342a mRNA, SprY might prevent the recruitment of the Ribosome to the RBS on the mRNA, and therefore downregulates the translation of SAOUHSC_1342a.

"A small regulatory RNA alters Staphylococcus aureus virulence by titrating

RNAIII activity" (Published article)

The second identified target for SprY is RNAIII, one of the major regulators of S. aureus virulence. IntaRNA prediction showed a potential base pairing between SprY and the 13 th stem-loop of RNAIII. We then verified this predicted interaction between SprY and RNAIII in vitro by performing EMSA using synthetic RNAs. We also challenged the sequence specificity of the interaction by introducing point compensatory mutations to create SprY and RNAIII alleles (SprYmA and RNAIIImA) unable to bind the wild type sRNAs.

Furthermore, the stem-loop 13 of RNAIII has been demonstrated to control the expression of several virulence factors such as hla, rot, coa, lytM, spa, SA1000, SA2353, SA209, etc. [START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF][START_REF] Chevalier | Staphylococcus Aureus RNAIII Binds to Two Distant Regions of Coa MRNA to Arrest Translation and Promote MRNA Degradation[END_REF][START_REF] Chunhua | The Expression of LytM Is Down-Regulated by RNAIII in Staphylococcus Aureus[END_REF][START_REF] Geisinger | Inhibition of Rot Translation by RNAIII, a Key Feature of Agr Function[END_REF]Huntzinger et al. 2005;[START_REF] Morfeldt | Activation of Alpha-Toxin Translation in Staphylococcus Aureus by the Trans-Encoded Antisense RNA, RNAIII[END_REF]. We suggested that through this interaction with RNAIII, SprY might affect RNAIII regulation of its targets. To verify this hypothesis, we studied the expression of two RNAIII targets: ecb and rot mRNAs. To study the mRNA level of these targets, we performed qPCR using total RNA extraction from HG003 S. aureus strains overexpressing SprY. We also tested ecb and rot translational expression by double plasmid system as described in [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF]. This experiment consists in designing a mRNA-gfp translational gene fusion under the control of a constitutive promoter PtufA in pCN33 vector resulting in pCN33-PtufA-ecb/rot-gfp. We then transduced these plasmids in HG003 harboring pICS3, pICS3-sprY and pICS3-sprYmA which is sprY mutated in the interaction zone with RNAIII. The overexpression of SprY impacts significantly the fluorescence of Ecb-GFP and Rot-GFP, while SprYmA did not affect the fluorescence intensity. Since SprYmA does not bind RNAIII, these results implied that the interaction between SprY and RNAIII is needed for the regulation of ecb and rot by SprY. Taken together, our data disclose an sRNA acting as a sponge for RNAIII and showed that SprY also impacts on the hemolytic activity and involving in the virulence of S. aureus. These results are presented in the article accepted for publication in NAR (Le Huyen et al. 2021).

Plasmids pRMC2

Shuttle vector, Cm r (cat194), with tetracycline inducible promoter (Corrigan and Foster, 2009) pRMC2-ms2 pRMC2 with ms2 tag under control of an tetracycline inducible promoter This study pRMC2-sprY-ms2 pRMC2 with sprY fused to ms2 tag under control of an tetracycline inducible promoter This study pICS3 Shuttle vector, Cm r (cat194), pC194 replicon [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF] pICS3-sprY pICS3 with sprY of HG003 under control of its endogenous promoter This study pICS3-sprYmA pICS3 with sprYmA of HG003 under control of its endogenous promoter This study pICS3-PamiA-rnaIII pICS3 with rnaIII of HG003 under control of a consitutive promoter amiA [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF] pCN33 Low-copy-number shuttle vector, Em r (ermC), pT181 cop-wt repC (Charpentier et al., 2004) pCN33-PtufA-ecbgfp pCN33 with ecb of HG003 fused to gfp under control of PtufA promoter [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF] pCN33-PtufA-rot-gfp pCN33 with rot of HG003 fused to gfp under control of PtufA promoter This study (A) The sRNA sequence is shown with poly-U rich terminator tail (underlined). (B) Predicted secondary structure of SprY mRNA from HG003 strain is based on Mfold [START_REF] Zuker | Mfold Web Server for Nucleic Acid Folding and Hybridization Prediction[END_REF][START_REF] Rouillard | OligoArray 2.0: Design of Oligonucleotide Probes for DNA Microarrays Using a Thermodynamic Approach[END_REF] and LocaRNA [START_REF] Raden | Freiburg RNA Tools: A Central Online Resource for RNA-Focused Research and Teaching[END_REF][START_REF] Periasamy | Phenol-Soluble Modulins in Staphylococci[END_REF]Willl, S. et al., 2007).

The potential SprY 3'-end corresponds to the 3 rd hairpin (H3) and represents a stem-loop of the Rho-independent terminator family. (C) Interaction between SprYmA and RNAIIImA (compensatory mutation) was predicted by IntaRNA software [START_REF] Busch | IntaRNA: Efficient Prediction of Bacterial SRNA Targets Incorporating Target Site Accessibility and Seed Regions[END_REF]. et al. 1994), lytM [START_REF] Ramadurai | Characterization of a Chromosomally Encoded Glycylglycine Endopeptidase of Staphylococcus Aureus[END_REF], spa (Huntzinger et al. 2005), SA1000, SA2093 and SA2353 [START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF] and mgrA mRNA [START_REF] Gupta | Erratum: RNAIII of the Staphylococcus Aureus Agr System Activates Global Regulator MgrA by Stabilizing MRNA[END_REF]. The synthesis of Rot is regulated at the translational level by RNAIII [START_REF] Geisinger | Inhibition of Rot Translation by RNAIII, a Key Feature of Agr Function[END_REF]; this study). The AUG codons are underlined and the interaction zone between RNAIII and SprY is colored in red.

III. Regulation network between SprY and SprX2

A. Comparison of sprY and sprX2 expressions As we mentioned before, spry gene is located in immediate proximity to sprx2 gene in phage Φ12 of HG003 strain (Figure 35). In addition, SprX, was also defined as a bona fide sRNA (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF]) and studied for its contribution to virulence and antibiotics resistance of bacteria [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Bronwlee | The Small RNA SprX Regulates the Autolysin Regulator WalR in Staphylococcus Aureus[END_REF][START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF][START_REF] Kathirvel | Enhancement of the Pathogenicity of Staphylococcus Aureus Strain Newman by a Small Noncoding RNA SprX1[END_REF]. This observation insisted us to take an interest in expression of these two genes. We demonstrated that sprX2 and sprY are expressed independently regardless of their proximity in a cluster of sRNAs (Figure 2C Next, we wonder if sprX2 and sprY are also express differently in various stress conditions.

For this study, HG001 strain was cultivated until the exponential growth phase (3h) and then it was subjected for 30 min and 2h30 to the following stresses: a poor environment (NZM), thermal shocks (42°C and 15°C), ionic stress (0.5 mM dipyridyl, which corresponds to iron depletion), 10 mM H2O2, 1M NaCl, pH 5.5 (addition of 5N HCl), anaerobic culture and serum. The expression level of both sRNAs was detected by Northern Blot and was quantified by ImageTools 7.0, compared to SprY level in LB at 37°C (Ctrl). Our data showed a clear different expression profile of SprX2 and SprY in temperature stresses. Indeed, at 42°C, SprX2 decreased by 0.6-fold after 30 min and by 0.8-fold after 2h30, while SprY levels increased 2.1-fold after 30 min and 1.3-fold after 2h30 of heat stress (Figure 45).

Moreover, at 15°C, while SprX2 expression level did not showed much fluctuation after exposing to heat stress, SprY expression decreased after 2h30 of stress exposure (Figure 45). Altogether, these results showed that the expression levels of SprX and SprY2 do not undergo the same variation according to stress.

B. In silico prediction of interaction between SprY and SprX2

The opposite expression profile of sprX2 and sprY in certain conditions and close localization leads to the hypothesis that SprX2 and SprY might interact with each other.

We therefore verified potential interaction between these two sRNAs in silico by IntaRNA [START_REF] Busch | IntaRNA: Efficient Prediction of Bacterial SRNA Targets Incorporating Target Site Accessibility and Seed Regions[END_REF]Mann, Wright, and Backofen 2017). The analysis indicates potential base-pairing between SprX2 and SprY involving the 5' of SprX2

(from the 14 th to the 30 th nts) and the 2 nd hairpin of SprY (from the 34 th to the 50 th nts) (Figure 46A). However, we did not detect complex formation between SprX2 and SprY by EMSA (Figure 46B).

C. Regulation mechanisms of SprY and SprX2

Several targets of SprX has been identified and validated in the last decade [START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF][START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF][START_REF] Kathirvel | Enhancement of the Pathogenicity of Staphylococcus Aureus Strain Newman by a Small Noncoding RNA SprX1[END_REF]. Since sprY is located in immediate proximity of sprX2 gene and is showed to possess an interesting profile of expression comparing to sprX2, we wonder if SprY sRNA would affect the expression of these targets of SprX2.

Impact of SprY and SprX2 on spoVG expression

In [START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF], the authors have demonstrated that SprX sRNA interacts with the RBS of yabJ-spoVG mRNA and inhibits the translation of the second downstream gene, spoVG. First, to test the potential interaction of these two sRNAs with spoVG mRNA, we carried out EMSA using synthetic RNAs. Complex formation between SprX2 sRNA and spoVG mRNA was observed while no complex formation between the mRNA with SprY was detected (Figure 47A). Next, we tested the impact of SprX2 and SprY on the translational level of spoVG by Western blot assay. Our results showed that the overexpression of SprX2 reduced the expression of SpoVG, which is correlated with the results in [START_REF] Eyraud | A Small RNA Controls a Protein Regulator Involved in Antibiotic Resistance in Staphylococcus Aureus[END_REF]. On the contrary, overexpression of SprY increased the protein expression (Figure 47B) regardless of the absence of interaction between SprY and spoVG mRNA, which suggests that another intermediate factor may be involved.

Impact of SprY and SprX2 on ecb expression

According to [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF], SprX2 has been demonstrated to downregulate ecb at translational level. We showed that SprY affects ecb expression by impacting RNAIII function (Le Huyen et al. 2021). Here, we tested the effect of both SprY and SprX2 on ecb expression in the same conditions and the same genetic background. HG003 harboring pICS3 or pICS3-sprY were then co-transformed with pCN33-PtufA-ecb-gfp [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF]. A co-transduction of pCN33-PtufA-ecb-gfp with pICS3-PamiA-rnaIII was used as positive control of the regulation system. Bacterial growth of all strains used for this experiment was essentially equivalent (Figure 48B). Overproduction of SprX2 and RNAIII reduce the fluorescence of strain containing pCN33-PtufA-ecb-gfp, which represent a decrease of ecb translational expression levels (Figure 48A) as described in [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF][START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF]. On the contrary, the overexpression of SprY leads to a significant increase of the fluorescence intensity of Ecb-GFP, corresponding to the result in Le Huyen et al. 2021.

SprX2 and SprY affect hemolytic activity of HG003 strain

Furthermore, sprX2, one of the two copies of sprX in HG003 (alias rsaOR) is a homologue of sprX in S. aureus N315 strain [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF] and it has been showed that SprX affect hemolysis of S. aureus Newman strain [START_REF] Kathirvel | Enhancement of the Pathogenicity of Staphylococcus Aureus Strain Newman by a Small Noncoding RNA SprX1[END_REF]. Hence, we tested the effect of SprX2 and SprY on the hemolytic activity of the bacteria in human blood. We used HG003, HG003 ΔsprX2, HG003 ΔsprY strains and HG003 WT containing either pICS3 or pICS3-sprX2 or pICS3-sprY. Although the absence of sprX2 did not affect hemolysis compared to WT, sprY deletion led to an increase of hemolysis in human blood (Figure 49). Moreover, overexpression of sprX2 (HG003 pICS3-sprX2) led to a considerable increase the hemolytic activity of S. aureus, while overproduction of SprY reduced significantly the hemolytic activity (Figure 38 and Figure 6 in Le Huyen et al. 2021).

Taken together, our data showed that SprY and SprX2 possess opposite effect on common targets such as ecb and spoVG and affect differently the hemolysis induced by S. aureus.

