WO2010020694A1 - Polynucleotides which down-regulate the expression of the sbi protein - Google Patents

Abstract

The invention relates especially to a particular isolated polynucleotide, to a composition comprising same and to the nontherapeutic use thereof for inhibiting the expression of the gene encoding the Sbi protein.

Description

 POLYNUCLEOTIDES REGULATING NEGATIVELY THE EXPRESSION OF

SBI PROTEIN

The present invention relates to polynucleotides capable of downregulating the expression of the Sbi protein and to their application, in particular for the detection, prevention and / or treatment of a staphylococcal infection.

Staphylococcal infections are widespread throughout the world, affecting both humans and animals.

The bacteria of the genus Staphylococcus were discovered by Louis Pasteur in 1880. To date, about twenty species have been identified. Of these, the most commonly encountered in human and / or animal pathology are Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus hyicus, Staphylococcus intermedius and Staphylococcus aureus.

These commensal species are called opportunistic pathogens since they can cause infections under particular conditions. Thus, Staphylococcus epidermidis may be responsible for infections of the skin, nasal and also of endocarditis and localized infections in immunocompromised patients. Staphylococcus saprophyticus has been identified as responsible for some urinary tract infections. Staphylococcus hyicus and Staphylococcus intermedius may be responsible for various infections in animals such as exudative piglet dermatitis (S. hyicus) or furunculosis of the dog (S. intermedius).

The most pathogenic species of Staphylococcus bacteria is Staphylococcus aureus (S. aureus). S. aureus is a member of human commensal flora. However, if mucosal barriers are breached or the host's immune defenses are altered, S.aureus can become an opportunistic pathogen and one of the leading causes of nosocomial infections and community-acquired infections in humans and humans. animals (Lowy, 1998). The pathogenesis of S.aureus-associated disease usually begins with a local infection (eg, infection of a wound, boil, cellulitis), then can be followed by systemic spread (bacteremia), followed by metastatic infections (by example endocarditis, osteomyelitis and septic arthritis). S. aureus is also one of the pathogens most frequently isolated from bovine mastitis in both the United States and Europe. Affected cows then have a drop in milk production which has serious economic consequences for farms. The study of the molecular mechanisms allowing staphylococcal infection to develop and persist in the colonized organism has shown that one of the main causes of success of staphylococcal infections, in particular S. aureus, is their ability to produce a large number of virulence factors. Some of these factors are exported to the surface of cells and others are excreted in the extracellular environment of the host.

Thus, for example, S. aureus is able to bypass the defenses of the host by producing traps of the immune response of the infected host such as the SpA protein ("Staphylococcal protein A", Moks et al., 1986 ) and Sbi protein ("S. aureus IgG binding protein"). Sbi protein was originally identified in S. aureus as an IgG immunoglobulin binding protein (Zhang et al., 1998). It has been shown later that its expression is induced by the presence of human IgG (Zhang et al, 2000) and that it precipitates with human IgG (Atkins et al., 2008).

It is now established that the Sbi protein also has the ability to interact with components of the innate immune system of the host. Indeed, Sbi is a secreted protein that traps the complement system by fixing and titrating the complement C3 factor (Burman et al, 2008, Upadhyay et al, 2008), the central molecule of the three complement activation pathways.

Thus, the Sbi multifunctional protein directly interferes with the host's adaptive immune system via its first two IgG binding domains (Sbi-I and Sbi-II, Atkins et al, 2008) and modulates the innate immune system of the host. host by using the alternative pathway of complement activation via its third and fourth domains (Sbi-III and Sbi-I V, Burman et al., 2008), in particular by the interaction between its fourth domain and the C3 protein of the complement (Upadhyay et al, 2008).

So far, the most common treatment for staphylococcal infection is antibiotics such as penicillins (penicillin G, methicillin, oxacillin cephalosporins), macrolides and tetracyclines.

However, with the widespread use of antibiotics, the spread of bacterial strains of the genus Staphylococcus, particularly S. aureus, highly resistant to antibiotics, including methicillin (SRM), has increased significantly in recent years and is a clinical challenge. and global epidemiology, especially in hospitals.

With a view to treatment but also to preventing staphylococcal infection, there is a need in both humans and animals to have new classes of antistaphylococcal agents as alternatives to antibiotics. The inventors have now isolated polynucleotides which make it possible to solve all or part of the problems mentioned above. These polynucleotides have the ability to negatively regulate the expression of the Sbi protein. The inventors have demonstrated that these polynucleotides hybridize specifically, without the aid of associated chaperone protein, with a particular sequence that they have located at the 5 'end of the messenger RNA (mRNA) encoding the Sbi protein. . By analyzing this particular sequence, the inventors have identified the "shine-Dalgarno" sequence predicted to interact with the 16S ribosomal RNA and the translation initiation codon of the mRNA encoding the Sbi protein. The inventors have shown that the specific hybridization of these polynucleotides with this particular sequence of the mRNA encoding the Sbi protein, prevents the binding of 7OS ribosomes to said mRNA.

The inventors have demonstrated that these polynucleotides make it possible to reduce the amount of Sbi proteins present in the intracellular medium but also the amount of Sbi proteins released into the extracellular medium and thus to reduce the ability of bacteria of the genus Staphylococcus to interfere with the system. immunity of the host by interacting with some of its components (IgG, C3 protein). These polynucleotides are therefore particularly useful for the prevention and / or treatment of staphylococcal infections, in particular antibiotic-resistant strains. In addition, the polynucleotides according to the invention have the advantage unlike putative treatments, to have a targeted action on bacteria of the genus Staphylococcus thus optimizing their action and limiting the risk of side effects. Thus, according to a first aspect, the subject of the invention is a first isolated polynucleotide comprising a sequence chosen from the group comprising: the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and fragments thereof; and the sequence ID No. 2, the derived sequences having at least 70% identity with the sequence SEQ ID No. 2 and the fragments thereof; and the sequence complementary to the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with said complementary sequence and fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID NO: 3.

For the purposes of the present invention, the term "polynucleotide" is intended to mean a polymer composed of several nucleotides linked by phosphodiester bonds, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). The polynucleotides according to the invention may be a DNA or an RNA. DNA has the advantage of being more stable than RNA.

The sequence ID No. 1 corresponds to the following sequence: embedded image in which the nucleotide at position 23 can be c or t; the nucleotide at position 24 may be c or t; and the nucleotide at position 36 may be a or t.

The term "a, t, c, g, u" is used here to denote, respectively, the nucleotides adenine, thymine, cytosine, guanine and uracil. In particular, the first isolated polynucleotide according to the invention comprises a sequence chosen from the group comprising:

the sequence SEQ ID No. 1, in which the nucleotides in positions 23, 24 and 36 are t;

the sequence SEQ ID No. 1, in which the nucleotides in positions 23 and 24 are c and the nucleotide in position 36 is t; and the sequence SEQ ID No. 1, in which the nucleotides in positions 23, 24 are c and the nucleotide in position 36 is a.

The sequence ID No. 2 corresponds to the following sequence: embedded image in which the nucleotide at position 23 can be c or u; the nucleotide at position 24 may be c or u; and the nucleotide at position 36 may be a or u.

In particular, the first isolated polynucleotide according to the invention comprises a sequence chosen from the group comprising: the sequence SEQ ID No. 6, the derived sequences having at least 70% identity with the sequence SEQ ID No. 6 and the fragments of these; and the sequence SEQ ID No. 7, the derived sequences having at least 70% identity with the sequence SEQ ID No. 7 and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID NO: 3. In particular, the first isolated polynucleotide according to the invention is an RNA comprising a sequence chosen from the group comprising:

the sequence SEQ ID No. 2, in which the nucleotides in positions 23, 24 and 36 are u;

the sequence SEQ ID No. 2, in which the nucleotides in positions 23 and 24 are c and the nucleotide in position 36 is u; and

the sequence SEQ ID No. 2, in which the nucleotides at positions 23, 24 are c and the nucleotide at position 36 is a; and

the sequence SEQ ID No. 7, in which the nucleotides at positions 44, 45 and 57 are u;

the sequence SEQ ID No. 7, in which the nucleotides at positions 44 and 45 are c and the nucleotide at position 57 is u; and

the sequence SEQ ID No. 7, in which the nucleotides at positions 44, 45 are c and the nucleotide at position 57 is a.

The first iso forms sequences SEQ ID No. 2 and SEQ ID No. 7 as defined above (the sequence SEQ ID No. 2, in which the nucleotides in positions 23, 24 and 36 are u and the sequence SEQ ID No. 7, in which the nucleotides at positions 44, 45 and 57 are u) has been identified in particular in S. aureus strain N315.

The second isoform of the sequences SEQ ID No. 2 and SEQ ID No. 7 as defined above

(The sequence SEQ ID No. 2, in which the nucleotides at positions 23 and 24 are c and the nucleotide at position 36 is u and the sequence SEQ ID No. 7, in which the nucleotides at positions 44 and 45 are c and the nucleotide at position 57 is u) has been identified in particular in S. aureus strains MRSA252, Newman.