These results suggest that the two sRNAs might share a possible link in the regulation of S. aureus virulence and potential in antibiotic resistance. aureus strain where it resides as a prophage next to Φ11 and Φ13 [START_REF] Ye | Sequence Analysis and Comparison of Int and Xis Genes from Staphylococcal Bacteriophages L54a and Φ11[END_REF]. Furthermore, Φ12 (or ΦSa2), is conserved in several other S. aureus strains than NCTC8325 [START_REF] Iandolo | Comparative Analysis of the Genomes of the Temperate Bacteriophages Φ11, Φ12 and Φ13 of Staphylococcus Aureus 8325[END_REF][START_REF] Pantůček | Identification of Bacteriophage Types and Their Carriage in Staphylococcus Aureus[END_REF][START_REF] Sass | Lytic Activity of Recombinant Bacteriophage Φ11 and Φ12 Endolysins on Whole Cells and Biofilms of Staphylococcus Aureus[END_REF], such as MW2 [START_REF] Baba | Genome and Virulence Determinants of High Virulence Community-Acquired MRSA[END_REF], MSSA476, MRSA252 [START_REF] Holden | Complete Genomes of Two Clinical Staphylococcus Aureus Strains: Evidence for the Evolution of Virulence and Drug Resistance[END_REF]), USA300 [START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF], Newman and RF122 strain [START_REF] Herron-Olson | Molecular Correlates of Host Specialization in Staphylococcus Aureus[END_REF]). We also remark that the lukS-PV and lukF-PV genes encoding a virulence factor, PVL, were found in N315 and USA300 ΦSa2 phage instead of sprY gene (Figure 50). Furthermore, bacteriophages have been demonstrated to encode different virulence factors and regulators such as genes coding for exfoliative toxin A (eta) [START_REF] Yamaguchi | Complete Nucleotide Sequence of a Staphylococcus Aureus Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C[END_REF], the cell-wall anchored protein SasX [START_REF] Li | MRSA Epidemic Linked to a Quickly Spreading Colonization and Virulence Determinant[END_REF], and the immune evasion cluster (IEC) composed of enterotoxin S (sea), staphylokinase (sak), the chemotaxis inhibitory protein (chp), and the staphylococcal complement inhibitor (scn) [START_REF] Van Wamel | The Innate Immune Modulators Staphylococcal Complement Inhibitor and Chemotaxis Inhibitory Protein of Staphylococcus Aureus Are Located on β-Hemolysin-Converting Bacteriophages[END_REF]

II. Verification of SAOUHSC_03046 expression regulated by SprY

The identification of all the potential targets of regulatory RNAs is crucial to unravel their functional roles. One of many ways to look for SprY potentially direct targets is through in silico predictions. By comparing results from predictions by TargetRNA2 [START_REF] Kery | TargetRNA2: Identifying Targets of Small Regulatory RNAs in Bacteria[END_REF] and CopraRNA [START_REF] Tjaden | TargetRNA: A Tool for Predicting Targets of Small RNA Action in Bacteria[END_REF]Wright et al. 2014) and biases the data. Future studies evaluating the expression of SAOUHSC_03046 in RN4220, which is cured of phages Φ11, Φ12 and Φ13 [START_REF] Kreiswirth | The Toxic Shoch Syndrome Exotoxin Structural Gene Is Not Detectably Transmitted by a Prophage[END_REF], might help us determine the effect of SprYmB independently from endogenous SprY.

To better understand the role of this regulation, we studied the nature of SAOUHCS_03046 gene which encodes for a helix-turn-helix domain-containing protein belonging to XRE family protein (Xenope response element) [START_REF] Ibarra | Global Analysis of Transcriptional Regulators in Staphylococcus Aureus[END_REF]. With a similarity in sequence with a known XRE protein (XdrA) in USA300 strain [START_REF] Mccallum | Transcriptional Profiling of XdrA, a New Regulator of Spa Transcription in Staphylococcus Aureus[END_REF], we suggested that SAOUHSC_03046 protein might also affect the expression of the same target of XdrA, which is spa gene. Our preliminary results showed that the absence of SAOUHSC_03046 provoked an increase of spa mRNA level compared to HG003 WT. Although more experiments are needed to reach statistical significance, our results suggest SAOUHSC_03046 would downregulate the expression of spa mRNA.

Interestingly, SprY overexpression showed an increase in the levels of spa transcript regardless of the expression of SAOUHSC_03046, suggesting that the effect of SprY on spa is not only mediated by SAOUHSC_03046. Indeed, spa expression is regulated by several factors such as agr/RNAIII system and Rot [START_REF] Gao | Regulatory Elements of the Staphylococcus Aureus Protein A (Spa) Promoter[END_REF]Huntzinger et al. 2005;[START_REF] Saïd-Salim | Global Regulation of Staphylococcus Aureus Genes by Rot[END_REF]; both of their expressions were affected by SprY (Le Huyen et al. 2021). The fact that SAOUHSC_03046 might also modulate spa gene expression will present a new layer of regulation network of S. aureus virulence.

III. Identification of SprY direct targets by MAPS technique

MS2 affinity purification coupled with RNA sequencing (MAPS), developed by Lalaouna and Massé, is a technique extensible used in the last couple of years. Initially used to identified identify proteins partners for RNAs in several Gram-negative bacteria [START_REF] Corcoran | Use of Aptamer Tagging to Identify in Vivo Protein Binding Partners of Small Regulatory RNAs[END_REF][START_REF] Said | In Vivo Expression and Purification of Aptamer-Tagged Small RNA Regulators[END_REF], MAPS was adapted to study RNA-RNA interaction [START_REF] Carrier | A Game of Tag: MAPS Catches up on RNA Interactomes[END_REF][START_REF] Lalaouna | Identification of Unknown RNA Partners Using MAPS[END_REF][START_REF] Silva | SraL SRNA Interaction Regulates the Terminator by Preventing Premature Transcription Termination of Rho MRNA[END_REF]). Recent modifications have been made to adapt this technic in Gram-positive like S. aureus (Lalaouna et al. 2019;[START_REF] Tomasini | The RNA Targetome of Staphylococcus Aureus Non-Coding RNA RsaA: Impact on Cell Surface Properties and Defense Mechanisms[END_REF], which unlocks more possibilities for identification of new targets for several sRNAs of S. aureus. In parallel, in our lab, we also performed MAPS to study RNAs targets for several staphylococcal sRNAs using inducible plasmid pRMC2 instead of pCN51 plasmid in [START_REF] Tomasini | The RNA Targetome of Staphylococcus Aureus Non-Coding RNA RsaA: Impact on Cell Surface Properties and Defense Mechanisms[END_REF] although the system remains the same (Ivain Lorraine thesis).

Here, in this work, we used MAPS approach to identify direct targets of SprY in normal growth condition in S. aureus. The analysis comparing RNAseq data from eluates of the ms2-sprY fusion and the control ms2 alone, we identified three potential interesting targets for SprY: rpmG1, rnaIII and SAOUHSC_1342a mRNAs (Le Huyen et al. 2021).

However, regardless of in silico predictions, SAOUHSC_03046 was not found through MAPS analysis. SAOUHSC_03046 expression profile has been studied in Madër et al. 2016 and its amount has been showed to be fairly low in most of culture conditions beside in CDM and RPMI media at exponential phase. The fact that our MAPS assay was done in LB could explain the absence of SAOUHSC_03046 in our RNA-seq analysis.

After testing the potential interaction in silico by IntaRNA and in vitro by EMSA, SprY was only predicted to bind to RNAIII (Le Huyen et al. 2021) and SAOUHSC_1342a mRNA.

rpmG1 mRNA enrichment by MAPS without direct interaction with SprY suggest that in vivo the formation of the complex may involve other factors. In this work we were looking for direct targets that form sRNA-sRNA or sRNA-mRNA complexes, for that reason rpmG1

was removed from the study.

RNAIII is an sRNA, thus our MAPS study revealed an sRNA-sRNA interaction in vivo. We then demonstrated that the binding of SprY on RNAIII prevents the latter to regulate the expression of his targets. Lalaouna and his collaborators studies using MAPS also helped them to identify RNAs acting as a sponge for sRNAs in Gram-negative bacteria. Their results showed that the 3' external transcribed spacers sequences of polycistronic transcripts (3' ETS), previously considered for a long time as "junk RNA", binds to RyhB and RybB sRNAs to reduce sRNAs excess and to prevent them from regulating their mRNAs targets [START_REF] Lalaouna | Identification of SRNA Interacting with a Transcript of Interest Using MS2-Affinity Purification Coupled with RNA Sequencing (MAPS) Technology[END_REF][START_REF] Ziebuhr | The End Is Not the End: Remnants of Trna Precursors Live on to Sponge up Small Regulatory RNAs[END_REF].

IV. SprY regulates SAOUHSC_1342a expression

SAOUHSC_1342a encodes for mechanosensitive channel of large conductance (MscL), which has been mostly studied in E. coli [START_REF] Bootha | The MscS and MscL Families of Mechanosensitive Channels Act as Microbial Emergency Release Valves[END_REF][START_REF] Ou | One Face of a Transmembrane Helix Is Crucial in Mechanosensitive Channel Gating[END_REF][START_REF] Wray | An Agonist of the MscL Channel Affects Multiple Bacterial Species and Increases Membrane Permeability and Potency of Common Antibiotics[END_REF][START_REF] Wray | Novel MscL Agonists That Allow Multiple Antibiotics Cytoplasmic Access Activate the Channel through a Common Binding Site[END_REF] and more recently in S. aureus [START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF]. Mechanosensitive channels (MS channels) function as an emergency release valves in response to membrane tension upon low osmotic stress. For instance, when the osmotic environment decreases, water flows in and threats bacterial cell integrity, MS channels will release cytoplasmic pressure by letting rapid efflux of cytosolic molecules from the cell [START_REF] Bootha | The MscS and MscL Families of Mechanosensitive Channels Act as Microbial Emergency Release Valves[END_REF]Haswell, Phillips, and Rees 2011). In bacteria, there are two families of MS channels: MS of large conductance (MscL) and MS of small conductance (MscS). Contrary to MscS, MscL are highly conserved between species, including pathogens. MscLs have been demonstrated to protect bacterium against osmotic forces and to also participate in the transport and uptake of peptides and antibiotics in E. coli [START_REF] Maurer | A High-Throughput Screen for MscL Channel Activity and Mutational Phenotyping[END_REF][START_REF] Ou | One Face of a Transmembrane Helix Is Crucial in Mechanosensitive Channel Gating[END_REF][START_REF] Wray | Novel MscL Agonists That Allow Multiple Antibiotics Cytoplasmic Access Activate the Channel through a Common Binding Site[END_REF]. Only recent studies has shown another role of MscL upon adhesion force in S. aureus [START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF]. MscL in S. aureus were shown to not only be opened by fluctuations of membrane tension due to osmotic force, but also by the adhesion force to surfaces. When the bacteria presents strong adhesion forces through adhering to substratum surface, it provokes deformation of staphylococcal cell wall and leads to opening of MscL [START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF]). However, the expression regulation of these MscL remains unclear. Here, we demonstrated that SprY potentially downregulates SAOUHSC_1342a translation, which could reveal partially the expression regulation of MscLs in S. aureus. The impact of SprY on SAOUHSC_1342a at translational level could be explained by the fact that through binding to the 5' end of SAOUHSC_1342a mRNA, SprY masks the RBS and prevents the Ribosome recruitment onto the mRNA. As for perspectives, we intend to test the effect of SprY on some phenotypes related to mechanosensitive channel gating in S. aureus by measuring the ability of uptaking fluorescent calcein or the antibiotic (dihydrostreptomycin) described in [START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF]. For example, the authors have demonstrated that at a high adhesion force, RN4220 wild-type strain showed more fluorescence intensity that in RN4220 deleted for mscL (RN4220 ΔmscL), which is correlated to the uptake of the fluorescent calcein in the bacteria through these mechanosensitive channels; or RN4220

WT allows more import of the antibiotic resulting in more dead cells than in RN4220

ΔmscL [START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF].

V. SprY acts as a sponge of RNAIII and alters its activity in the regulation of S.

aureus virulence

Here, we have demonstrated that the 5' end of SprY binds directly the 13 th stem-loop of RNAIII. Our study showed that SprY binding alters RNAIII function without affecting RNAIII stability and that SprY prevents the complex formation between RNAIII and its targeted mRNAs. By antisense base-paring, RNAIII controls the translation or/and the stability of various mRNA targets. The 3' region of RNAIII is responsible for repression of multiple mRNA targets [START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF]Huntzinger et al. 2005;[START_REF] Lebeau | Coagulase Expression in Staphylococcus Aureus Is Positively and Negatively Modulated by an Agr-Dependent Mechanism[END_REF][START_REF] Ramadurai | Characterization of a Chromosomally Encoded Glycylglycine Endopeptidase of Staphylococcus Aureus[END_REF], and the sequence corresponding to the interaction region with SprY overlaps with sites for binding several mRNA targets, such as rot, spa, coa, lytM, ecb, SA2093 and SA2353 (reviewed in (Delphine Bronesky et al. 2016)). We showed that SprY overproduction enhances rot and ecb mRNAs translation only when the interaction between SprY and RNAIII takes place (Le Huyen et al. 2021). Taken together, we present here case of a sRNA that acts as a sponge to fine-tune the sophisticated RNAIII regulation network (Le Huyen et al. 2021).

Recently, reports have described novel RNA functions modulating the action of sRNAs; some of them act as RNA decoys [START_REF] Figueroa-Bossi | Erratum: Caught at Its Own Game: Regulatory Small RNA Inactivated by an Inducible Transcript Mimicking Its Target (Genes and Development (2009) 23 (2004-2015[END_REF][START_REF] Overgaard | Switching off Small RNA Regulation with Trap-MRNA[END_REF] or as RNA sponges (reviewed in [START_REF] Figueroa-Bossi | Sponges and Predators in the Small RNA World[END_REF][START_REF] Miyakoshi | Cross Talk between ABC Transporter m RNA s via a Target m RNA -derived Sponge of the G Cv B Small RNA[END_REF]. They are identified to impact indirectly the expression of several genes without subtilis, P. aeruginosa (reviewed in [START_REF] Denham | The Sponge RNAs of Bacteria -How to Find Them and Their Role in Regulating the Post-Transcriptional Network[END_REF]). Since the degradation is not a necessitate aftermath of duplex formation, a sponge RNA can act by titrating a regulatory sRNA and by competing with their true targets like tRNA precursor [START_REF] Lalaouna | Identification of SRNA Interacting with a Transcript of Interest Using MS2-Affinity Purification Coupled with RNA Sequencing (MAPS) Technology[END_REF].

Unlike chb-ChiX regulation, the interaction between 3'ETS tRNA spacer sequences with RybB or RyhB sRNAs does not promote the degradation of the sRNA but prevent the sRNA from regulating its targets. Until now, in S. aureus, only one case of RNA has been suggested to be RNA sponge is RsaI (or RsaOG): the interaction between RsaI and RsaG was hinted to block RsaG from regulating its own targets (D. [START_REF] Bronesky | A Dual SRNA in Staphylococcus Aureus Induces a Metabolic Switch Responding to Glucose Consumption[END_REF].

However, to our knowledges, the findings presented here are the to identify a sRNA with a sponge activity for RNAIII in S. aureus. This observation suggests that SprY could reduce the toxicity of S. aureus by preventing the function of RNAIII and therefore, could switch the bacteria to a phenotype less virulent.

VI. How sRNAs gene cluster contribute to S. aureus during bacterial growth and infection?

During my thesis project, analysis of SprY and SprX2 expressions have shown some interesting remarks on these two sRNAs. Despite the immediate proximity of sprY and sprX2 genes in HG003 strain, the absence or overproduction of one sRNA does not affect the expression of the other. Moreover, we showed that they possess an opposite expression profiles: SprY accumulates overtime while it is the contrary for SprX2. Not only that they express differently during bacterial growth, but they also respond oppositely under temperature stress. SprX2 was shown to decrease under 42°C and to express more at 15°C while SprY decreases at 15°C and expresses more at 42°C. Maybe the stress affect the transcription and/or the degradation of sRNA (F. [START_REF] Repoila | Signal Transduction Cascade for Regulation of RpoS: Temperature Regulation of DsrA[END_REF] and it would be interesting to test the stability of SprY and SprX2 in different stresses to verify this hypothesis. Furthermore, SprY affect the expression of spoVG in the opposite way than SprX2. While SprX2 reduces the translation of spoVG by direct binding to the RBS of the mRNA, SprY provokes an increase of SpoVG amount in an unknown mechanism that does not implicate direct interaction between SprY and spoVG mRNA. By the same token, SprY has an opposite effect than SprX onto the expression of ecb [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF]). SprX2

regulates ecb expression by direct binding [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF] whereas SprY regulates ecb through affecting RNAIII function (Le Huyen et al. 2021). Thus, two sRNAs located in the same cluster and regulate oppositely a common target through different mechanisms.

Moreover, both sRNAs SprX2 and SprY also contribute to the virulence of S. aureus but in opposite way: SprX2 favorizes the hemolytic activity of S. aureus while SprY restrains it.

In general, organization of genes in clusters is trait for genes expressing macromolecules with similar functions [START_REF] Vanderpool | Dual-Function RNA Regulators in Bacteria[END_REF]. In addition to protein gene clusters [START_REF] Jongerius | Staphylococcal Complement Evasion by Various Convertase-Blocking Molecules[END_REF][START_REF] Mccarthy | Staphylococcus Aureus Innate Immune Evasion Is Lineage-Specific: A Bioinfomatics Study[END_REF], sRNAs have been demonstrated to be expressed from clusters such like the case of S. aureus in [START_REF] Bronsard | SRNA and Cis-Antisense SRNA Identification in Staphylococcus Aureus Highlights an Unusual SRNA Gene Cluster with One Encoding a Secreted Peptide[END_REF]). Although several sRNAs cluster genes have been discovered, their

functions are yet to be elucidated [START_REF] Felden | When Eukaryotes and Prokaryotes Look Alike: The Case of Regulatory RNAs[END_REF], whether they possess shared biological functions or different roles in the bacterial survival or the virulence.

Interestingly, sprY and sprX2, located in a direct adjacency in a cluster and possess opposite expression profiles during growth. We wonder if this reversed profile could

S. aureus RN4220 electro-competent cells preparation and transformation

A pre-culture of S. aureus RN4220 strain was inoculated in BHI at 37°C with agitation at 160 rpm. The next day, the bacterial culture was diluted to twentieth in 10 mL TSB (or BHI) and cultivated for 5 hours in the same condition. Then, the culture was diluted again in 100 mL TSB (or BHI) to OD600 final of 0.5 and incubated for another 30 minutes before being placed on ice for 10 minutes. The bacteria are then centrifuged at 3500 rpm, 4°C

for 10 minutes. Then, the pellets were washed twice with sterile water (50mL then 5mL) and then once with 2mL of iced 0.5M sucrose. After centrifugation, the washed pellets were resuspended in 250 μL of 0.5M sucrose + 10% glycerol, were aliquoted and stocked in -80°C.

For RN4220 transformation, 50 μL of competent bacteria were incubated with 100ng of plasmids for 5 minutes at room temperature before electroporation at 2500V. 4 μL of DMSO was added into the mix for cryopreservation. The bacteria were then incubated for at least 1 hour in BHI medium at 37°C, and then spread on a BHI dish supplemented with the appropriate antibiotic.

Phage preparation and transduction

To prepare phage containing the plasmid of interest, a pre-culture of RN4420 transformed with the plasmid was cultivated overnight in TSB/ BHI with appropriated antibiotic at 37°C with agitation. The next day, 200 μL of the pre-culture was diluted in 500 μL of TSB/ BHI with CaCl2 10mM and 100 μL of that dilution was mixed with 100 μL of phage 80α and incubated for 5 hours at 28°C. Phages containing the plasmid of interest were then collected through filter of 0.45 μm. In parallel, 100μL of the dilution without phage was also incubated at 28°C as a negative control. We used RN4220 strain to prepare phage-containing vectors expressing sRNA or target-gfp fusion.

As for phage transduction, we used S. aureus HG003 strains to transduce sRNA expressing vector and/ or to co-transform with the target-gfp fusion. For this, 100 μL of HG003 preculture with 10mM CaCl2 was incubated with 20 μL of phage 80α containing plasmid of interest for 30 minutes at 37°C, before adding 900 μL of BHI + 10mM Sodium Citrate and incubating for at least 1 hour at 37°C with agitation. The culture was then spreading on a BHI box supplemented with the appropriate antibiotic.

B. Stress conditions

The stress conditions studied and samples are from Alex Eyraud's thesis in 2014. 10 mL of the pre-cultures of strain HG001 (WT) the day before are diluted to hundredth in 600 mL of LB medium in order to obtain an OD of 0.1 then incubated at 160 rpm at 37 ° C for 3 hours until the exponential phase (OD600 = 2). Then, 50 mL of culture are distributed in Erlens to undergo the following stresses: a poor medium (NZM), thermal shocks (42°C and 15°C), ionic stress (0.5 mM dipyridyl), which corresponds to iron depletion), without agitation and anaerobic. The 50 mL of control culture and stress temperatures are directly placed at 37°C, 15°C and 42°C respectively. For ionic stress and in poor medium, the 50 mL of culture are first centrifuged for 10 minutes at 4000 rpm and at room temperature, washed with water and then respectively taken up in 50 mL of NZM medium and LB medium supplemented with dipyridyl (0.5 mM). After 30 minutes, 2h30 and 4h, the total RNAs are extracted as described in [START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF].

C. Plasmids constructions

Table 5 lists all the primers used. To construct the sRNA-expressing vectors, we used pICS3 (which is pRMC2 without the anhydrotetracycline inducible promoter) (described in [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF].

As for double plasmids system, we used pCN33 to express SprY target fused with reporter gen (gfp) under a constitutive promoter PtufA (pCN33-PtufA-3046-gfp and pCN33-PtufA-1342a-gfp). HG003 strains carrying each of the target-gfp fusions and the sRNA plasmids were grown on BHI agar plates supplemented with 10 µg/mL chloramphenicol and erythromycin. The fluorescence measurements of the co-transduced HG003 strains was performed as previously described [START_REF] Ivain | An in Vivo Reporter Assay for SRNA-Directed Gene Control in Gram-Positive Bacteria: Identifying a Novel SRNA Target in Staphylococcus Aureus[END_REF].

II. Methods

A. Proteins extractions, Western blots and Mass Spectrometry S. aureus strains were grown until exponential phase (2 hours) or stationary phase (6 hours) in BHI at 37°C, with agitation at 160 rpm, and the cells were then pelleted for 10 min at 4°C (8000 g). The total proteins and extracellular proteins extractions were prepared according to [START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF] (Jackson). Western blots were revealed using the Amersham ECL Plus detection Kit. Signals were visualized using LAS 4000 (GE Healthcare).

B. Toeprint assay

All RNAs were transcribed from PCR-generated DNA using MEGAscript T7 kit (Ambion).

The template for transcription was amplified using HG003 genomic DNA and forward primers containing T7 promoter sequences (Table 5). RNAs were labelled at 5′-end using [γ-32P] ATP (Amersham Biosciences) and T4 polynucleotide kinase (Invitrogen). Labelled and unlabeled RNAs were purified on a 5% acrylamide urea gel, eluted in Elution buffer (20mM Tris-HCl pH 7.5, 250mM NaCl, 1mM EDTA, 1% SDS) at 37°C, eluted, ethanol precipitated, quantified by Qubit (Thermo Fisher Scientific) and stored at -80°C.

For Toeprint assay, 5 pmoles of RNA target with 10 µL of DNA probes labelled were denaturized in 50 mM Tris HCl pH 7.5, 60 M NH4Cl, 1 mM DTT for 2 min at 80°C, followed by refolding as described previously. The ribosomes were reactivated for 15 min at 37° C, followed by a dilution of 1/100 in 20mM Tris HCl pH 7.5, 20 mM MgCl2, 60 mM NH4Cl, 1 mM DTT and incubated for 15 min at 37°C. Various concentrations (0, 0.5, 1, or 2 pmoles) of purified 70S ribosomes were added to each sample and incubated for 5 minutes at 37°C. The mixture was supplemented by 10mM MgCl2 (final concentration) and 10 pmoles of uncharged tRNAfMet, followed by another incubation for 15 min at 37°C. The cDNA was synthesized with 4U of AMV RT (NEB) for 15 minutes at 37°C. Reactions were stopped by the addition of 15 µL of loading buffer II (Ambion) and heated for 10 min at 65°C. The cDNAs were loaded and separated onto 8% polyacrylamide/8M urea gels.

Sequencing ladders were generated with the same 5'-ended primer.

C. Statistical analysis

For the statistical analysis of the data, a check of the normality of the data as well as the equality of variances was carried out. Then, according to the results of these two tests, (Lindsay et Holden 2006). Le pangénome de S. aureus regroupe environ 7 500 gènes dont environ 1 500 appartiennent au core génome [START_REF] Tettelin | Comparative Genomics: The Bacterial Pan-Genome[END_REF]).

-Le core génome représente environ 75 % du chromosome et est très conservateur dans toutes les souches séquencées. Ainsi, celui-ci rassemble tous les gènes conservés au sein d'une espèce codant pour les fonctions de base de la croissance des bactéries telles que le métabolisme des bactéries, la synthèse des protéines et la réplication des acides nucléiques [START_REF] Bossi | Competing Endogenous RNAs: A Target-Centric View of Small RNA Regulation in Bacteria[END_REF][START_REF] Lindsay | Staphylococcus Aureus: Superbug, Super Genome?[END_REF].

-Le reste du génome est variable, composé de gènes dits accessoires (ou MGE pour éléments génomiques mobiles) que les bactéries acquièrent par transfert horizontal, avec un % de G+C différent du génome central [START_REF] Lawrence | Amelioration of Bacterial Genomes: Rates of Change and Exchange[END_REF]. Les MGE représentent environ 10 à 20 % d'un chromosome de S. aureus, y compris les plasmides, les transposons, les bactériophages, les îlots de pathogénicité des staphylocoques et les chromosomes des cassettes staphylococciques. Ils jouent notamment un rôle dans la virulence, dans la résistance aux antibiotiques, dans la pathogénicité et l'adaptabilité dans différentes conditions environnementales de S. aureus (revue dans [START_REF] Lindsay | Staphylococcus Aureus: Superbug, Super Genome?[END_REF].