The third isoform of the sequences SEQ ID No. 2 and SEQ ID No. 7 as defined above has been identified in particular in S. aureus strains NCTC8325, Mu50, Mu3, MSSA476, JH1, JH9, MW2, USA300 TCH1516, USA300. FPR3757. The sequence SEQ ID No. 3 corresponds to the following sequence:

5 '-gcauacaauaaauuuaauauguaaaaagaaagggaauacacaugaaaaauaaa-3'

The inventors have identified in this sequence SEQ ID No. 3, the first nucleotide g corresponding to the 5 'end of the mRNA encoding the Sbi protein, the nucleotides gaaaggg located at position 28 to 34 corresponding to the sequence "Shine- Dalgarno "predicts interaction with 16S ribosomal RNA and 42-44 positional nucleotides aug corresponding to the translation initiation codon. For the purposes of the present invention, the term "Sbi protein" is intended to mean the protein of sequence SEQ ID No. 5 and its homologous proteins. Here is meant by "homologous protein", the evolutionarily related proteins. The homologous proteins may have structural homology and / or homology of amino acid sequences between them. The amino acid sequences of the homologous proteins may have at least 50% identity with each other, preferably at least 70% identity and most preferably at least 90% identity. The homologous proteins of the Sbi protein may have an amino acid sequence having at least 50% identity, preferably at least 70% identity and most preferably at least 90% identity with the sequence SEQ ID No. 5 . In particular, homologous proteins of the Sbi protein have the ability to bind specifically to complement C3 protein and / or IgG immunoglobulins. Examples of proteins homologous to the Sbi protein include the Efb-C complement inhibitors (Hammel et al., Nat ImmunoL, 2007), Ehp (Hammel et al., J. Biol Chem., 2007). and SCIN (Rooijakkers et al, 2007). Three-dimensional structure studies by NMR (Nuclear Magnetic Resonance) spectroscopy have shown that the Sbi-IV domain of the Sbi protein of sequence SEQ ID No. 5 has a 3-helix beam conformation similar to that of the complement inhibitors Efb- C, Ehp and SCIN (Upadhyay et al, 2008).

Here is meant by "homologous sequence", the sequences related in evolution; the homologous sequences may have at least 50% identity between them, preferably at least 70% identity and most preferably at least 90% identity.

The term "bacteria of the genus Staphylococcus" in the sense of the present invention, the set of bacterial strains of the genus Staphylococcus.

In particular, the bacteria of the genus Staphylococcus according to the invention are chosen from the group comprising bacterial strains of the genus Staphylococcus whose genome contains: a gene encoding the Sbi protein of sequence SEQ ID NO: 5 or a homologous protein; and / or a gene encoding an RNA comprising the sequence SEQ ID No. 3 or a sequence homologous to the sequence SEQ ID No. 3 and the fragments thereof; and / or a gene comprising the sequence SEQ ID No. 4 or a sequence homologous to the sequence SEQ ID No. 4 and the fragments thereof.

These strains can be easily identified by those skilled in the art, for example, by screening techniques of libraries (genomic DNA or cDNA of bacteria of the genus Staphylococcus) using probes or primers specific to a sequence belonging to bacterial strains of the genus Staphylococcus as defined above. Such probes or primers can be selected from the polynucleotide sequences according to the invention. For example, a probe comprising a sequence as defined according to the first polynucleotide according to the invention may be used. In particular, those skilled in the art can use the method for detecting, in vitro, mRNA of the gene encoding the Sbi protein according to the invention.

In particular, the bacteria of the genus Staphylococcus according to the invention are strains of Staphylococcus aureus.

The term "sequence complementary to a second sequence" in the sense of the present invention, a sequence whose set of contiguous nucleotides have the ability to form Watson-Crick pairings of gc, cg, au, ua, a- t, ta and possibly gu-type wobble pairings with the set of contiguous nucleotides of the second sequence.

The term "identity" refers to the identity between two polynucleotide sequences. The identity can be determined by comparing the nucleotides present at one position in each of the polynucleotide sequences aligned for that purpose. When, at a given position, a nucleotide is identified as identical on the two aligned sequences, said sequences are said to be identical at this position. The percentage identity between two polynucleotide sequences is a function of the number of identical nucleotides at the different positions on the two aligned nucleotide sequences. Different programs can be used to align polynucleotide sequences and thus calculate the percentage identity between these sequences, such as FASTA or BLAST.

By "sequence derived from a second sequence" is meant a sequence having at least 70% identity, preferably at least 80% identity and most preferably at least 90% identity with a second sequence.

The fragments of sequences of the first polynucleotide according to the invention may have a size of between 35 and 53, preferably between 35 and 50, and most preferably between 36 and 45 nucleotides. Preferably, the fragments of sequences of the first polynucleotide according to the invention comprise the nucleotides located from position 14 to position 49 in the sequence SEQ ID No. 1 or the sequence SEQ ID No. 2, said nucleotides corresponding to the localized nucleotides of the position at position 70 in the sequence SEQ ID No. 6 or the sequence SEQ ID No. 7. The specific hybridization ability of a first polynucleotide sequence with a second polynucleotide sequence can be determined in vitro by tests well known to those skilled in the art. By way of example, mention may be made of the test described in C. Pichon & B. Felden

(2005, with the example of the sprA regulatory RNA and its SA2216 mRNA target), which comprises the following steps:

a first polynucleotide sequence is radioactively labeled, purified and increasing amounts of a second unlabeled polynucleotide sequence are added and analyzed by electrophoresis gel under native conditions. Obtaining a migration delay on this gel involves the formation of a complex between these two polynucleotide sequences, by hybridization;

the effect of adding an excess of 100 to 2000 times, in molar ratios, of a third polynucleotide sequence (for example a transfer RNA mixture) on the formation of said complex between the first two sequences , is analyzed;

the effect of adding an excess of 100 to 2000 times, in molar ratios, of the first unlabeled polynucleotide sequence on the formation of said complex between the first two sequences is analyzed;

the hybridization between the first two polynucleotide sequences will be considered specific if (i) said complex preformed between the first two polynucleotide sequences remains stable in the presence of this excess of the third polynucleotide sequence (ex: tRNA) and if (ii ) said preformed complex is destabilized in the presence of small amounts of the first unlabeled polynucleotide sequence.

Those skilled in the art can easily determine the derived sequences and the fragments which have the capacity to hybridize specifically to the sequence ID No. 3 according to the first isolated polynucleotide according to the invention, by implementing a specific hybridization test. as described above.

In particular, the derived sequences and fragments which have the capacity to hybridize specifically to the sequence ID No. 3 according to the first polynucleotide isolated according to the invention, also have the capacity to negatively regulate the expression of the Sbi protein. .

The term "reduction or downregulation of protein expression" is used herein to refer to a decrease of at least 20%, in particular at least 35% and most preferably at least 50% of the level. of expression of a protein with respect to its level of expression in the absence of a first polynucleotide according to the invention. The reduction of the expression of a protein can be determined by techniques well known to humans of the Trade such as "Western-Blot", using at least one antibody specific for said protein.

The present invention also encompasses the polynucleotides according to the invention, synthetic, semisynthetic, recombinant and their analogues. The analogs of the first polynucleotide according to the invention have the capacity to hybridize specifically with the sequence

SEQ ID NO: 3. In particular, the analogs of the first polynucleotide according to the invention also have the capacity to negatively regulate the expression of the Sbi protein.

For the purposes of the present invention, the term "analogue" means polynucleotides comprising at least one chemical modification at the level of their sugar, phosphate, their base or their 5 'and 3' ends; said modification can be carried out by chemical or enzymatic synthesis.

Indeed, the polynucleotides according to the invention may comprise at least one chemical modification intended in particular to increase their stability, their resistance to degradation by endo and exonucleases, to promote their ability to enter a cell of interest and / or the case of the first polynucleotide according to the invention to increase its capacity to negatively regulate the expression of the protein Sbi. The increase of the capacity of the first polynucleotide according to the invention to negatively regulate the expression of the protein Sbi can be obtained, for example, by a chemical modification allowing the increase of the formation and / or the stability of a protein. complex between the first polynucleotide according to the invention and the sequence SEQ ID No. 3, by specific hybridization. Thus, the polynucleotides according to the invention may comprise modified nucleotides in terms of their sugar, phosphate, base and / or may have modifications, particularly post-transcriptionally, at their 5 'and 3' ends. Such modified nucleotides may be chosen from the group comprising: 2'-O-methylnucleotides, 2'-O-methoxyethylnucleotides, deoxynucleotides such as 2'-deoxynucleotides and 2'-deoxy-2'-fluoronucleotides, nucleotides with peptide bonds, fluorochromes and radioactive elements.

The polynucleotides according to the invention may also comprise, at their 5 'and / or 3' ends, structural elements (such as stable helices) reinforcing their stability and / or their resistance to degradation. The polynucleotides according to the invention can be obtained by methods well known to those skilled in the art, such as by screening methods of libraries (genomic DNA or cDNA of bacteria of the genus Staphylococcus) by probes or primers. specific of said polynucleotides (selected for example from the polynucleotide sequences according to the invention), but also by chemical syntheses (Beaucage SL., 2008) or by enzymatic reactions (Sherlin LD et al., 2001).

The subject of the invention is also a first expression cassette comprising a DNA sequence chosen from the group comprising: the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and fragments thereof; and the sequence SEQ ID No. 6, the derived sequences having at least 70% identity with the sequence SEQ ID No. 6 and the fragments thereof; and a DNA sequence that can be transcribed into a first polynucleotide isolated according to the invention, said polynucleotide being an RNA.