S. aureus est une bactérie à Gram positif fréquemment trouvée dans la flore commensale de la peau et des muqueuses de l'Homme et de l'animal [START_REF] Williams | Healthy Carriage of Staphylococcus Aureus: Its Prevalence and Importance[END_REF]. 20 à 30 % de la population humaine sont des porteurs à long terme de S. aureus qui peuvent être de virulence qui interviennent dans l'attachement de la bactérie au substrat, l'évasion du bouclier immunitaire de l'hôte, l'invasion tissulaire, provoquant une septicémie et des syndromes toxiniques.

S. aureus possède un large arsenal de facteurs de virulence, contenant de composants pariétaux et extracellulaires impliqués dans la virulence de la bactérie [START_REF] Chavakis | The Anti-Inflammatory Activities of Staphylococcus Aureus[END_REF][START_REF] Costa | Microbial pathogens and strategies for combating them: science[END_REF]Foster 2019). La diversité de ces facteurs différents paramètres pour définir les ARNs [START_REF] Bronsard | SRNA and Cis-Antisense SRNA Identification in Staphylococcus Aureus Highlights an Unusual SRNA Gene Cluster with One Encoding a Secreted Peptide[END_REF][START_REF] Livny | High-Throughput, Kingdom-Wide Prediction and Annotation of Bacterial Non-Coding RNAs[END_REF][START_REF] Mraheil | Comparative Genome-Wide Analysis of Small RNAs of Major Gram-Positive Pathogens: From Identification to Application[END_REF][START_REF] Pichon | Small RNA Genes Expressed from Staphylococcus Aureus Genomic and Pathogenicity Islands with Specific Expression among Pathogenic Strains[END_REF]. Dès lors, de plus en plus de prédictions bio- aureus par in silico. analyse (W. [START_REF] Liu | Assessment of Bona Fide SRNAs in Staphylococcus Aureus[END_REF]. Plus récemment, l'équipe de Dr. Carroll a créé un rapport de mis à jour de l'annotation du génome de S. aureus, comprenant des annotations pour 303 ARNs connus dans USA300, s'associant à des ensembles de données RNA-Seq accessibles au public afin de récupérer les informations perdues sur l'expression, la stabilité et le potentiel de codage des peptides. [START_REF] Sorensen | Reading between the Lines: Utilizing RNA-Seq Data for Global Analysis of SRNAs in Staphylococcus Aureus[END_REF].

La découverte et la caractérisation progressive des ARNs bactériens ont mis en évidence divers mécanismes de régulation. Ils peuvent agir sur différentes cibles : les acides nucléiques (ARN et ADN) ou les protéines. Selon leur mécanisme d'action, ils ont été séparés en plusieurs classes : riboswitchs, ARN régulant les protéines, ADN ou ARN par appariements de bases. Ils peuvent également agir à différentes étapes : lors de la réplication ou de la réparation de l'ADN ou de l'expression génique, ou à différents encore dans la virulence comme, SprD [START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF][START_REF] Chabelskaya | Dual RNA Regulatory Control of a Staphylococcus Aureus Virulence Factor[END_REF], SprX [START_REF] Kathirvel | Enhancement of the Pathogenicity of Staphylococcus Aureus Strain Newman by a Small Noncoding RNA SprX1[END_REF]Ivain et al. 2017, Buchad and[START_REF] Bronwlee | The Small RNA SprX Regulates the Autolysin Regulator WalR in Staphylococcus Aureus[END_REF] 3. Ensuite, nous avons étudié l'implication de SprY dans la virulence et l'activité hémolytique de S. aureus par la méthode déjà utilisée pour montrer l'importance de SprD dans [START_REF] Chabelskaya | A Staphylococcus Aureus Small RNA Is Required for Bacterial Virulence and Regulates the Expression of an Immune-Evasion Molecule[END_REF]. ), and to understand its cellular function. First, we studied the expression profile of SprY sRNA during bacteria growth and in stress conditions. Next, we identified a few potential direct targets for SprY by in silico predictions and in vivo by MAPS (MS2-Affinity Purification Coupled With RNA Sequencing Approach). Among all predicted targets, we are drawn into SAOUHSC_03046 encoding for a potential transcriptional regulator of the XRE family proteins, SAOUHCS_1342a encoding for a mechanosensitive channel and RNAIII, which is the major riboregulator of S. aureus virulence. In addition, SprY is shown to interact with mRNA of SAOUHSC_03046 and SAOUHSC_1342a at their RBS (Ribosome Binding Site) and to prevent the translation initiation.

IV. Expression de

The characterization of interaction between SprY and RNAIII showed that SprY acts as a sponge for RNAIII and alters expression regulation of several RNAIII targets. SprY also reduces the hemolytic activity of S. aureus. Altogether, our study showed that a regulatory RNA can act as a sponge for another regulatory RNA and contributes to the pathogenicity of S. aureus during infection.

IFigure 1

 1 Figure 1. (A) Sir Robert Koch (December 11, 1843 -May 27, 1910), a German physician-scientist, well known for his pioneer studies in bacteriology, specifically in tuberculosis. (B) Sir Alexander Ogston (April 19, 1844-February 1, 1929), a Scottish surgeon, famous for his discover of major cause of wound infection. (C) Sir Anton Julius Friedrich Rosenbach, (December 16, 1842 -December 6, 1923), a German physician and microbiologist, credited for differentiating Staphylococcus aureus and Staphylococcus epidermidis.

Figure 2 .

 2 Figure 2. (A) Micrographs of scanning electron and transmission electron microscopy of S. aureus. characterized by grape-like clusters, intact membrane, and plentiful cytoplasmic contents. (B) S. aureus grows in blood agar (Z. Zhang et al. 2013).

Figure 3 .

 3 Figure 3. Horizontal gene transfer between bacteria (A) Transformation occurs when naked DNA is released on lysis of an organism and is taken up by another organism. (B) In transduction, genes are transferred from one bacterium to another by means of bacteriophages and can be integrated into the chromosome of the recipient cell. (C) Conjugation occurs by direct contact between two bacteria: plasmids form a mating bridge across the bacteria and DNA is exchanged (Furuya and Lowy 2006).

Figure 4 .

 4 Figure 4. ACME as an element associated at the downstream of SCCmec element in USA300 strain.Genes encoding resistance determinants shown in green, cassette chromosome recombinase in red, transposases in yellow, arginine deiminase pathway (arc) cluster in orange, oligopeptide permease (opp-3) cluster in purple, and other genes in black. In USA300, same region contains two genetic elements-type I ACME and type IV SCCmec element. Various genetic elements are demarcated by sets of direct-repeat (DR) and inverted-repeat (IR) sequences characteristic of SCC elements that integrated precisely into orfX. DR sequences indicated by black arrows and IR sequences by red arrows[START_REF] Diep | Complete Genome Sequence of USA300, an Epidemic Clone of Community-Acquired Meticillin-Resistant Staphylococcus Aureus[END_REF]).

Figure 5 .

 5 Figure 5. Overview of phage integration and excision. Phage genome is in blue and host chromosome in grey (from Fogg et al. 2014).

Figure 6 .

 6 Figure 6. The SaPI1 genomic map. Colors: red represents interference genes; aqua -terS; orange -accessory genes; blue -regulatory genes; yellow -int/xis; gray -hypotheticals; purplereplication (Richard P. Novick 2019).

Figure 7 .

 7 Figure 7. Timeline of antibiotics discovery and clinic applications. The antibiotics are colored per their source: green = actinomycetes, blue = other bacteria, purple = fungi and orange = synthetic. At the bottom of the timeline are key dates relating to antibiotic discovery and antimicrobial resistance, including the first reports of drug resistant strains methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), vancomycin-resistant S. aureus (VRSA) and plasmid-borne colistin resistance in Enterobacteriaceae (Hutchings, Truman, and Wilkinson 2019).

  Figure 8. Antibiotics classification

  They are either non-carriers (approximately 20% of the population), persistent-carriers (20-25%) or intermittent carriers (55-60%), which have a transient Staph and strains vary frequently.

Figure 9 .

 9 Figure 9. Overview of staphylococcal diseases

Figure 10 .

 10 Figure 10. Cell wall-anchored proteins (CWA). (A) CWA proteins are divided into four groups (MSCRAMMs, NEAT motif family, Three-helical bundle family and G5-2 repeat family). (B) CWA proteins contribute to Staphylococcus aureus virulence at different levels. Iron-regulated surface determinant (Isd) binds haemoglobin and extracts and transports haem across the cell wall and membrane into the cytoplasm, where iron is released. Protein A acts as a superantigen for B lymphocytes and disrupts adaptive immune responses. Through binding to and activating tumor necrosis factor receptor 1 (TNFR1) on host epithelial cells, protein A also triggers the synthesis of cytokines (IL-6) and causes disruptive inflammation, which contributes to pathogenesis (from Foster et al. 2014).

1.

  At low density of bacteria, Staphylococcus aureus favors the colonization within the host cell tissues by employing various cell surface factors, which are involved in tissue adhesion and immune evasion. These surface proteins include Cell wall-anchored proteins (CWA), Secreted expanded repertoire adhesive molecules (SERAMs), capsular polysaccharides, cell wall components, and Staphyloxanthin. 1.1. CWA proteins CWA are classified into four basic groups, based on the structural-functional motifs

Figure 13 .

 13 Figure 13. Two component system SrrA/ SrrB. (A) Genomic organization of srrA and srrB genes (J. M. Yarwood, McCormick, and Schlievert 2001). (B) Mode of action of SrrA/ SrrB. SrrB is found in the membrane while SrrA is in the cytoplasm. Upon signalling, SrrB is auto-phosphorylated and transfers a phosphate to SrrA. SrrA~P binds the spa, srr, tst and agr promoters. The green arrows indicate the positive regulation of SrrA, and the red one for repressive regulation (Adapted from Pragman and Schlievert 2004).

  transcription factor family can regulate agr, including SarA and MgrA (positively) as well as other regulators like CodY (negatively). The other two-component systems also influence agr expression including SaeRS and SrrAB (reviewed in (Jeremy M Yarwood and Schlievert 2003)).

Figure 15 .

 15 Figure 15. Model of CodY regulation. An important intracellular concentration of BCAAs and GTP activates CodY activity. Genes shown in boldface are direct targets of CodY-mediated regulation.Other genes regulated by CodY indirectly through its effect on the agr locus (Adapted from[START_REF] Boisset | Staphylococcus Aureus RNAIII Coordinately Represses the Synthesis of Virulence Factors and the Transcription Regulator Rot by an Antisense Mechanism[END_REF][START_REF] Camargo | Staphylococcus Aureus -Probing for Host Weakness?[END_REF][START_REF] Majerczyk | Direct Targets of CodY in Staphylococcus Aureus[END_REF]).

Figure 16 .

 16 Figure 16. Model of CcpA regulation. Catabolite control protein a (CcpA) binding to its cognate cis-acting cre site mediates additional tst repression and restrains the production of TSST-1. At high concentration of glucose, CcpA also regulates another sRNA, RsaI, and its mRNA targets (Adapted from Diego O. Andrey et al. 2015; C. Caballero, 2018).

Figure 17 .

 17 Figure 17. Model of Fur regulation in E. coli. In iron-rich environments, Fur functions as a dimer, bound to Fe2+ and represses RyhB expression, resulting in repression of target genes and in restraining the iron uptake (Adapted from Faulkner and Helmann 2011).

Figure 18 .

 18 Figure 18. The SarA protein family. Inter-regulation of transcription factors of the Sar family. Arrows represent activation and barred lines interrupted arrows for repression (Adapted from JuneckoJ et al. 2012 and Bronner S et al. 2004).

Figure 19 .

 19 Figure 19. Riboswitches predicted mechanisms. (A) Transcription termination, (B) Translation initiation, and (C) Splicing control (in eukaryotes) are the most common mechanisms. (D) Transcription interference or possibly antisense action, (E) Dual transcription and translation control, and (F) Ligand-dependent self-cleaving ribozyme action are more rare mechanisms observed or predicted in some bacterial species (from Breaker, 2012).

Figure 20 .

 20 Figure 20. Model for the regulation of 6S RNA function and RNA polymerase availability by pRNA synthesis (Cavanagh and Wassarman 2014).

Figure 21

 21 Figure 21. (A) Carbon storage regulator (CsrA) regulation mechanism. In the absence of CrsB, CsrA protein binds to GGA motifs contained in, or in the vicinity of, the Shine-Dalgarno sequence (SD) of various mRNA targets (e.g., glgCAP involving in glycogen metabolism, pgaABCD involving in biofilm attachment), leading to inhibition of translation initiation and generally to mRNA degradation. In the presence of CsrB, CsrA protein is sequestered by CsrB sRNA and allow CsrA targets to be transcript (from Nameki , Someya and Kawai, 2013) (Ranganathan, Zhao, and Simon 2013).