For the purposes of the present invention, the term "expression cassette" means a cassette comprising a promoter and a transcription termination region in which a DNA sequence of interest is operably linked to the promoter in order to allow the transcription of said DNA sequence. It can be any promoter or derived sequence, inducible or not, allowing the transcription of a polynucleotide according to the invention. In particular, the promoter is chosen from promoters allowing the transcription of a polynucleotide according to the invention in at least one bacterial strain of the genus Staphylococcus. By way of example of such promoters, mention may be made of the bacteriophage T7 promoter (Milligan JF et al., 1989). The subject of the invention is also a first recombinant vector comprising a first isolated polynucleotide according to the invention or a first expression cassette according to the invention.

The vectors that may be used may be viral vectors, such as bacteriophages, or non-viral vectors, such as plasmid vectors. As examples of plasmid vectors that can be used according to the invention, mention may be made of all the pCN-derived vectors described in Charpentier et al. (2004). Some of these vectors, for example pCN51, allow the transcription of a DNA sequence of interest into RNA through the use of a cadmium inducible promoter.

In particular, the plasmid vector may be chosen from the group comprising the plasmids pCN35 and pCN38 described in Charpentier et al. (2004). These plasmids have the advantage of being maintained and thus allow the expression of polynucleotides according to the invention in transformed bacteria of the genus Staphylococcus. Indeed, the inventors have shown that S. aureus bacteria, transformed with the vector pCN38 comprising the sequence SEQ ID No. 6 according to the invention, are capable of maintaining this plasmid during successive bacterial generations after transplanting and thus to allow the expression of a polynucleotide according to the invention. In particular, the vector according to the invention is a bacteriophage.

Preferably, said bacteriophage is capable of infecting at least one bacterial strain of the genus Staphylococcus. The term "bacteriophage capable of infecting a bacterial strain of the genus Staphylococcus" means any bacterial virus capable of introducing part of its genetic material into the genome of a bacterial strain of the genus Staphylococcus (prophage) or to allow transitory expression of this genetic material in the recipient bacterium without chromosomal integration.

Bacteria of the genus Staphylococcus are infected by three types of phages: obligatory, temperate and chronic lyrical (Nicholas H. Mann, 2008). All of these phages can be used as a vector according to the invention. In the context of the prevention and / or treatment of staphylococcal infection, the phages that can be used as vectors according to the invention are preferably broad-spectrum and capable of infecting as many as possible bacterial strains of the genus Staphylococcus responsible for infections in humans and / or animals. By way of example of such bacteriophages, mention may be made of the obligate lytic phage φ812, phages K and φMR1. The use of bacteriophage as a vector according to the invention has the advantage of optimizing the prevention and / or the treatment. Staphylococcal infection or a disorder related to at least one bacterium of the genus Staphylococcus. Indeed, in addition to the negative regulatory effect of the first polynucleotide according to the invention on the expression of the Sbi protein in vitro and in vivo, the bacteriophage makes it possible to lyse the bacteria during its lytic cycle. In addition, the use of bacteriophage as a vector according to the invention has the advantage that the bacteriophage used is potentially effective against various bacterial strains of the genus Staphylococcus, even those resistant to antibiotics (Nicholas H. Mann, 2008) and not have side effects, including being devoid of adverse effects on the eukaryotic cells of the infected host. According to another aspect, the subject of the invention is a first host cell transformed by a first expression cassette according to the invention or a first vector according to the invention. The host cell may be in particular a prokaryote and advantageously a bacteriophage as described above. The subject of the invention is also a composition comprising at least one component selected from the group comprising a first isolated polynucleotide according to the invention, a first expression cassette according to the invention, a first vector according to the invention and a first cell. host according to the invention. Said composition may be useful in particular as a tool in biology in order, for example, to select probes or primers specific for the polynucleotides according to the invention or else to identify potential agents capable of modulating (activating or inhibiting) the interaction between the first and the second or third polynucleotides according to the invention.

Advantageously, the composition according to the invention is a pharmaceutical composition. The pharmaceutical compositions according to the invention have the advantage unlike putative drugs to have a targeted action on bacteria of the genus Staphylococcus, including strains resistant to antibiotics, optimizing their action and limiting the risk of side effects.

In particular, said components are present in the pharmaceutical composition according to the invention in therapeutic amounts (active and non-toxic amounts). Such therapeutic amounts may be determined by those skilled in the art by routine tests comprising the evaluation of the effect of the administration of said components mentioned above on pathologies and / or disorders that one seeks to prevent and / or to be treated by the administration of said pharmaceutical composition according to the invention, for example a staphylococcal infection and on toxicity. For example, such tests can be implemented by quantitatively and qualitatively analyzing the effect of the administration of different amounts of said aforementioned components on a set of markers (biological and / or clinical) characteristic of these pathologies and / or of these disorders, as well as by bacterial counting in samples of biological samples. In addition, the therapeutic index of the pharmaceutical composition according to the invention can be determined by tests well known to those skilled in the art.

The polynucleotides according to the invention can be administered directly, preferably using a suitable vehicle to facilitate their entry into the cell of interest and / or protect them from degradation by endo- or exo-nucleases . Examples of such usable vehicles include microspheres, liposomes, nanoparticles.

The polynucleotides according to the invention may also be administered via a vector such as advantageously a bacteriophage as described above. The pharmaceutical compositions according to the invention may be administered by different routes compatible with the desired effect and preferably orally, parenterally, subcutaneously, topically, intraocularly, sublingually and most preferably intraocularly.

The pharmaceutical compositions according to the invention can be administered in one or more times or in continuous release.

The pharmaceutical compositions according to the invention can be in various forms well known to those skilled in the art and adapted to the routes and modes of administration used. In particular, when the pharmaceutical compositions are administered to animals, they may be included in food compositions (for example in the form of powders or granules).

According to another aspect, the subject of the invention is also a first isolated polynucleotide according to the invention, or a first expression cassette according to the invention, or a first vector according to the invention or a first host cell according to the invention. for its application as a medicament for the prevention and / or treatment of a staphylococcal infection. The invention also relates to the use of a component selected from the group comprising a first isolated polynucleotide according to the invention, a first expression cassette according to the invention, a first vector according to the invention, a first cell. host according to the invention, for the preparation of a medicament for the prevention and / or treatment of a staphylococcal infection. Staphylococcal infections include all infections that cause bacteria of the genus Staphylococcus. These infections include all pathologies and / or disorders that can be improved and / or avoided by reducing the expression of the Sbi protein.

In particular, staphylococcal infections include Staphylococcus aureus infections including superficial or deep folliculitis.

(such as boils, anthrax and staphylococcal sycosis), staphylococcal syndrome of scalded children, wound infections, boils, bacteraemia, endocarditis, osteomyelitis or septic arthritis.

According to another aspect, the subject of the invention is also a product containing: at least one component selected from the group comprising a first isolated polynucleotide according to the invention, a first expression cassette according to the invention, a first vector according to the invention, a first host cell according to the invention; and at least one other antistaphylococcal agent, as a combination product for simultaneous, separate and / or extended use for the prevention and / or treatment of a staphylococcal infection.

The term "antistaphylococcal agent" in the sense of the present invention, an agent for controlling at least one bacterial strain of the genus Staphylococcus, in particular against at least one bacterial strain of the genus Staphylococcus selected from the group comprising bacterial strains of the genus Staphylococcus whose genome contains: a gene coding for the "Sbi" protein of sequence SEQ ID No. 5 or a homologous protein; and / or a gene encoding an RNA comprising the sequence SEQ ID No. 3 or a derived sequence having at least 70% identity with the sequence SEQ ID No. 3 and the fragments thereof; and / or a gene comprising the sequence SEQ ID No. 4 or a derived sequence having at least 70% identity with the sequence SEQ ID No. 4 and the fragments thereof; and most particularly against at least one strain of Staphylococcus aureus.

The antistaphylococcal agent may be selected from the group consisting of: antibiotics for preventing and / or treating local infections such as fusidic acid, mupirocin; antibiotics for preventing and / or treating systemic infections such as rifampicin, fluoroquinolones, fusidic acid, trimethoprim-sulfamethoxazole, glycopeptides; and bacteriophages such as φ812, K and φMR1 1.

The combination products according to the invention comprising another antistaphylococcal agent have the advantages of optimizing the prevention and / or treatment of a staphylococcal infection and of being able to reduce the therapeutic amount of each component.

According to another aspect, the subject of the invention is a second isolated polynucleotide comprising a sequence chosen from the group comprising: the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with the sequence SEQ ID No. 3 and fragments thereof; and the sequence SEQ ID No. 4, the derived sequences having at least 70% identity with the sequence SEQ ID No. 4 and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2.

In particular, the second isolated polynucleotide according to the invention comprises a sequence chosen from the group comprising: the sequence SEQ ID No. 8, the sequences derived having at least 70% identity with the sequence SEQ ID No. 8 and the fragments of these; and the sequence SEQ ID No. 9, the derived sequences having at least 70% identity with the sequence SEQ ID No. 9 and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2.

In particular, said derived sequences and said fragments having the capacity to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2, according to the second polynucleotide according to the invention, comprise the nucleotides gaaaggg located at position 28 to 34 (corresponding to the sequence "Shine-Dalgarno") and nucleotides aug located in position 42 to 44 (corresponding to the translation initiation codon of the mRNA encoding the protein Sbi) in the sequence SEQ ID No. 3 or SEQ ID No. 4.