  They have been demonstrated to regulate different metabolic pathways, biofilm formation, motility, virulence circuitry of pathogens, quorum sensing and stress response systems (Cavanagh and Wassarman 2014; Trotochaud and Wassarman 2005; Ul Haq, Müller, and Brantl 2020). 3. sRNAs interacting with DNAs CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Proteins) is part of a new type of prokaryotic regulatory RNAs involved in

Figure 22 .

 22 Figure22. The three stages of CRISPR/ Cas system. During Adaptation, Cas1-Cas2 complex selects part of the foreign DNA to integrate into the host's CRISPR array. Next, for the processing stage, the CRISPR locus is transcribed into a single pre-crRNA (CRISPR RNA) which will be matured to produce a collection of small crRNA. In the interference stage, the mature crRNAs associates with Cas nucleases to cleave the foreign nucleic acid upon binding of the crRNA to the target sequence[START_REF] Hille | The Biology of CRISPR-Cas: Backward and Forward[END_REF]).

Figure 23 .

 23 Figure 23. Mechanism of altered transcription initiation by nanoRNAs. NanoRNAs (2-5 nts) can either be degraded by Orn ribonuclease or used by RNA polymerase to initiate transcription. Compared to transcription from NTP (i.e., de novo), initiation by nanoRNAs could potentially alter the expression of target genes by modifying the levels of transcripts, by altering the sequence of the 5 'end of the transcripts, or by modifying the phosphorylation state of the 5 'end of the transcripts (from Nickels and Dove 2011).

Figure 24 .

 24 Figure 24. Schematic view of RNAI, an antisense sRNA regulation in E. coli. RNAI is encoded on the opposite strand of the gene encoding RNAII, has beens demonstrated to inhibit primer formation and consequently prevent RNA-DNA hybrid for plasmid ColE1 replication (Brantl 2007).

Figure 25 .

 25 Figure 25. Type I toxin antitoxin system regulation. Type I toxin and antitoxin genes are independently transcribed from their own promoters. Their antitoxins are unstable cis-encoded antisense sRNAs that interact with toxin-encoding mRNAs by pairing, therefore preventing toxin mRNA translation and/or inducing its degradation (from Camille Riffaud, Pinel-Marie, and Felden 2020).

Figure 26 .

 26 Figure 26. RyhB sRNA acts as trans-encoded sRNA in E. coli. (A) RyhB sRNA, with the help of Hfq chaperone protein, binds to sodB mRNA and prevents the translation of this mRNA target by the recruitment two RNAses (Troxell and Hassan 2013). (B) RyhB sRNA also activates the translation of shiA mRNA by enhancing the Ribosome binding to the RBS of the mRNA (from Prévost et al. 2007).

Figure 27 .

 27 Figure27. RNase E-and RNase III-dependent mRNA degradation mediated by ncRNAs (from[START_REF] Repoila | Small Regulatory Non-Coding RNAs in Bacteria: Physiology and Mechanistic Aspects[END_REF]

Figure 28 .

 28 Figure 28. Molecular mechanisms of ProQ-dependent sRNA in Salmonella Typhimurium. hupA gene encodes the α-subunit of bacterial histone-like protein HU. ProQ assists the interaction between RaiZ sRNA and hupA mRNA to prevent the translation of the mRNA by masking the binding site of the 30S ribosome subunit (from Smirnov et al. 2017)

Figure 29 .

 29 Figure 29. Overview of Spr sRNAs genomic localization. Schematic view of the genome of S. aureus N315 with three pathogenicity islands (SaPI1-SaPI3, in black rectangles), a staphylococcal cassette chromosome (SCC), a bacteriophage (ΦN315), and the location of the detected sRNA genes (from Pichon and Felden 2005).

Figure 30 .

 30 Figure 30. RNAI mechanisms in control the replication of pT181. The promoter Prep directs synthesis of the RepC initiator (green ellipse), which initiates replication by binding to its cognate dso. Replication of the plasmid pT181 is controlled by RNAI (red) that is complementary to the 5' UTR of the repC mRNA (from Pluta and Espinosa 2018).

Figure 31 .

 31 Figure 31. RNAIII activity and temporal expression of S. aureus virulence factors during growth.At low cell density (during the exponential phase), the agr system is not induced: the level of expression of RNAIII is low and the mRNAs allowing tissue colonization (sa1000, spa, sa2353 and rot) are expressed. At high cell density (during the stationary phase), the agr system self-induces via quorum-sensing (QS). The expression of RNAIII allows the inhibition of translation of the above-mentioned target RNAs and the activation of the expression of hla. In addition, the coding phase of RNAIII is translated to produce hemolysin delta. Induction of the agr system by QS enables the coordinated response of the entire S. aureus population. This response allows the destruction of host tissue and promotes colonization by the bacteria (Adapted from[START_REF] Repoila | Small Regulatory Non-Coding RNAs in Bacteria: Physiology and Mechanistic Aspects[END_REF].

Figure 32 .

 32 Figure 32. Regulatory activity of RNAIII. RNAIII is directly involved in the regulation of 12 mRNAs by either activating or inhibitory mechanisms (from Matthias Gimpel and Sabine Brantl, 2016).

Figure 33 .

 33 Figure 33. An overview of Spr sRNAs involved in S. aureus pathogenicity. All sRNAs are framed in black. The red line represents the repression regulation, and the green arrow indicates the positive regulation. (1) Germain-Amiot et al. 2019; (2) Hirschhausen et al. 2010, Le Pabic et al. 2015; (3) Chabelskaya et al. 2010; (4) Chabelskaya et al. 2014; (5) Kathirvel et al. 2016; (6) Ivain et al. 2017; (7) Eyraud et al. 2014; (8) Buchad and Nair 2021.

Figure 34 .

 34 Figure 34. PSM-mec activity. Through base-pairing, sRNA Psm-mec RNA interacts with the agrA mRNA within the ORF region and inhibits translation of agrA mRNA (from Matthias Gimpel and Sabine Brantl, 2016).

102 RESULTSFigure 35 .

 10235 Figure 35. Genomic localization of sprX and sprY in HG003 strain. Schematic representations of the organization of the genetic loci of sprX and sprY. The coding phases, the sprX and sprY genes and the nucleotides (nts). Hp stands for hypothetical protein.

Figure 36 .

 36 Figure 36. Analysis of sprY1, sprY and sprY3/ srn_9342 genes. (A, C) Alignment of the sprY sequence of with sprY1 sequence (A) and with sprY3/ srn_9342 sequence (C), from 5' to 3'. Identical nucleotides between the three copies are marked with a star. The sequence of the conservative 3' end of sprY gene sequences is underlined. The sequences underlined correspond to sequences of primers using in Northern blots assays to detect separately the expression of 3 copies of spry gene in HG003 strain. (B, D) Total RNA extraction of HG003 was performed and used for Northern Blot assay. Analysis of sprY1 expression (B) and of sprY and sprY3/srn_9342 (D) expressions in HG003 strain. Northern Blot was carried out using total RNA extraction of HG003 S. aureus strain at exponential phase (E) and at stationary phase (S).

Figure 37 .

 37 Figure37. Analysis of sprY expression in different S. aureus strains (A)(Le Huyen et al. 2021) and clinical isolates (B). Total RNA extraction was performed at exponential phase (E) and at stationary phase (S). Northern Blot was carried out using labelled DNA probes for SprY. As loading controls, the blots were also probed for tmRNA.

Figure 38 .

 38 Figure 38. Expression of sprX2 and sprY in RN4220 S. aureus strain. (A) Schematic organization of the genetic loci of sprX2 and sprY in HG003 strain. Different plasmids constructions were made to overexpress sprX2 and/ or sprY in RN4220 strain and are numerated from 1 to 4 ((1): pICS3-sprY, sprX2; (2): pICS3-sprx2p1; (3): pICS3-sprX2p4 and (4): pICS3-sprY). (B) and (C) Northern Blot analysis of SprX2 and SprY expressions in RN4220 containing different plasmids. Another transcript located in the downstream of sprX2 gene was detected by Madër et al., 2016 and named S627. newcopy2 black arrow indicates primers used in Northern Blot to detect the expression of sprX2 and primer SprX2C for sprY expression.

Figure 39 .

 39 Figure 39. SprY interacts with saouhsc_03046 mRNA. (A) Interaction between SprY and saouhsc_03046 was predicted by IntaRNA software (Busch et al., 2008). The nucleotides underlined and in red correspond to the mutations in the sprY and sprYmB sequences. (B) Schematic presentation of SprY predicted secondary structure and the region of SprY that interacts with saouhsc_03046 mRNA (shown in red line). (C) Complex formation between SprY and saouhsc_03046 mRNA was analyzed by native gel retardation assays of purified labelled SprY (SprY* and SprYmB*) with increasing concentrations of saouhsc_03046 mRNA (0.1, 0.5 and 2 nM for SprY*; 2.5 and 5 µM for SprYmB*). Complex formation between SprY and RNAIII (2.5 and 5 µM) is used as positive control for shift assay. (D) Toeprint assay of saouhsc_03046 mRNA. SprY specifically prevents ribosome loading on the saouhsc_03046 translational initiation site. Here we show toeprint assays on saouhsc_03046 mRNA in the presence of increasing concentrations of SprY (3, 15 and 75 nM). '+' indicates the presence of purified 70S ribosomes. An arrow indicates the location of the experimentally determined toeprint. U, A, G and C refer to the saouhsc_03046 mRNA sequencing ladders. Two potentials translational initiation are framed in yellow and red.

Figure 40 .

 40 Figure 40. saouhsc_03046 expression regulated by SprY in HG003 S. aureus strain. (A) Analysis of saouhsc_03046 transcript level in HG003 (WT) strain containing empty plasmid (pICS3) or overexpressing sprY or sprYmB (pICS3-sprY; pICS3-sprYmB) by qPCR, using RNA extraction of those strains at early exponential growth phase. The mRNA expression level of saouhsc_03046 in qPCR is normalized with the control gene (gyrB) and is calculated with 2-ΔΔCt relative quantification. (B) Northern Blot analysis of sprY and sprYmB expressions in HG003 strain containing pICS3 or pICS3-sprY or pICS3-sprYmB using labelled DNA recognizing sprY and sprYmB. (C) S. aureus HG003 carrying the pCN33-PtufA-03046-gfp fusion plasmid co-transformed with pICS3 or pICS3-sprY or pICS3-sprYmB plasmids were grown on BHI agar plates. They were supplemented with chloramphenicol (10 µg/ml) and erythromycin (10 µg/ml). The images were obtained by scanning fluorescence on plates (right panel) and in visible light (left panel). (D) The translational initiation level of saouhsc_03046 under SprY regulation were studied by using gfp gene reporter. S. aureus strain (WT) containing the pCN33-PtufA-03046-gfp fusion plasmids co-transformed with pICS3, pICS3-sprY or pICS3-sprYmB. The fluorescent intensity (D) and growth of these strains at OD600 (E) were measured every 10 minutes over 20 hours in a Biotek microplate reader. All statistical analyses were performed using Student's t test. The error bars correspond to the average values from three independent experiments. Statistical significance is indicated by bars and asterisks as follows: *, P< 0.05; **, P< 0.01; ***, P< 0.005.

Figure 41 .

 41 Figure 41. Predicted regulation of spa gene by SAOUHSC_03046 protein. Sequence alignement between saouhsc_03046 (in HG003 strain) with sausa300_2460 (in USA300 strain) (A) and sausa300_1797 (B) using Clustal Omega (Madeira F et al., 2019). The stars indicate the similarities between the two sequences.

Figure 42 .

 42 Figure42. spa mRNA expression regulated by SAOUHSC_03046 in HG003 S. aureus strain. Analysis of spa transcript level in HG003 (WT) strain and strain deleted for SAOUHSC_03046 (HG003 Δ3046) containing empty plasmid (pICS3) or overexpressing sprY (pICS3-sprY) by qPCR, using RNA extraction of those strains at early exponential phase (2h) and stationary phase (6h). The mRNA expression level of spa in qPCR is normalized with the control gene (gyrB) and is calculated with 2-ΔΔCt relative quantification.

Figure 43 .

 43 Figure 43. SprY interacts with saouhsc_1342a mRNA. (A) Interaction between SprY and saouhsc_1342a was predicted by IntaRNA software (Busch et al., 2008). The nucleotides underlined and in red correspond to the mutations in the sprY and sprYmB sequences. (B) Schematic presentation of SprY predicted secondary structure and the region of SprY that interacts with saouhsc_1342a mRNA (shown in red line). (C) Complex formation between SprY and saouhsc_1342a mRNA was analyzed by native gel retardation assays of 0.025 pmoles of purified labelled SprY (SprY*) (left panel) and 0,025 pmoles of SprYmB* (right panel) with increasing concentrations of saouhsc_03046 mRNA (0.05, 0.25 and 0.5 µM for SprY; 0.25 and 0.5 µM for SprYmB ). Complex formation between SprY or SprYmB with RNAIII (0.5 µM) is carried out as a positive control for the assay.