The fragments of sequences of the second polynucleotide according to the invention may have a size of between 14 and 50, preferably between 20 and 45 and most preferably between 41 and

45. Preferably, the fragments of sequences of the second polynucleotide according to the invention comprise the 41 nucleotides located from position 1 to position 41 in the sequence SEQ

ID No. 3 or the sequence SEQ ID No. 4.

The analogues of the second polynucleotide according to the invention have the capacity to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2.

The subject of the invention is also a third isolated polynucleotide, comprising: a sequence containing nucleotides 1 to 41 of one of the sequences SEQ ID No. 3 or

SEQ ID NO: 4, coupled at its 3 'end to a coding sequence of a gene of interest.

The term "coupled sequences" in the sense of the present invention, the binding of two sequences by phosphodiester bonds. The binding can be carried out by chemical or enzymatic synthesis or by PCR construction (polymerase chain reaction). In particular, the third polynucleotide according to the invention comprises a coding sequence of a gene of interest coupled at its 5 'end to the 3' end of a sequence containing nucleotides 1 to 41 of one of the SEQ sequences. ID No. 3 or SEQ ID No. 4. The term "a sequence containing nucleotides 1 to 41 of one of the sequences SEQ ID No. 3 or SEQ ID No. 4" within the meaning of the present invention, a sequence containing the 41 nucleotides located from position 1 to the position 41 in the sequence SEQ ID No. 3 or the sequence SEQ ID No. 4. For the purposes of the present invention, the term "coding sequence" is intended to mean a nucleotide sequence which directly defines the amino acid sequence of a protein or fragment of the corresponding protein encoded by a gene of interest. A coding sequence of a gene of interest notably comprises the codons for initiation and termination of the translation of the mRNA of this gene into protein. Preferably, in the third polynucleotide according to the invention, the 5 'end of the translation initiation codon of the mRNA of the gene of interest is directly linked by phosphodiester bond to nucleotide c located at position 41 in the one of the sequences SEQ ID No. 3 or SEQ ID No. 4. Thus, in the case where the translation initiation codon of the mRNA of the gene of interest is aug, nucleotide a of this initiation codon is linked to nucleotide c at position 41 of one of the SEQ sequences. ID No. 3 or SEQ ID No. 4 to form the sequence of nucleotides caug.

The gene of interest may be any gene which encodes a protein or a protein fragment of interest whose expression it is desired to regulate, in particular negatively by the presence of a first polynucleotide according to the invention. The expression of the protein or protein fragment of interest encoded by the gene of interest according to the third polynucleotide according to the invention can thus be negatively regulated by the presence of a first polynucleotide according to the invention.

In particular, a coding sequence of a gene of interest may be a sequence coding for at least one domain of the Sbi protein, said domain being capable of binding specifically to the complement C3 protein and / or to IgG immunoglobulins.

The third polynucleotide according to the invention may further comprise at least one non-coding sequence of said gene of interest.

The subject of the invention is also a second expression cassette comprising a DNA sequence chosen from the group comprising: the sequence SEQ ID No. 4, the derived sequences having at least 70% identity with the sequence SEQ ID No. 4 and fragments thereof; and a DNA sequence that can be transcribed into a second or third polynucleotide according to the invention, said polynucleotide being an RNA.

The subject of the invention is also a second recombinant vector comprising a second expression cassette according to the invention. The subject of the invention is also a second host cell according to the invention transformed by a second expression cassette according to the invention or a second vector according to the invention.

The subject of the present invention is also a pharmaceutical composition according to the invention further comprising: a third isolated polynucleotide according to the invention in which said coding sequence is a sequence encoding at least one domain of the Sbi protein, and / or

an expression vector comprising a third isolated polynucleotide according to the invention wherein said coding sequence is a sequence coding for at least one domain of the Sbi protein, said domain of the Sbi protein being capable of binding specifically to the complement C3 protein and / or IgG immunoglobulins.

The term "domain of the Sbi protein which is capable of binding specifically to the complement C3 protein" refers to the Sbi-IV domain as described in Upadhyay et al. (JBC, June 11, 2008), namely the domain including amino acids 198 to 266 of the sequence SEQ ID No. 5, domains derived from the Sbi-IV domain, and fragments thereof. Said domains derived from the Sbi-IV domain, and said fragments having the capacity to bind specifically to the complement C3 protein.

The term "domain of the Sbi protein which is capable of binding specifically to IgG immunoglobulins" means the Sbi-I and Sbi-II domains as described in Upadhyay et al. (JBC, June 11, 2008), namely the Sbi-I domain including amino acids 42 to 94 of the sequence SEQ ID No. 5, the Sbi-II domain incorporating amino acids 92 to 156 of the sequence SEQ ID No. 4, domains derived from the Sbi-I and Sbi-II domains, and fragments thereof. Said domains derived from the Sbi-I and Sbi-II domains, and said fragments having the capacity to bind specifically to IgG immunoglobulins. Such pharmaceutical compositions according to the invention have the advantage of allowing the modulation of the immune system of the host, especially over time. Indeed, the components of such pharmaceutical compositions can be administered in a simultaneous, separated and / or spread over time to modulate the immune system. Such pharmaceutical compositions may make it possible to vary the expression of the Sbi protein as a function of the quantity of the components (first and third polynucleotides according to the invention) and / or as a function of time and thus to vary the response of the immune system. adaptive and / or innate host (IgG, C3 protein).

The present invention also relates to a method for detecting, in vitro, the presence of bacteria of the genus Staphylococcus in a biological sample, comprising the detection of at least one sequence chosen from the group comprising:

the sequences as defined according to the first polynucleotide according to the invention; and the sequences as defined according to the second polynucleotide according to the invention; with at least one appropriate detection means of said sequence.

Said means for detecting an appropriate sequence may be any pair of specific primers for the amplification of said sequence (for example by PCR or RT-PCR), or any specific probe of said sequence. The sequences of the polynucleotides according to the invention may be useful for selecting such primers and probes.

In particular, the sequences as defined according to the first polynucleotide according to the invention may be useful for selecting probes allowing the detection of sequences as defined according to the second polynucleotide according to the invention and vice versa. Thus, according to a particular embodiment of said method of detecting, in vitro, the presence of bacteria of the genus Staphylococcus in a biological sample, the detection of said sequence can comprise the following steps:

1. bringing said biological sample into contact with at least one probe comprising a sequence chosen from the group comprising: the sequences as defined according to the first polynucleotide according to the invention:

the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and the fragments thereof; and

the sequence ID No. 2, the derived sequences having at least 70% identity with the sequence SEQ ID No. 2 and the fragments thereof; and the sequence complementary to the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with said complementary sequence and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID NO: 3; and the sequences as defined according to the second polynucleotide according to the invention:

the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with the sequence SEQ ID No. 3 and the fragments thereof; and

the sequence SEQ ID No. 4, the derived sequences having at least 70% identity with the sequence SEQ ID No. 4 and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2; and

2. detecting the hybridization of said probe with said sequence with at least one means of appropriate detection of said hybridization. According to a particular embodiment of said method of detecting, in vitro, the presence of bacteria of the genus Staphylococcus in a biological sample, the detection of said sequence can comprise the following steps:

1. bringing said biological sample into contact with at least one pair of primers specific for said sequence under conditions allowing hybridization of the primers to said sequence;

2. the amplification of said sequence; and

3. highlighting the amplification of fragments of said sequence corresponding to the fragment flanked by the primers including these.

The primers specific for the sequences as defined according to the first or second polynucleotide according to the invention may be selected by those skilled in the art by routine tests from said sequences.

The subject of the present invention is also a method for detecting, in vitro, mRNA of the gene encoding the Sbi protein present in a biological sample, comprising the detection of the sequence SEQ ID No. 3, with at least one appropriate detection means. of said sequence. The method of in vitro detection of mRNA of the gene encoding the Sbi protein present in a biological sample according to the invention has the advantage over a detection method of the DNA encoding the Sbi protein to allow the detection of active infections Staphylococcal. Indeed, it has been shown that the expression of the Sbi protein is induced by the presence of human IgG (Zhang et al, 2000).

The sequences as defined according to the first polynucleotide according to the invention can be used as a probe to detect, in vitro, specifically and directly the mRNA of the gene encoding the Sbi protein present in a biological sample.

Thus, in another aspect, the subject of the invention is a method for detecting, in vitro, mRNA of the gene encoding the Sbi protein present in a biological sample, comprising the steps of: bringing said biological sample into contact with at least one probe comprising a sequence selected from the group consisting of:

the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and the fragments thereof; and

the sequence ID No. 2, the derived sequences having at least 70% identity with the sequence SEQ ID No. 2 and the fragments thereof; and the sequence complementary to the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with said complementary sequence and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID NO: 3; and detecting the hybridization of said probe with said mRNA of the gene encoding the Sbi protein with at least one means for detecting said appropriate hybridization.

The detection method according to the invention may further comprise, prior to the contacting step, at least one of the following steps: culturing of the biological sample, optionally after isolation of bacteria of the genus Staphylococcus on a medium selective, under appropriate conditions allowing the culture of bacteria of the genus Staphylococcus; centrifugation of the biological sample; lysis of bacteria of the genus Staphylococcus; extracting the polynucleotides present in said sample. The present invention also relates to a mRNA detection kit of the gene encoding the Sbi protein comprising: at least one probe comprising a sequence selected from the group consisting of:

the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and the fragments thereof; and

the sequence ID No. 2, the derived sequences having at least 70% identity with the sequence SEQ ID No. 2 and the fragments thereof; and

the sequence complementary to the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with said complementary sequence and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID NO: 3; and

at least one means for detecting the hybridization of said probe with said mRNA of the gene encoding the Sbi protein.

Said means of appropriate detection of the hybridization of said probe with a sequence, in particular with said mRNA of the gene encoding the Sbi protein, may be any means allowing the detection of a specific hybridization between two polynucleotides.

In particular, said detection means may be based on the marking of the probe. Unmarked sequences can be used directly as probes. However, the sequences can be labeled directly or indirectly by a radioactive element ( ³² P, ³⁵ S) or by a non-radioactive molecule (biotin, fluorophore) to obtain probes that can be used in many applications, in particular in detection methods as described above.

The probes can be in solution or immobilized on a support.

The sequence SEQ ID No. 3 can also be used to select oligonucleotide primers, in particular for PCR (from genomic DNA) and RT-PCR (from total RNA extract) techniques. These oligonucleotides may be used to detect, in vitro, specifically and in a direct manner, the mRNA of the gene encoding the Sbi protein present in a biological sample.

According to another aspect, the subject of the invention is a kit for regulating the expression of a gene of interest comprising: at least one first component selected from the group comprising a second or third isolated polynucleotide according to the invention, a second expression cassette, a second vector and a second host cell according to the invention; and at least one second component chosen from the group comprising a first polynucleotide according to the invention, a first expression cassette, a first vector and a first host cell according to the invention.

The subject of the invention is also the use, in particular non-therapeutic, of a first isolated polynucleotide according to the invention for inhibiting the expression of the gene encoding the Sbi protein, in particular for inhibiting the translation of the mRNA encoding the protein. Sbi.

The subject of the invention is also the use, in particular non-therapeutic, of a first polynucleotide isolated according to the invention in combination with a second and / or third polynucleotide isolated according to the invention for inhibiting the expression of a gene. of interest, especially for inhibiting the translation of an mRNA encoding a protein of interest.

Other advantages and features of the invention will become apparent from the following examples.

These examples are given for illustrative and not limiting.

For the purposes of the first invention, the term "SprD" means a first polynucleotide according to the invention corresponding to the second iso form of the sequence SEQ ID No. 7 (namely the sequence SEQ ID No. 7, in which the nucleotides in positions 44, 45 and 57 are u). SprD has been identified in particular in S. aureus strain N315.

Figure 1 illustrates the solution sprD structure and its cell location. (AC): Structural survey of sprD. Autoradiograms of 5 'lead labeled synthetic 5' dG cleavage products and S1 and Vl nucleases after long ( _λ4 ) and short (B) electrophoretic migrations. Lanes C, incubation controls; GL tracks, bands obtained by RNase Tl hydrolysis; AL tracks, bands obtained by RNAse U2 hydrolysis; AH tracks, bands obtained by alkaline hydrolysis. The sprD sequence is indexed at the right sides. (Q Secondary structure model of sprD of S. aureus strain N315, based on structural mapping, showing sounding data, triangles correspond to cuts by Vl, arrows with a circle are cuts by Sl the uncapped arrows are cut by lead acetate The intensity of the cuts and cleavages is proportional to the coloring density of the symbols: an empty symbol (without coloration), grayed out and full corresponds to a weak cut, medium and strong, respectively The structural domains H1-H4 and L1-L4 are indicated The framed nucleotide corresponds to the experimentally determined 5 'end (RACE) as well as to the transcription initiation site. by the two black hooks were deleted in the sprDΔ mutant. (D): Cell Location of SprD, tRNA, and RNAIII in vivo, by Northern

Blots of different cell fractions using labeled strand specific DNA probes that are specific for each RNA. The cytoplasmic fraction (C) contains the tRNAs, the ribosomal fraction (R) contains the three ribosomal RNAs. The membrane fraction (M) contains the ErbS and ATPase proteins, according to immunoblot immunoblot analysis of the immunoblotted C, R, and M cell fractions with anti-ErbS and anti-ATPase antibodies.

Figure 2 illustrates the regulation of in vivo expression of SprD. Northern blot analysis of SprD expression, using a labeled strand-specific DNA probe, in RN4220 and SH1000 bacterial strains, both lacking the sprD gene, transformed with the pCN38 or pCN51 vectors allowing the expression of DPRS. SprD expression in strain N315 is shown as a positive control. As loading controls, the blots were also probed for 5S rRNA.

Figure 3 illustrates the downregulation of sbi protein levels in vivo by sprD. ( _^ 4). SDS-PAGE analysis of exoproteins in RN4220 and SH1000 wild type strains and their isogenic sprD mutants prepared from the exponential growth phase (E). The SDS-PAGE gel was stained with Coomassie blue. The arrow highlights the reduced protein levels when sprD is expressed, at a molecular weight of about 45 kD, identified by mass spectrometry as the sbi protein, based on independent peptide sequences. Western immunoblot analysis of the extracellular (B) and intracellular (Q) fractions from the RN4220 and SH1000 bacterial strains expressing, or not, both the exponential (E) and stationary (S) phases, 50 μg of protein from the cell fractions were loaded on 15% SDS-PAGE, transferred to a polyvinylidene difluoride membrane and immunoblotted (immunoblotted) with anti-sbi antibody, followed by chemiluminescence detection.

Figure 4 illustrates that SprD is not involved in the induction of sbi protein levels by human serum. _(^ 4) Analysis by immunoblotting (Western Immunoblot) extracellular fractions from bacterial strains MRSA252 SH1000 and cultured in the presence or absence of human serum. The strain SH1000 expresses sprD (+), or not (-). Expression levels of protein A in each band are provided as an internal loading control. (B) Northern blot analysis of SprD expression using a labeled strand specific DNA probe. As loading controls, blots were also probed for 5S rRNA.

Figure 5 illustrates the formation of a complex between SprD and the mRNA encoding the Sbi protein. (A) The predicted pairing between sprD and sbi mRNA, based on (i) a prediction (ii) a mutational and delay analysis on native gel; (iii) a structural survey of sprD within the duplex. Structural domains from sprD that are unfolded during complex formation are indicated. The signs (-) and (+) correspond to nucleotides from sprD that are protected (-) or become cleaved (+) by Sl or Vl ribonucleases when binding to the mRNA encoding the Sbi protein. The black rectangle shows the 5 'end of the sbi mRNA as experimentally determined by RACE. The framed heptanucleotide sequence (GAAAGGG) is the predicted RBS (Ribosome Binding site) and the framed AUG is the initiation codon for translating sbi mRNA into protein. (B) Non-denaturing gel retardation assays of purified labeled RNA corresponding to 179 (sbi mRNA) or 118 (sbi) Δ61 nucleotides at its 5 'end, both with increasing amounts of purified unlabeled gDG, or with a sprD mutant lacking nucleotides 35-70 (sprD Δ), or with an excess of 100-2000 folds of purified non-labeled total RNA from yeast.

Figure 6 shows the mapping of the conformational change induced by the formation of a complex between sbi mRNA and sprD by structural probes. The autoradiograms of 5 'labeled 5' -dispD cleavage products by the ribonucleases Sl and Vl, in the presence (+) or absence (-) of an 8-fold molar excess of sbi mRNA derived from short electrophoretic migrations (left) and long (right). Lanes C, incubation controls; GL tracks, bands obtained by RNase Tl hydrolysis; AL tracks, bands obtained by RNase U2 hydrolysis; the sequence is indexed on the right sides. The area of matching between the two ARNs is indicated, based on mutational and survey data.

Figure 7 illustrates the inhibition by sprD of translation initiation of sbi mRNA via 7OS ribosomes. The localization by the toeprinting technique of the ribosome on sbi mRNA as described in the Materials and Methods section, hereinafter. +/- indicates the presence of ribosomes, wild type sprDs (lanes 2, 5, 6 and 7) or sprD-Δ (lanes 3, 8, 9 and 10). The concentrations of sprD and sprD-Δ were 0.4 μM (bands 5 and 8), 2 μM (bands 6 and 9) and 10 μM (bands 7 and 10). The "toeprints" (forward impressions) determined experimentally are indicated. U, A, G and C refer to sequencing scales of sbi mRNA.

EXAMPLES

I. Materials and methods

1.1 Strains and Plasmids

The strains of S. aureus and the plasmids used in this study are listed in Table 1. Strains of S. aureus were grown in the usual way at 37 ^° C. in a broth of infusion of brain and heart (BHI, Oxoid). If necessary, the inventors used chloramphenicol and erythromycin at a rate of 10 μg / ml. Strain DH5α ά'E. CoIi was used as a bacterial host for the construction of the plasmid. Strains of E. CoIi were grown in a Luria-Bertani (LB) medium. In plasmid pCN38-SprD, the gene encoding SprD is expressed under the control of its own promoter. To construct this plasmid, sprD with 40 nucleotides upstream and 35 nucleotides downstream was amplified by PCR from genomic RNA of S. aureus strain N315 as a fragment of 217 base pairs with Pst I and EcoR I flanking restriction. The PCR product was cloned between these two sites in the low copy number vector pCN38 (Charpentier et al, 2004).