Figure 44 .

 44 Figure 44. saouhsc_1342a expression-regulated by SprY in RN4220 S. aureus strain. (A) The translational initiation level of saouhsc_1342a under SprY regulation were studied using S. aureus RN4220 carrying the pCN33-PtufA-1342a-gfp fusion plasmid co-transformed with different plasmids (pICS3 or pICS3-sprY or pICS3-sprYmB). The fluorescent intensity (A) and the growth of these strains at OD600 (B) were measured every 10 minutes over 20 hours in a Biotek microplate reader. All statistical analyses were performed using Student's t test. The error bars correspond to the average values from three independent experiments. Statistical significance is indicated by bars and asterisks as follows: *, P< 0.05; **, P< 0.01; ***, P< 0.005.

Figure 1S .

 1S Figure 1S. sprY and sprX2 localization in HG003 strain. (A) Visualization of RNA seq data in the Artemis tool. The read alignment views in Artemis showing RNA-Seq data for NCTC8325 S. aureus strain (W Liu et al., 2018). The framed pink reads correspond to SprX2 transcripts. The framed reads on the right of SprX2 correspond to SprY transcripts, also known as S629 (Mäder et al., 2016). The framed reads on the left of SprX2 suggest a presence of another potential sRNA, which already defined as bona fide RNA, called S628 in W Liu et al., 2018. (B) The 5' end of SprY determined by Reverse transcription (RT). The arrow indicates the 5' end of the sprY gene determined experimentally. A, T, C and G refer to the sRNA sequencing ladders.

Figure 2S .

 2S Figure 2S. sprY and sprX2 expression in HG003 strain. Northern Blot analysis of sprY and sprX2 expression profiles in HG003 during growth, using labelled DNA probes specific for SprY and SprX2. As loading controls, the blots were also probing for tmRNA.

Figure 3S .

 3S Figure 3S. SprY sequence and structure predicted.

Figure 5S .

 5S Figure 5S. (A) Interaction between SprY and RNAIII was predicted by IntaRNA software (Busch et al., 2008). Different mutants of SprY and RNAIII were constructed. The nucleotides underlined and bolded in red correspond to the mutations in the sprY and rnaIII sequences. (B) Complex formations were analysed by native gel retardation assays of purified labelled SprY (SprY*) with an increasing concentration of RNAIII wild-type (left panel) and RNAIII muted in 13rd loop (right panel) (0.08, 0.4, 0.16, 0.32 µM).

Figure 6S .

 6S Figure 6S. WT, ΔsprY strains with or without empty plasmid (pICS3) were growth in BHI liquid in 96 wells-plate and the graphs correspond to OD 600nm measurements from Biotek. The left panel correspond to strains in double plasmid system with pCN33-rot-gfp (A) and the right panel with pCN33-ecb-gfp (B). All statistical analyses were performed using Student's t test. The error bars correspond to the average values from three independent experiments. Statistical significance is indicated by bars and asterisks as follows: *, P< 0.05; **, P< 0.01.

Figure 7S .

 7S Figure 7S. WT, ΔsprY and ΔrnaIII strains containing empty plasmid (pICS3), or over-expression of sprY or sprYmA (pICS3-sprY or pICS3-sprYmA) or rnaIII (pICS3-PamiA-rnaIII) were growth in BHI liquid in 96 wells-plate and the graphs correspond to OD 600nm measurements from Biotek. The left panel correspond to strains in double plasmid system with pCN33-rot-gfp (A) and the right panel with pCN33-ecb-gfp (B).

Figure 8S .

 8S Figure 8S. (A) HG003 WT strain were growth in BHI, TSB, NZM and RPMI liquid in 96 wells-plate and the graphs are presented in semi-log (OD 600) measurements from Biotek. (B) The quantification absolute was carried out using RNA extraction was performed at 2h and 6h of growth. The table represents the number of copies of SprY or RNAIII per ug of RNA extraction sample.

Figure 9S .

 9S Figure 9S. Schematic presentation of the interaction region between RNAIII stem-loop 13 and mRNAs encoding for the transcriptional regulatory protein Rot and virulence factors : coa (Lebeau

Figure 45 .

 45 Figure 45. Analysis of the expression levels of SprX2 and SprY sRNAs under stress conditions in the strain of S. aureus. (A) Analysis of the expression of sprX and sprY in strain HG001 S. aureus strain after 30 min and 2h30 of culture in medium depleted (NZM), under conditions at low temperature (15°C) or at high temperature (42°C), depleted in iron (Dipyridyl), oxidative stress (H202), in high salt concentration (NaCl), in acid medium (HCl), in 10% of human serum or in anaerobic manner compared to the expression of sprX and sprY at 37°C in LB rich medium (Ctrl). (B) The quantification of SprX2 and SprY expressions at 30 min and at 2h30 after exposing to temperature stress was performed by ImageQuant Tool 7.0 and is normalized to tmRNA.

  in Le Huyen et al. 2021). Interestingly, sprY and sprX2 possess opposite expression profiles during growth (Figure supplementary 2S in Le Huyen et al. 2021). SprY accumulates during bacterial growth whereas SprX2 expresses early at exponential phase and decreases over time.

Figure 46 .

 46 Figure 46. Analysis of predicted interaction of SprY with SprX2. (A) Interaction between SprY and SprX2 was predicted by IntaRNA software[START_REF] Busch | IntaRNA: Efficient Prediction of Bacterial SRNA Targets Incorporating Target Site Accessibility and Seed Regions[END_REF]. (B) Complex formation between SprY and SprX2 was analyzed by native gel retardation assays of purified labelled 0,025 pmoles of SprX2* with increasing concentrations of SprY (0.5, 2 and 5 µM). Complex formation between SprX2 and RNAIII (5 nM) is used as positive control for shift assay.

Figure 47 .

 47 Figure 47. SpoVG expression under SprY and SprX2 regulation. (A) Complex formation betweensRNA and the target mRNA was analyzed by native gel retardation assays of purified labelled 0,025 pmoles of SprX2* or SprY* with increasing concentrations of spoVG mRNA (0.5, 2 and 5 µM). (B) HG003 harboring pICS3 or pICS3-sprX2 or pICS3-sprY were grown in BHI at 37°C and total intracellular proteins extractions were carried out at 2h and 6h of growth. Western Blot analysis of SpoVG protein expression was performed using polyclonal antibodies against SpoVG. Coomassie stained gel was used as loading control.

Figure 48 .

 48 Figure 48. Expression of ecb under SprY and SprX2 regulation. S. aureus HG003 (WT) carrying the pCN33-PtufA-ecb-gfp fusion plasmid co-transformed with different plasmids (pICS3 or pICS3-sprX2 or pICS3-sprY or pICS3-PamiA-rnaIII). The fluorescent intensity (A) and the growth of these strains at OD600 (B) were measured every 10 minutes over 20 hours in a Biotek microplate reader. All statistical analysis were performed using Student's t test. The error bars correspond to the average values from three independent experiments. Statistical significance is indicated by bars and asterisks as follows: *, P< 0.05; **, P< 0.01; ***, P< 0.005.

Figure 49 .

 49 Figure 49. Hemolytic activity of S. aureus regulated by de SprX2 et SprY in human blood. The supernatants of different strains of HG003 (WT, ΔsprX2, ΔsprY) and HG003 WT containing empty plasmid (pICS3) or overexpressing sprX2 or sprY (pICS3-sprX2 or pICS3-sprY) were collected after 6 hours of growth in BHI at 37°C. Hemolysis was performed by incubating cell culture supernatants with Human blood samples at a ratio 50:50 at 37°C for 1h. The hemolytic activity was observed in a flat-bottom 96-well microtiter plate at OD 540nm. The blood samples prepared in PBS1X represent as negative control and in Triton 0.1% as 100% hemolysis. All statistical analysis from three independent experiments were performed using Student's t test . Statistical significance is indicated by bars and asterisks as follows: *, P< 0.05; **, P< 0.01; ***, P< 0.005.

Figure 50 .

 50 Figure 50. Overview of genomic localization of sprY gene in NCTC8325 strain compared to lukF/S-PV gene in USA300 strain. The red frame indicates the corresponding zone of two genes (sprY and lukF/S) and the black arrow shows sprY sequence in NCTC8325 strain.

  are the well-studied phage-encoded virulence factors in S. aureus. In addition, few sRNAs involved in the virulence regulation like SprD[START_REF] Chabelskaya | Dual RNA Regulatory Control of a Staphylococcus Aureus Virulence Factor[END_REF] Chabelskaya, Gaillot, and Felden 2010a) or SprF1/ SprG1 (Pinel-Marie, Brielle, and Felden 2014) were also found encoded in staphylococcal phages. Here, we have shown the presence of yet another sRNA, SprY, phage encoded that affects the pathogenicity of S. aureus by modulating the hemolytic activity of S. aureus. The differential expression of sprY in S. aureus commonly used laboratory strains and clinical isolates that we showed suggests that sprY could provide S. aureus with adaptative advantages in particular infection environments. Further experiments in a larger scale are needed to confirm this hypothesis such as the assessment of the hemolytic activity of different S. aureus strains in the absence or presence of sprY gene.

  affecting the stability nor inducing the cleavage of the RNA of interest. Moreover, utilization of high-throughput technologies such as MAPS, CLIP-seq, etc. that were developed for identification of direct targets for sRNAs allow the discovery of RNA that mimic mRNA targets and bind to regulatory sRNA altering their function (reviewed in[START_REF] Denham | The Sponge RNAs of Bacteria -How to Find Them and Their Role in Regulating the Post-Transcriptional Network[END_REF]). The term "sponge" RNA came from eukaryotic regulatory RNA studies demonstrating the titration of regulatory RNA thus affecting its function. Later, it was shown that the natural eukaryotic and prokaryotic coding and noncoding RNAs could have sponge-like activity to mimic primarily targets of regulatory sRNA. The first sponge RNA in prokaryotes has been identified in Salmonella enterica[START_REF] Figueroa-Bossi | Erratum: Caught at Its Own Game: Regulatory Small RNA Inactivated by an Inducible Transcript Mimicking Its Target (Genes and Development (2009) 23 (2004-2015[END_REF]. ChiX sRNA represses the synthesis of ChiP, however, the presence of another mRNA transcript, chitosugars (chb), lead to the titration of ChiX through base pairing and as consequence allows the production of ChiP. Subsequently the mechanism of RNA sponge-like activity has been recently described in different bacteria suchlike E. coli, B.

  Figure 51. Overview regulation network of S. aureus virulence by several sRNAs. This schema represents a partial regulation network of S. aureus

  the parametric tests of Student (for paired or unpaired data) and of Welch or the nonparametric test of Mann-Whitney were carried out, on at least three independent experiments, to evaluate the significance. Data were expressed as mean ± standard deviations.

  de virulence implique que la pathogénicité de S. aureus est un processus complexe, nécessitant une expression organisée au cours des différentes étapes de l'infection (donc la colonisation, l'évasion immunitaire, la croissance et division cellulaire, et la dissémination bactérienne). En effet, S. aureus régule d'abord positivement l'expression de gènes codant pour des protéines de surface impliquées dans l'adhésion et la défense contre le système immunitaire de l'hôte ; et ce n'est que tardivement dans l'infection qu'il commence à réguler à la hausse la production de toxines qui facilitent la propagation des tissus. Pour contrôler la production des déterminants de la virulence pendant l'infection, S. aureus utilise plusieurs systèmes de régulation qui répondent à la densité cellulaire bactérienne (quorum sensing) et aux signaux environnementaux (par exemple, le pH, l'osmolarité et la disponibilité des nutriments, la température et la tension d'oxygène). Ces systèmes de régulation peuvent être divisés en cinq grandes catégories : les systèmes de transduction du signal à deux composants, les régulateurs transcriptionnels globaux, la famille de protéines SarA, les facteurs sigma et le groupe le plus récent est celui des ARN régulateurs (régARN).II. Des ARN régulateurs chez S. aureusA ce jour, il existe trois principaux types d'ARN « classiques » : les ARN messagers (ARNm), les ARN de transfert (ARNt) et les ARN ribosomiques (ARNr). Les ARNm sont traduits en protéines, tandis que les ARNt et les ARNr ont des rôles lors de la traduction de l'ARNm. Les ARNt et les ARNr sont des ARN non codants, c'est-à-dire qu'ils ne disposent pas des informations nécessaires à la synthèse d'une protéine. Cependant, un autre type d'ARN non codant a été identifié, à savoir les ARN régulateurs (régRNA). Ils permettent aux bactéries de réguler l'expression de différents facteurs impliqués dans l'adaptation aux changements environnementaux. En général, les ARN régulateurs sont définis comme des molécules stables entre 50 et 500 nts de taille et leur annotation dépend des techniques abordées et/ou de la localisation des ARNs étudiés. Au début des années 2000, seulement quelques ARN régulateurs avaient été identifiés en partie grâce aux techniques disponibles telles que les approches computationnelles, qui permettaient l'identification d'ARN en utilisant

  informatiques ainsi que l'explosion des techniques à haut débit (RNA-seq, DNA microarrays, clonage shotgun) ont permis l'identification de nombreux ARN[START_REF] Altuvia | Regulatory Small RNAs: The Key to Coordinating Global Regulatory Circuits[END_REF][START_REF] Hüttenhofer | Experimental Approaches to Identify Non-Coding RNAs[END_REF][START_REF] Kazantsev | Bacterial RNase P: A New View of an Ancient Enzyme[END_REF][START_REF] Moore | The TmRNA System for Translational Surveillance and Ribosome Rescue[END_REF]. Cependant, l'absence d'un génome consensuel et entièrement annoté de S. aureus a ajouté au problème de la nomenclature des ARNs. En effet, il existe des situations où un ARN étudié dans un travail a été nommé différemment dans une autre publication, par exemple, RsaOW est nommé Teg17[START_REF] Bohn | Experimental Discovery of Small RNAs in Staphylococcus Aureus Reveals a Riboregulator of Central Metabolism[END_REF][START_REF] Guillet | Emerging Functions for the Staphylococcus Aureus RNome[END_REF] ou SprX alias RsaOR et Teg15 (W. Liu et al. 2018). Pour surmonter ce problème, Sassi et al. 2015 a fourni une base de données d'ARN régulatrices de staphylocoques (SRD) qui rassemble une liste d'ARNs identifiés et validés expérimentalement au fil des années. Beaucoup plus d'ARNs ont été identifiés et il devient difficile de poursuivre la mise à jour. En effet, Madër et ses collaborateurs ont récemment fourni une analyse globale de la régulation transcriptionnelle et des ARN non-codants chez S. aureus (Mäder et al. 2016) et l'équipe de Dr. Bouloc a fourni une nouvelle réévaluation d'environ 50 sRNA authentiques chez S.