E. coli DH5α Sambrook et al. 1989

S. aureus

RN4220 Restricted Derivative Derivative of 8325-4 Kreiswirth et al. 1983

SHl 000 8325-4 rsbU rsbU functional derivative Horsburgh et al. 2002

Plasmids pCN51 Charpentier et al. 2004 pCN38 Charpentier et al. 2004 pCN51-sprD This study pCN38-sprD This study Table 1

1.2 Cell splitting

Cell fractions of S. aureus strain N315 were prepared as described (Chesneau et al., 2005) with some modifications. 300 to 400 ml of bacterial cultures were grown to the exponential phase (OD 600 = 2.0). The protoplasts were collected by lysostaphin lysing of the cell walls (100 μl of stock solution 1 mg / ml) in 10 ml of Tris-HCl pH = 7.5 with 30% sucrose. Then, the protoplasts were lysed by osmotic shock in 25mM HEPES pH 7.6, 10mM MgCl _2, 3mM NH ₄ Cl, 4mM betamercaptoethanol and 10mM Vanadyl ribonucleoside complex (BioLabs). Centrifugations were performed at 30,000 g for 30 minutes (P30: membrane fraction) and 100,000 g for 3 hours (PlOO: ribosomal fraction, S100: cytoplasmic fraction).

1.3 Protein isolation, mass spectrometry and western immunoblots

For the preparation of the total protein extracts and the extracellular extracts, the bacteria were cultured until the exponential (OD600 = 2.0) or stationary (OD600 = 5.0) growth phases and the cells were separated from the supernatant by centrifugation for 10 minutes at ^{40 °} C. (8000 g). For purification of the extracellular proteins, the supernatants were collected, filtered (0.45 μm sterilized filter) and precipitated with 10% trichloroacetic acid. The precipitates were washed with ice-cold acetone and loaded onto SDS-PAGE according to the Laemmli method, 1970. For total protein extractions, 2 ml pellets of cultures were washed with TE (50 mM EDTA, 50 mM Tris pH 7.5), and resuspended in 0.2 ml. ml of the same buffer containing 0.1 mg / ml of lysostaphin. After incubation at 37 ^° C. for 10 minutes, the samples were boiled for 5 minutes in sample buffer, analyzed by SDS-PAGE and stained with Coomassie blue R-250. The proteins of interest were extracted by SDS-PAGE ID, digested with trypsin and the resulting peptides were identified by Maldi-Toff mass spectrometry and RP-HPLC / NanoLC / ESI-MS-MS. For immunoblot analyzes, the proteins were transferred to a PVDF membrane (Immobilon-P, Millipore). Western blots analyzes were performed using a polyclonal anti-Sbi antibody (donated by Dr. van den Elsen, University of Bath, UK) and also anti-EbpS and anti-ATP-ase antibodies, as described in Downer et al. ., (2002). The signals were visualized using a STORM 840 Phosphor-Imager (Molecular Dynamics) imager and quantified using Image-QuantNT 5.2 software.

1.4 Isolation of V RNA and Northern blots

Total RNAs were prepared from the exponential (OD600 = 2.0) or stationary (OD600 = 5.0) growth phases of various S. aureus strains as described in Oh and So (2003). For 5S RNAs and other RNAs, Northern blots were run with 5 μg of denaturing PAGE denaturing gel-denatured total RNA as described in Pichon and Felden (2005). For sbi mRNA, Northern blots were performed as described in McCallum et al. (2006).

1.5 '-RACE Assay, In Vitro Transcription and RNA 5' End Marking 5'-RACE assays were performed according to the method of Antal et al, 2005 with the primers described in Table 2 Wild-type and mutant RNAs for structural probing, gel retardation assays and toeprinting experiments were transcribed from PCR fragments generated from genomic DNA with the primers described in Table 2. All fragments contain a T7 RNA polymerase promoter sequence. To produce the DNA template encoding the sprD RNA deletion mutant, sprDΔ, mutagenized oligonucleotides (Table 2) were used in a two-step protocol. First, "sprDforTR" and "T7sprD_delrev" DNAs produced an "upstream" fragment by PCR and both "sprDrevTR" and "T7sprD_delfor" DNAs generated a "downstream" DNA fragment. The upstream and downstream fragments overlap partially. The mixture of the two purified fragments by PCR (200 ng) was used as a template in a third PCR reaction using the two DNA oligonucleotides "sprDforTR" and "sprDrevTR" to produce a fragment coding for sprDΔ. All RNAs were produced by in vitro transcription using MEGAscript (Ambion). Radiolabeled RNA transcripts were produced by adding [α ³² -P] UTP to the transcription reaction mixture. RNA labeling at their 5 'ends was performed as described in Antal et al. (2005). The labeled and unlabeled RNAs were purified on 8% PAGE gel, eluted, precipitated with ethanol and stored at -80 ^° C. before use.

DNAs Sequences Uses

Antil640 ggcgctccttgaaaacgccc sprD northern / 5 'RACE

Antisprd3 gcccgaaaaggagcaatacat sprD 5 'RACE

Sbineg5 tcactcgcttttgcttcccca sbi 5 'RACE

Sbineg3 tcgcgtaatgttttgatgtattgg

SprD5PstI ttaaatçtgçagagataatgcaatagtagccat pCN38-sprD

SprD3 EcoRI taaattggaattççgacataccttattgtatttatag sprDforTR taatacgactcactataggggcgttttcaaggagcgcc sprD mRNA sprDrevTR aaggtaagcaccgaaatgcttac transcription

T7sprD_delfor gcgcctttcattttttatgtttgcgctttccaaatcaa

T7sprD_delrev ttgatttggaaagcgcaaacataaaaaatgaaaggcgc

SBIforTR taatacgactcactataggcatacaataaatttaatatgtaa Sbi mRNA

SBIrevTR gttgttttgagttgtttggtgct transcription

SBIdeltaTR taatacgactcactataggcgaagttgctagttggggcagca

Table 2

1.6 Gel delay tests and probe of RNA structure in solution

Gel delay tests between SprD and sbi mRNA were performed as described in Antal et al. (2005). From the gel retardation assays, 0.4 pmole of 5 'labeled or truncated wild-type sbi mRNA (SbiΔβ1) were incubated with various concentrations (from 1.6 to 20 pmol) of untagged or truncated wild type (sprDΔ). For structural analysis in solution, RNA duplexes between sbi mRNA and sprD were prepared by incubating 0.4 pmol of labeled sprD and 1.6 pmol of sbi mRNA in a buffer containing 10 mM Tris-HCl (pH 7.5), 6 mM NaCl, 10 mM EDTA and 5 mM dithiothreitol for 15 minutes at 25 ^° C. Structural analyzes of a 5'-end labeled sprD renatured, either alone or in a complex with its target, sbi mRNA, were performed as described in Antal et al. (2005). The digestions were carried out at 25 ^° C. for 15 minutes in the presence of 2.5 μg of total yeast tRNA with the following enzymes: 0.2 or 1 unit of S1 nuclease and 10 ^-4 or ⁵ × 10 ⁵ units of RNase V1. Lead (II) cleavages were performed with 0.2 or 0.4 mM Pb acetate in 25 mM Hepes (pH 7.5), 7 mM Mg acetate and 35 mM potassium acetate for 10 minutes at 25 ^° C. Alkaline hydrolysis bands were obtained by incubating labeled dWD in 0.1 M NaOH and 2 mM EDTA at room temperature. 90 ⁰ C for 3 minutes. The reactions were terminated by ethanol precipitation, the RNA pellets were washed twice in 80% ethanol and dissolved in Ambion II loading buffer. The RNA samples were denatured for 5 minutes at 95 ^° C. before separation on 8% polyacrylamide / 8 M urea sequencing gels in TBE × 0.5. The gels were dried, exposed and visualized using a STORM 840 Phosphor-Imager imager.

1.7 Toeprinting tests

The toeprinting tests were performed as described in Hartz et al. (1988), with minor modifications. The hybridization mixtures contained 0.2 pmol of unlabeled sbi mRNA and 1 pmol of 5 'end labeled "SBIrevTR" primer in a buffer containing 10 mM Tris-acetate (pH 7.5) of 60 mM NH ₄ Cl and 1 mM DTT. For ribbon inhibition assays on sbi mRNA by sprD, different concentrations of wild-type or mutant sprD (sprDΔ) were added before adding the purified 7OS subunits from E. Coli. The 7OS ribosomes were renatured for 15 minutes at 37 ^° C. and then diluted in the reaction buffer in the presence of 1 mM MgCl ₂ (to promote the dissociation of the subunits) 4 pmoles of 7OS ribosomal subunits were added in each test, incubated for 5 minutes and the concentration of MgCl ₂ was adjusted to 10 mM (to promote the reassociation of the subunits). After 5 minutes of incubation, 10 pmol of ^{unfilled tetraFMEt} was added and the incubation was continued for another 15 minutes. CDNA was synthesized with 2 IU AMV RT (Biolabs) per run for 15 minutes. Reactions were terminated by adding 10 μL loading buffer II (Ambion). The cDNA products were loaded and separated on 8% polyacrylamide / 8M urea gels. The "toeprints" (forward imprints) were localized on the sbi mRNA sequence by sequencing the DNA with the same labeled DNA primer at the 5 'end. The gels were dried and analyzed as described above.

II. Results

II.1 The SprD Structure and its Cell Location Are Both Compatible With Targeted mRNA Control In a first step, the 5 'and 3' ends of sprD were determined. Its end

5 'was identified using a 5'-RACE. From several DNA clones, the 5 'end of sprD was mapped at position G2007185 of the sequence of S. aureus N315 (Kuroda et al., 2001) extracted from the genome database of European Molecular Biology Laboratory (EMBL, European Molecular Biology Laboratory). Its length was deduced from Northern blots on polyacrylamide gels (see Figure ID), which led to a predicted propeller of nine base pairs ending in a U ₆ extension acting as a Rho-independent terminator at the 3 'end (H4-L4, Figure IC). SprD RNA contains 142 nucleotides.

12 SprD sequences from the genomes of S. aureus strains N315, MRSA252, Newman, NCTC8325, Mu50, Mu3, MSSA476, JH1, JH9, MW2, USA300 TCH1516 and USA300 FPR3757 have been aligned and have very high sequence identity, except at positions 44 and 45 (u or c) and 57 (u or a) of the sequence SEQ ID No. 7. Sequence variation is too weak to establish a secondary structure of sprD by phylogenetic analysis. Therefore, its conformation has been analyzed by structural probes in solution, an approach that has contributed to the establishment of the structures of many RNAs (Felden 2007). A gDNA transcript was labeled at its end and its conformation in solution was probed by RNase VI, which cleaves double-stranded RNA or stacked nucleotides, and by Sl nuclease and lead, which both cleave single RNAs. strand. The reactivity with respect to its probes has been studied for each nucleotide (the two panels A and B of FIG. 1 are representative). Five independent experiments were carried out and their data are summarized on a model of secondary structure that they support (Figure IC). Specific double-strand breaks from U28-U30, G39-C40, A54, A62-A64, U79-U80, A89, G91, A125-A127, and the absence of cleavage by S1 nuclease and lead at the level of G1-U8, G14-U21, G39-C42, G49-C52, G76-U92, U101-U109, G13-U19, G121-U129 and G134-C142 suggest that four RNA helices form in solution: a Hl of 8-bp, a H 2 of 4-bp, a H3 of 17-bp and a H4 of 9-bp (Figure IC). The H2 helix could be further extended from 4 to 13 base pairs, but its lower portion (U28-U38 / A54-A64) is severely cut by both single-stranded (U28-U38, G55- A64) and double-stranded (U28-U30, A62-A64), which implies significant instability in solution. Sl cleavages at U9-G13 (L1), U44-C48 (L2), A95-U100 (L3), C132-G133 (L4) and lead cleavage at Al 1 -A12 (L1) , U46-C48 (L2) and U94 (L3) are consistent with the existence of four loops (L1-L4) respectively capping helices H1 to H4. Based on the presence of several Sl and lead cleavages and the absence of VI cleavages, the U22-U27 and U65-C75 nucleotide extensions fold as single strands of RNA in solution. Lead cuts at Ul 10 -IU 12 support the presence of an internal bulge within H3, with Al 1 being single stranded. There is no detectable degradation site within the sprD sequence. In summary, the sprD structure consists of two folded 5 '(H1) and 3' (H3-H4) ends, which flank a large (54 nucleotides in length corresponding to sequence SEQ ID No. 2) domain. unstable and largely unfolded (with the exception of H2), which may be available for interactions with dedicated mRNA targets (see below).

Cell fractionation was performed and the cell location of sprD was analyzed by Northern blots. Cell lysates from cells of S. aureus strain N315 were fractionated by successive centrifugations to obtain membrane (M), cytoplasmic (C) and ribosomal (R) fractions (for details see the Materials and Methods section). Methods). In accordance with Western blot immunoblot analysis, fraction M contains two membrane proteins, ErbS and ATPase, while these two proteins are absent from R and C fractions (Figure ID). The R fraction contains the three ribosomal RNAs (rRNA) and the fraction C contains the transfer RNAs (tRNA, Figure ID). Transfer-messenger RNA (mtRNA) is responsible for monitoring the translation and release of ribosomes blocked in eubacteria (Moore and Sauer 2007), but this function has not been reported in S. aureus. As shown here, S. aureus mtRNA is present in both C and R fractions in vivo (Figure 1D), as is expected from an RNA that binds to, and releases blocked ribosomes. The effector of the S. aureus, RNAIII, coding system for lta-hemo lysine, is mainly associated with ribosomes in vivo, with only a minor cytoplasmic fraction that is available for the binding of its dedicated target mRNAs (Boisset et al. al, 2007). SprD is predominantly in the cytoplasm, consistent with target mRNA regulatory functions, but a minor fraction is associated with ribosomes (Figure ID).

II.2 Activation and deactivation of in vivo expression of SprD

To study the function of SprD, the inventors took advantage of the lack of expression of SprD in strains RN4220 (laboratory strain, Kreiswirth et al, 1983) and SH1000 (virulent strain, Horsburgh et al, 2002), as demonstrated by Northern blots in both the exponential (E) and stationary (S) phases (Figure 2). The sprD gene, with its upstream predicted promoter sequence, was cloned into two low E. coli-S copy vectors. aureus, pCN38 and pCN51 (Charpentier et al., 2004) and transformed into strains RN4220 and SH1000. In both strains, at both E and S phases, these two vectors express in their expected size and at levels of expression comparable to their endogenous expression levels in strain N315 (FIG. 2). In both strains of S. aureus, there are no detectable growth phenotypes regardless of whether or not sprD is produced (the growth curves are superimposable, not shown).

II.3 SprD negatively (down) negatively affects Sbi protein levels in vivo The inventors made a comparison of total protein extracts from S. aureus strains, expressing or not, with the purpose of revealing differences in the expression levels of the selected proteins that could be extracted and identified by mass spectrometry. (SM). For this purpose, total proteins from extracellular and intracellular extracts at both E and S phases were prepared from RN4220 and SH1000 strains expressing or not, sprD. Remarkably, a meticulous comparison of the extracellular protein extracts of the two strains by SDS-PAGE reveals a protein band of about 45 kD whose level decreases in the presence of sprD at phase E (Figure 3A). This result is observed for both the coding vectors for sprD, and for both strains of S. aureus, with a more pronounced reduction in protein levels for the SH1000 strain (Figure 3A). The protein (s) of this band were eluted, a triptych digest was prepared and the protein fragments were subjected to Maldi-Toff Mass Spectrometry. Twenty-five independent peptides were identified, all corresponding to the amino acid sequence of a single Sbi protein (Table 3). A second, independent confirmation of the downregulation of Sbi protein by SprD was obtained by studying endogenous Sbi protein expression in both extracellular (Figure 3B) and intracellular ( FIG. 3C) by Western blots, using a polycolonal antibody against the Sbi protein. As expected from a cell surface protein, sbi is identified in the extracellular fraction, but is also detected in the intracellular medium of the bacteria. The protein is strongly expressed in phase E but is also detected in phase S. When sprD is expressed, in both intra- and extra-cellular media, expression of the sbi protein is significantly reduced both in RN4220 and SH1000 cells and both E and S phases, in agreement with data from SDS-PAGE gels and mass spectrometry. In phase S, sprD also reduces the level of expression of another extracellular protein band that has an apparent molecular weight of about 80 KD. From the mass spectrometry data, it appears that it contains two proteins, the precursor of extracellular triacylglycerol lipase and intracellular formate acetyltransferase (a contamination of the extracellular fraction by an intracellular protein), respectively eleven and seven peptide sequences have have been detected for these proteins.

Observed Mr (expected) Mr (calculated) Peptides

452.2365 902.4584 902.4385 K. YLTDTYK. S

493.2919 984.5692 984.5604 K.QLDALVAQK.D

539.7876 1077.5606 1077.5454 K.AIKDFQDNK.A

542.8126 1083.6107 1083.5964 K.LLGYYQSLK.D

559.8298 1117.6450 1117.6284 K.AFYQVLHLK.G

561.7985 1121.5825 1121.5716 K. VDDKNGYLAK. S

562.8242 1123.6338 1123.6237 K.VEVPQIQSPK.V

565.8000 1129.5855 1129.5801 R.EVNKAPMDVK.E 566.2913 1130.5680 1130.5641 R.EVNKAPMDVK.E + Deamidation (NQ)

569.3008 1136.5871 1136.5713 R.AQEVFSESLK.D

589.2702 1176.5259 1176.5127 K.YYYNTYYK.Y

609.3096 1216.6047 1216.6047 K.LNEKDSIENR.R

613.3108 1224.6071 1224.5986 R.NYVTESINTGK.V

639.8308 1277.6470 1277.6615 K. SAAYEANSKLPK.D

653.3601 1304.7057 1304.6976 K.SYIQPLKVDDK.N

697.8796 1393.7447 1393.7353 R.VAQQNAFYNVLK.N

734.3853 1466.7560 1466.7252 R.AQEVFSESLKDSK.N

746.3830 1490.7515 1490.7365 R.SQQVWVESVQSSK.A

773.4359 1544.8572 1544.8409 K.GAIDQTVLTVLGSGSK. S

775.9313 1549.8481 1549.8364 R.RVAQQNAFYNVLK.N

793.4562 1584.8978 1584.8551 K.VLYTFYQNPTLVK.T

846.4664 1690.9183 1690.9253 K.VEAPQIQSPQIEKPK.A

852.9039 1703.7933 1703.7863 K.HQTTQNNYVTDQQK.A

919.0168 1836.0191 1835.9992 K. YKGAIDQTVLTVLGSGSK. S

1018.5161 2035.0177 2034.9857 K.NDNLTEQEKNNYIAQIK.E +

Table 3

II.4 SprD is not involved in the regulation of sbi expression levels by human serum

The level of Sbi protein on the surface of S. aureus cells is low but increases significantly after growth in the presence of human serum, the serum component responsible for the induction of Sbi synthesis being IgG (Zhang et al. al., 2000). Since sprD downregulates sbi protein levels (FIG. 3), the inventors have assumed that sprD may be involved in the regulation of sbi protein levels by IgG from human serum. A reasonable hypothesis could be that human IgG downregulates, which in turn raises sbi protein levels to act as "environmental detection" receptors. In both SH1000 and MRSA252 strains of S. aureus, the presence of human serum increases the level of sbi protein, as shown by immunoblotting analysis using polyclonal antibodies against sbi (Figure 4A). The presence or absence of sprD, however, has no detectable influence on the stimulatory effect of human serum on sbi protein levels (Figure 4A). On the other hand, the levels of sprD RNA are essentially similar in the presence or absence of human serum as shown by Northern blots (Figure 4B). SprD is therefore not involved in the mechanism of induction of sbi expression by human IgG.