  SprY dans des conditions de croissance bactérienneLors des études sur l'ARN SprX2 et les séquences voisines, nous avons identifié un autre ARN que nous avons appelé SprY. Situé sur le phage Φ12 entre le gène sprX2 et le gène SAOUHSC_01515 codant pour le peptidoglycane hydrolase putative, le gène spry a également été identifié comme un transcrit d'un ARN par Madër et ses collaborateurs, désigné S629[START_REF] Mäder | Staphylococcus Aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions[END_REF]. Peu de temps après, le laboratoire de Dr. Bouloc a réalisé une étude dans laquelle cet ARN a été validé comme un véritable régARN(W Liu et al. 2018). Nous avons déterminé le début de la transcription du gène sprY et la taille de l'ARN SprY d'environ 125 nucléotides(Le Huyen et al. 2021). Nous avons également montré une structure secondaire prédite de SprY composée de trois boucles de tige et la seulement ouvert par les fluctuations de la tension membranaire dues à la force osmotique, mais aussi par la force d'adhérence aux surfaces. Lorsque la bactérie présente de fortes forces d'adhésion en adhérant à la surface du substrat, cela provoque une déformation de la paroi cellulaire du staphylocoque et conduit à l'ouverture de MscL[START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF]. Cependant, la régulation de l'expression de ces MscL reste incertaine. Ici, nous avons démontré que SprY régule potentiellement à la baisse la traduction de SAOUHSC_1342a, ce qui pourrait révéler partiellement la régulation de l'expression de MscLs dans S. aureus. Nous avons l'intention de tester l'effet de SprY sur certains phénotypes liés au contrôle de canal mécanosensible chez S. aureus en mesurant la capacité d'absorption de la calcéine fluorescente ou de l'antibiotique (dihydrostreptomycine) décrit dans[START_REF] Carniello | Role of Adhesion Forces in Mechanosensitive Channel Gating in Staphylococcus Aureus Adhering to Surfaces[END_REF]. Par exemple, les auteurs ont démontré qu'à une force d'adhésion élevée, la souche de type sauvage RN4220 présentait plus d'intensité de fluorescence que dans RN4220 supprimée pour mscL (RN4220 ΔmscL), ce qui est corrélé à l'absorption de la calcéine fluorescente dans les bactéries à travers ces canaux mécanosensibles. o ARNIII (article accepté d'être publié dans NAR) La deuxième cible identifiée pour SprY est l'ARNIII, l'un des principaux régulateurs de la virulence de S. aureus. La prédiction par IntaRNA a montré un appariement de bases potentiel entre SprY et la 13è tige-boucle de ARNIII. Nous avons ensuite vérifié cette interaction prédite entre SprY et ARNIII in vitro en effectuant une EMSA à l'aide des ARN synthétiques. Nous avons également analysé la spécificité de l'interaction en introduisant des mutations compensatoires ponctuelles pour créer des allèles SprY et ARNIII (SprYmA et ARNIIImA) incapables de se lier aux ARNs de type sauvage. De plus, il a été démontré que la tige-boucle 13 de ARNIII contrôle l'expression de plusieurs facteurs de virulence tels que hla, rot, coa, lytM, spa, SA1000, SA2353, SA209, etc. (Boisset et al. 2007; Chevalier et al 2010 ; Chunhua et al. 2012 ; Geisinger et al. 2006 ; Huntzinger et al. 2005 ; Morfeldt et al. 1995). Nous avons suggéré qu'à travers cette interaction avec ARNIII, SprY pourrait affecter la régulation RNAIII de ses cibles. Pour vérifier cette hypothèse, nous avons étudié l'expression de deux cibles d'ARNIII : les ARNm ecb et rot. Pour étudier le niveau d'ARNm de ces cibles, nous avons effectué une qPCR en utilisant l'extraction d'ARN total à partir de souches HG003 S. aureus surexprimant SprY. Nous avons également testé l'expression traductionnelle de ecb et rot par un système à double 239 plasmide comme décrit dans Ivain et al. 2017. Cette expérience consiste à concevoir une fusion de gène traductionnel ARNm-gfp sous le contrôle d'un promoteur constitutif PtufA dans le vecteur pCN33 résultant en pCN33-PtufA-ecb/rot-gfp. Nous avons ensuite transduit ces plasmides dans HG003 hébergeant pICS3, pICS3-sprY et pICS3-sprYmA qui est muté sprY dans la zone d'interaction avec ARNIII. La surexpression de SprY impacte significativement la fluorescence de Ecb-GFP et Rot-GFP, tandis que SprYmA n'affecte pas l'intensité de fluorescence. Étant donné que SprYmA ne se lie pas à l'ARNIII, ces résultats impliquent que l'interaction entre SprY et RNAIII est nécessaire pour la régulation de l'ecb et de la pourriture par SprY. Nos données révèlent un ARN agissant comme une éponge pour l'ARNIII et ont montré que SprY a également un impact sur l'activité hémolytique et impliqué dans la virulence de S. aureus. Ces résultats sont présentés dans l'article accepté pour publication dans le NAR (Le Huyen et al. 2021). VI. Possible lien entre sprX2 et sprY: deux gènes de ARN régulateurs localisés dans le même cluster Au cours de mon projet de thèse, l'analyse des expressions SprY et SprX2 a montré quelques remarques intéressantes sur ces deux ARNs (Figure 4). Malgré la proximité immédiate des gènes sprY et sprX2 dans la souche HG003, l'absence ou la surproduction d'un ARN n'affecte pas l'expression de l'autre. De plus, nous avons montré qu'ils possèdent des profils d'expression opposés : SprY s'accumule dans le temps alors que c'est le contraire pour SprX2. Non seulement ils s'expriment différemment au cours de la croissance bactérienne, mais ils réagissent également de manière opposée sous un stress thermique. Il a été montré que SprX2 diminuait sous 42°C et s'exprimait davantage à 15°C tandis que SprY diminuait à 15°C et s'exprimait davantage à 42°C. Peut-être que le stress affecte la transcription et/ou la dégradation des ARNs. De plus, SprY affecte l'expression de spoVG de manière opposée à SprX2. Alors que SprX2 réduit la traduction de spoVG par liaison directe au RBS de l'ARNm (Eyraud et al. 2014), SprY provoque une augmentation de la quantité de SpoVG par un mécanisme inconnu qui n'implique pas d'interaction directe entre SprY et l'ARNm de spoVG. De même, SprY a un effet inverse de SprX sur l'expression de ecb (Ivain et al. 2017). SprX2 régule l'expression d'ecb par liaison directe (Ivain et al. 2017) tandis que SprY régule ecb en affectant la fonction de l'ARNIII (Le Huyen et al. 2021). Ainsi, deux ARNs situés dans le même cluster et régulent à l'opposé une cible commune par des mécanismes différents. De plus, les deux ARNs Titre : Etude de nouveaux ARN régulateurs impliqués dans la virulence de Staphylococcus aureus Mots clés : Staphylococcus aureus, ARN régulateur, virulence, ARNIII, ARN éponge Résumé : Staphylococcus aureus est une bactérie à Gram positif fréquemment trouvée dans la flore commensale de la peau. Elle est capable de s'adapter aux changements environnementaux grâce à la reprogrammation rapide de l'expression génique modulée par des ARN régulateurs, parmi d'autres facteurs. Au cours de ma thèse, mes travaux ont porté sur un nouvel ARN régulateur, appelé SprY (alias S629), avec l'objectif de comprendre sa fonction biologique. Dans un premier temps, nous avons étudié le profil d'expression de l'ARN SprY au cours de la croissance bactérienne et dans des conditions de stress. Ensuite, nous avons identifié plusieurs cibles directes pour SprY par des prédictions in silico et in vivo par MAPS (MS2-Affinity Purification Coupled With RNA Sequencing Approach). Parmi toutes les cibles identifiées, nous nous sommes intéressés au gène SAOUHSC_03046 codant pour un régulateur transcriptionnel des protéines de la famille XRE, SAOUHCS_1342a codant pour la protéine des canaux mécanosensibles et ARNIII, qui est le riborégulateur principal de la virulence de S. aureus. Nous avons démontré que SprY interagit avec les ARNm de SAOUHSC_03046 et SAOUHSC_1342a au niveau de leur RBS (Ribosome Binding Site) et empêche l'initiation de la traduction de ces cibles. La caractérisation de l'interaction entre SprY et ARNIII a montré que SprY agit comme une éponge pour ARNIII et modifie la régulation de l'expression de plusieurs cibles de ce riborégulateur. SprY réduit également l'activité hémolytique de S. aureus pendant l'infection. L'ensemble de ces résultats ont montré que SprY joue le rôle d'éponge pour ARNIII et sa contribution dans la pathogénicité de S. aureus. Title : Characterization of novo regulatory RNAs implicated in the virulence of Staphylococcus aureus Keywords : Staphylococcus aureus, regulatory RNA, virulence, RNAIII, RNA sponge Abstract : Staphylococcus aureus is a Grampositive coccus frequently found in the commensal flora of the skin and possesses an adaptive ability to environmental changes. This bacteria's capacity of fast gene expression reprogramming is mediated by regulatory RNAs among other factors. During my PhD program, my work aims to characterize a new regulatory RNA, called SprY (alias S629

  . Another example of multidrug resistance Staph is Vancomycin Resistant Staphylococcus aureus (VRSA) (Appelbaum 2006; Gardete and Tomasz 2014) discovered in 2002.

Table 1_Cell

 1_Cell surface proteins (Adapted from[START_REF] Chavakis | The Anti-Inflammatory Activities of Staphylococcus Aureus[END_REF] Foster 2019b; Foster and Geoghegan 2014)

Table 2_Secreted factors

 2_Secreted

Table Supplementary 2

 Supplementary . Primers used in this study

	N°Name	Sequences	Utilization	References
	1 T7 sprY for	TAATACGACTCACTATAGGGTATAGGGAATCTTACAGTTAT	EMSA	This study
	2 T7 sprY rev	AAATAGGCAAGTACCGAAGTACC	EMSA	This study
	3 T7 sprYmA for	TAATACGACTCACTATAGGGTATAGGTAATCTTAAGGTTATTAAATA ACTTATT	EMSA	This study
	4 T7 rnaIII for	TAATACGACTCACTATAGGGCCTAGATCACAGAGATGT	EMSA	(Chabelskaya et al., 2014)
	5 T7 rnaIII rev	CAAAAGGCCGCGAGCTTGGGA	EMSA	(Chabelskaya et al., 2014)
	6 T7 rnaIII mL13 for	CATTATTTGATAAATAAAAAACTGTAAAACATTCCCTTA	EMSA	This study
	7 T7 rnaIII mL13 rev	TAAGGGAATGTTTTACAGTTTTTTATTTATCAAATAATG	EMSA	This study
	8 T7 rnaIIImA for	TAAGATTGAAAAAATAACCTTAAAACATTACCTTAATAATAAGTATG GTCGTG	EMSA	This study
	9 T7 rnaIIImA rev	AGGTTATTTTTTCAATCTTATTTTGGGCATGTATTTAATTATGAAAAAA ATTT	EMSA	This study
	10 pICS2-ms2 (KpnI/ SacI) for	AAATAAGCTTGATGGTACCCGTACACCATCAGGGTACGTTTTTCAGA CACCATCAGGGTCTGGAGCTCGAATTCACTGGCCG	MAPS	This study
	11 pICS2-sprY-ms2 (KpnI) for	GTATAATTAAAATAAGCTTGATGGTACCTATAGGGAATCTTACAGTT ATT	MAPS	This study
	12 pICS2-sprY-ms2 (EcoRI) rev	GTAAAACGACGGCCAGTGAATTCCGAAGGCTAGCTATAACATAAAA AAATA	MAPS	This study
	13 pICS3	AAAGCTTGCATGCCTGCAGGGTACCGAATTCGGCGCCTGATGCGGTA TTT	Cloning	(Ivain et al., 2017)
	14 PamiA for	GAAAATTTGTTTGATTTTTAATGGATAATGTGATATAATGG	Cloning	(Ivain et al., 2017)
	15 pICS3-PstI-sprY for	GTATAATTAAAATAAGCTTGATGGTACCTATAGGGAATCTTACAGTT ATT	Cloning	This study
	16 pICS3-NarI-sprY rev	GACACCATCAGGGTCTGGAGCTCTATAGGGAATCTTACAGTTATT	Cloning	This study
	17 pICS3-PstI-sprYmA for	AGGTAATCTTAAGGTTATTAAATAACTTATTGGATGCATGTATTTATT CCTATACACTTT	Cloning	This study
	18 pICS3-NarI-sprYmA rev	AATACATGCATCCAATAAGTTATTTAATAACCTTAAGATTACCTATAA TTAATGTAGCAA	Cloning	This study
	19 PtufA rev	TCTCTCATGATAGTTTCTCAC	Cloning	This study
	20 pCN33-rot-gfp for	GAGAAACTATCATGAGAGAAGATCTGTATATAAATTATAAAATTAAT ATG	Cloning	This study
	21 pCN33-rot-gfp rev	CCTTAGTCTTCTCTTTGCTTCTAGAGATATCTTCTCTAGACATTTTGTAT TCG	Cloning	This study
	22 SprX2	TACGGGAATGCTAAAGTCAT	Northern Blot (Ivain et al., 2017)
	23 SprY	CGAAGTACCTGCCAGTTACG	Northern Blot	This study
	24 ms2	AACGTACCCTGATGGTGTACG	Northern Blot	This study
	25 RNAIII	ATTATTAAGGGAATGTTTTA	Northern Blot (Ivain et al., 2017)
	26 tmRNA	ACACGCTTAATGAGCTCGGG	Northern Blot (Ivain et al., 2017)
	27 SprY qPCR for	TGGATGGATGTTAATATTCCTATACAC	RT-qPCR	This study
	28 SprY qPCR rev	TACCGAAGTACCTGCCAGTTACG	RT-qPCR	This study
	29 RNAIII qPCR for	GAATTTTGTTCACTGTGTCGATAATCCATTT	RT-qPCR	This study
	30 RNAIII qPCR rev	GAAGGAGTGATTTCAATGGCACAAGATAT	RT-qPCR	This study
				134

Table Supplementary

 Supplementary

3. Whole list of RNAs co-purified with MS2-SprY (Raw data available under accession no. GSE166499)

  , we decided to study SAOUHSC_03046 which is the only common gene coming out among the top 15 (Annex 1 and Annex 2). Our study showed the presence of base pairing between SprY and this mRNA in vitro, and also confirmed regulation of its expression in vivo. SprY interacts with the 5' UTR of SAOUHSC_03046 mRNA masking the RBS, and consequently blocking its translation. SprY overexpression does not affect the mRNA level of SAOUHSC_03046 but SprYmB, which does not bind the target, unexpectedly upregulates SAOUHSC_03046 at mRNA level and at translational level. These results lead us to wonder if the regulation mechanism of SprY on SAOUHSC_03046 involves other intermediate factors or maybe if SprYmB overexpression competes with SprY endogenous expression in the HG003 strain

  All plasmids and primers used are listed in Table4 and Table 5. Cloning experiments were carried out at 50°C for 15 minutes, using Gibson Assembly Master Mix (New England Biolabs). Then, 100μL of competent bacteria are placed in the presence of approximately

	I. Materials
	A. Bacterial strains and growth condition
	All bacterial strains and plasmids used in this work are listed in Table 4. Mutated strain 100 ng of plasmids for 15 minutes on ice before undergoing a thermal shock for 35
	seconds at 42°C. After being put back on ice for 2 minutes, the bacteria are incubated for HG003 for saouhsc_03046 (HG003 Δ3046) was made as described in Monke et al. 2015, using primers 14 to 17 in Table 5. All other mutant strains are provided by Dr. Bouloc from Institute for Integrative Biology of the Cell (I2BC), University Paris-Saclay (Le Lam et 1 hour in SOC (SOB medium + 20 mM Glucose) medium at 37 ° C. and then spread on LB dishes supplemented with 50 μg/mL ampicillin. The plasmids were purified from MATERIALS & al. 2017). overnight cultures in LB broth supplemented with Ampicillin 10ug/mL and
	minipreparations were performed using Miniprep Extraction Kit (Qiagen). The plasmid
	Brain Heart Infusion (BHI, AES Chemunex), Luria Broth (LB, MO BIO), Tryptic Soy Broth (TSB, Oxoid), Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Thermo sequence was verified by Sanger sequenced by using BigDye Terminator v3.1 Cycle sequencing Kit, using a 3130x1 capillary electrophoresis genetic analyzer (Applied METHODS Fischer Scientific) and NZM (Sigma) media were used and supplemented, if necessary, Biosystems).
	with 100 μg/mL of ampicillin (amp) and 10 μg/mL chloramphenicol (cat) or erythromycin
	constant agitation at 160 rpm and followed by measuring the optical density at 600 nm
	(OD600).
	1. DH5-α E. coli competent cells preparation, transformation, and plasmids
	purification
	A culture of E. coli is cultivated in 400mL of SOB medium (86mM NaCl; 2.5mM KCl; 2%
	tryptone; 0.5% yeast extracts) and incubated for 2 to 3 days at 18°C with vigorous shaking
	(220 rpm) until the culture reaches an OD600 = 0.5. Once the OD600 is reached, the
	culture is incubated for 10 minutes on ice then centrifuged for 10 minutes at 2500g, 4 °
	C and the pellets are resuspended in 80mL of cold TB buffer (10mM PIPES; 15mM CaCl2;
	250mM KCl; 55mM MnCl2; pH 6 ,7). After a new incubation on ice for 10 minutes and a
	new centrifugation for 10 minutes at 2500g, 4°C, the pellets are gently resuspended in

(erm) unless otherwise specified. A pre-culture was inoculated with a colony isolated on a BHI dish at 37°C. The next day, the cultivation was carried out by dilution to one hundredth of this pre-culture in medium. Bacterial growth was cultivated at 37 ° C, with 20mL of cold TB before adding glycerol (7% final). After a final 10 minutes incubation on ice, the cells are aliquoted to 1mL/ microtube and stored at -80°C.

Introduction de Staphylococcus aureus

  Staphylococcus aureus est caractérisé comme une coque à Gram positif non mobile et non sporulée, qui se développe en colonies rondes, jaune doré et lisses avec un diamètre de 0,8 à 1 µm, dans de l'agar au sang[START_REF] Merghni | Antioxidant and Antiproliferative Potential of Biosurfactants Isolated from Lactobacillus Casei and Their Anti-Biofilm Effect in Oral Staphylococcus Aureus Strains[END_REF]. CDS). La plupart des CDS du génome de S. aureus ont une fonction qui leur est assignée, basée sur une homologie significative avec des gènes d'autres espèces

	Les premiers génomes de S. aureus achevés étaient ceux de N315 et Mu50 en 2001
	(Kuroda et al. 2001). Au cours des deux dernières décennies, les séquences génomiques
	d'environ 500 souches de Staphylococcus ont été complétées et annotées (Gill 2009). Les
	génomes sont présentés dans des chromosomes circulaires d'environ 2,8 millions de
	paires de bases avec une faible composition en GC (32 %) et codent pour environ 2 700
	séquences codantes (

I.

  ou Teg41[START_REF] Zapf | The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus Aureus[END_REF], etc.III. Objectif de ma thèseRécemment, nous avons identifié dans le phage Φ12 un gène codant pour un nouvel ARN régulateur exprimé à proximité de l'ARN SprX2 et l'avons appelé SprY. Sa localisation dans une région du génome contenant de nombreux facteurs de virulence suggère que ce régARN pourrait être impliqué dans la virulence de S. aureus. L'objectif de ma thèse est de caractériser le profil d'expression de SprY, d'identifier ses cibles, ses mécanismes d'action et les rôles physiologiques de SprY dans la souche HG003 S. aureus.1. Premièrement, nous avons étudié le profil d'expression de SprY dans la souche sauvage HG003 ainsi que des souches mutantes de HG003, dans des différentes conditions de croissance bactérienne.2. Deuxièmement, nous recherchions des potentielles cibles directes de SprY par deux approches différentes : l'une est des prédictions in silico via CopraRNA, et l'autre approche est une méthode à haut débit « MS2 », basée sur la purification des complexes ARNs avec son ARN cible in vivo.

traduite de l'ARNm sarT et favorise sa dégradation[START_REF] Xue | ArtR, a Novel SRNA of Staphylococcus Aureus, Regulates α-Toxin Expression by Targeting the 5′ UTR of SarT MRNA[END_REF]. AgrA s'est avéré se lier au promoteur et réprimer l'expression de l'ARN, ArtR. En résumé, S. aureus développe de multiples voies pour réguler sa virulence en modulant directement la production de toxines ou en contrôlant la fonction d'agr et de son effecteur, l'ARNIII.Ainsi, notre travail montre à quel point la régulation des facteurs de virulence est multiforme au cours de l'infection à S. aureus et découvre une autre couche d'ARNs impliquée dans la pathogénicité de S. aureus.

SAUSA300_2460 and ~55% identity of SAUSA300_1797 (xdrA gene) in USA300 strain (Figure 41). Since XdrA is showed to activate spa translation in USA300 strain [START_REF] Mccallum | Transcriptional Profiling of XdrA, a New Regulator of Spa Transcription in Staphylococcus Aureus[END_REF], we suggested that SAOUHSC_03046 protein could also affect spa gene expression in HG003 strain. Our preliminary results showed an increase of in spa mRNA level in HG003 deleted for SAOUHSC_03046 (HG003 Δ3046) compared to WT strain (Figure 42). The overproduction of SprY also increased spa mRNA levels, which correlates with the fact that SprY overexpression reduced SAOUHSC_03046 expression at translational level (described in Figure 41). However, SprY overexpression showed similar impact on spa mRNA level in the presence or absence of SAOUHSC_03046, which suggests that other regulatory factors besides SAOUHSC_03046 may be involved may be involved in the regulation of SprY on spa mRNA.

B. Identification of SprY targets by MAPS

In addition to in silico approaches to identify SprY targets, we also performed in vivo analysis through a MS2-RNA affinity purification coupled with RNA sequencing (MAPS) [START_REF] Carrier | A Game of Tag: MAPS Catches up on RNA Interactomes[END_REF][START_REF] Lalaouna | Identification of SRNA Interacting with a Transcript of Interest Using MS2-Affinity Purification Coupled with RNA Sequencing (MAPS) Technology[END_REF]Tomasini et al. 2017a).

MS2 tagged version of SprY was expressed under the control of an inducible promoter in HG003 strain and RNAs in complex with MS2-SprY were eluted and analyzed by RNAseq.

According to bioinformatic analysis of RNAs enriched in complex with SprY-MS2 compared with MS2 alone, we identified three potential targets for SprY: (1) rpmG1 mRNA, with an enrichment of 11.11-fold; (2) saouhsc_1342a mRNA, which was enriched by 7.59-fold; and (3) RNAIII, with 7.4-fold of enrichment (Le Huyen et al. 2021). However, no base pairing with a significant energy was predicted between SprY and rpmG1 mRNA by in silico analysis. Hence, we focus on testing SprY impact on the expression regulation of the two other RNAs.

SprY affects SAOUHSC_1342a mRNA expression at translational level

IntaRNA prediction showed the binding between SprY and 20 nts around the translational start site of SAOUHSC_1342a mRNA (Figure 43A andB). We therefore confirmed SprY interactions with SAOUHSC_1342a mRNA in vitro. A complex formation between SprY (D) Interaction between SprY and mRNA of saouhsc_1342a was predicted by IntaRNA software [START_REF] Busch | IntaRNA: Efficient Prediction of Bacterial SRNA Targets Incorporating Target Site Accessibility and Seed Regions[END_REF]. The start codon of saouhsc_1342a is framed in red.

Supplementary Data_ Le Huyen et al. 2021

Table Supplementary 1. Strains and plasmids

Strains

DISCUSSIONS & PERSPECTIVES