II.5 The regulation of sbi by SprD occurs by a direct interaction between the mRNA encoding the Sbi protein and the SprD RNA. The inventors have made it possible to answer the following two hypotheses: the regulation of the levels of the protein sbi by SprD is it performed directly or indirectly, through binding to an additional protein or to RNA regulators? In addition, the regulation Does sprD mediated occur at the level of the mRNA and / or at the level of the protein? To distinguish between these hypotheses and to understand regulation at the molecular level, putative pairing interactions between sprD and sbi mRNA were analyzed in silico, focusing on the starting site of the translation of the MRNA encoding the sbi protein. Bacterial RNAs can function as antisense RNAs by base pairing with target mRNAs (Majdalani et al, 2005). Complementary sites between SprD and the 5 'portion of sbi mRNA, including its AUG initiation codon and the upstream Shine and Dalgarno (SD) sequence were detected using FAS T A3 (Pearson 2000). The region of sprD predicted to be involved in the pairing is between H1 and H3, involving 43 nucleotides (in bold in Figure 1C, nucleotides of H2, L2, and H1 / H2 and H2 / H3 junctions), predicted for form 6 base pairs GC, 33 base pairs AU and 4 base pairs GU (Figure 5A) with the 5 'end of sbi mRNA. The 5 'end of sbi mRNA was determined experimentally using a 5'-RACE. From several DNA clones, the 5 'end of sprD was mapped at position G2476041 from the sequence of S. aureus N315 (Kuroda et al., 2001) extracted from the genome database of European Molecular Biology Laboratory (EMBL) (box G of Figure 5A). In vitro duplex formation between sprD (a transcript of a length of 142 purified nucleotides) and a synthetic sbi mRNA fragment of a length of 179 nucleotides containing its 5 'UTR sequence (41 nucleotides) followed by 46 codons has been studied by gel retardation tests. Both RNAs were separately and independently folded, cooled to room temperature, and labeled sbi mRNA was incubated with increasing concentrations of unlabeled sprD. The reactions were analyzed by gel electrophoresis using non-denaturing polyacrylamide gels (FIG. 5B). The "sbi-sprD mRNA" duplex is observed at a molar ratio of 1: 4 and almost all of the sbi protein is found in the complex with its RNA regulator at a molar ratio of 1:20. Therefore, in vitro, this "sbI-RNA mRNA" interaction is formed in the absence of the Sm protein-related protein Hfq, known to play a key role in many of these RNA transactions in some bacteria (Brennan and Link 2007). Hybridization between SprD and the sbi mRNA fragment is specific because a 100 to 2000-fold molar excess of yeast tRNAs does not displace sbi mRNA from a sbi spD-mRNA complex. preformed (Figure 5B). A deletion mutant of sbi mRNA was produced, SbiΔ61 lacking 61 nucleotides at its 5 'end including those predicted to be involved in the interaction with the sprD RNA. This deletion mutant becomes unable to hybridize to the SprD RNA, even at a molar ratio of 1:40 between SbiΔ61 and SprD, demonstrating that the first 61 nucleotides at the 5 'end of sbi are essential for produce hybridization with sprD, consistent with the proposed matching between the two NRAs. A mutant sprD containing a deletion of 36 nucleotides in the region predicted to interact with sbi mRNA (sprDΔ is lacking the nucleotides of positions 35 to 70, see Figure IC for more details) was not able to hybridize specifically to sbi mRNA (Figure 5B). It demonstrates the involvement of the H2, L2 and H2 / H3 junction domains from sprD in the recognition and specific hybridization of the mRNA encoding the Sbi protein.

IL 6 Study of the sbi sprD-mRNA complex by structural probes

To gain a deeper understanding of the interaction between sbi mRNA and sprD at a molecular level, a complex between the two RNAs was formed at a molar ratio of 1: 10 between sbi and labeled sprD and subjected to cleavages. by RNases Sl and Vl (Figure 6). In the presence of sbi mRNA (FIG. 4), Sl cleavages at positions U23-U30 (H1 / H2 and H2 junction), U37-G39 and U56-U61 (H2), U44-C48 (L2 loop) and A68 -U73 function H2 / H3) disappear in the sequence sprD, while the cuts Sl appear at the level of C51-U53 (H2). During the formation of the complex, cuts V1 at C40 and A54 disappear (the upper part of H2) while two cuts V1 appear at U44-U45 (L2). All of the experimentally identified structural changes are in and around the H2-L2 zone of sprD, with no detectable conformational changes for loop rods H1-L1, H2-L2 and H3-L3. These structural survey results provide strong experimental support for the secondary structure model of the interaction between the two RNAs shown in Figure 5A. Upon formation of the complex, the unstable H2 stem unfolds, allowing extensive but interrupted pairings from nucleotides 22 to 75. In the complex, the L2 loop folds as an RNA helix, as evidenced by appearance of the cuts Vl (U44-U45) and the disappearance of the cuts Sl (U44-C48). The relative instability in H2 solution in the sprD structure alone is well understood since H2 must unfold for extensive pairing with a complementary sequence from sbi mRNA (U28-C42 and U53-A64 from sprD pairs with A47-C34 and A23-U13 from sbi, respectively, Figure 5A). Specific hybridization of SprD to sbi mRNA leads to inhibition of the translation initiation of sbi mRNA into sbi protein (see Figure 7).

II.7 The sbi sprD-mRNA complex prevents binding of 7OS ribosomes

SprD, when interacting with sbi mRNA, covers the ribosome binding site

(RBS) and the AUG initiation codon (Figure 5A). Therefore, since the interaction sites of sprD with sbi mRNA coincide with the mRNA region that is covered by ribosomes during the initiation of translation (Hϋttenhofer and Nolier 1994), the inventors tested whether this complex was able to prevent 7OS ribosome binding by using "toeprinting" assays. The ternary initiation complex consisting of ribosomes 7OS ά'E. purified coli, the tRNA ^Met initiator and sbi mRNA blocked the elongation of a cDNA primer by reverse transcriptase (RT) and produced two "toeprints" of 14 and 17 nucleotides downstream of the initiation codon ( FIG. 7, band 4), which confirms experimentally the positioning of the start codon of sbi as represented in FIG. 5A. The presence of sprD reduces the interaction of 7OS ribosomes with sbi mRNA, in a concentration-dependent manner (Fig. 7, lanes 5-7). The addition of increasing amounts of a deletion mutant of sprD, spr-Δ, which can not form a complex with sbi mRNA in vitro (FIG. 5B), does not prevent the interaction of 7OS ribosomes with the Sbi mRNA. Therefore, sprD RNA inhibits translation of sbi mRNA by avoiding interaction of ribosomes with mRNA via direct antisense mating interaction with target sbi mRNA.

III. Discussion

The variety of clinical symptoms of staphylococcal infections is dependent on the expression of virulence factors that are under the control of specific intracellular regulatory proteins and RNA. The only known and well-characterized RNA regulatory agent for the pathogenesis of S. aureus is PARNIII, which controls the expression of a large number of virulence genes (Huntzinger et al., 2005, Boisset et al., 2007). The inventors have demonstrated that there is another RNA expressed in particular by S. aureus, SprD, which is also involved in the regulation of virulence genes. SprD acts as an antisense RNA and causes a direct repression of the translation of the mRNA encoding the sbi protein. The inventors have shown that the expression of the sbi protein is modulated by at least two regulators which are independent of each other (FIG. 4): a positive (upward) regulation triggered by the human IgG and a downregulation (downward) mediated by sprD, suggesting that the amount and timing of production of the sbi protein is restricted to a narrow window for bacteria of the genus Staphylococcus, including S. aureus, to successfully evade both adaptive immune system and innate immune system during human and animal infections.

The inventors have demonstrated the regulatory role and the mechanism of an RNA expressed in particular by S. aureus, SprD, and identified a target mRNA: the mRNA encoding the Sbi protein. SprD has at least one additional target mRNA that will be subjected to additional biochemical characterization by the inventors. The central domain (nucleotides 22-75 of sequence SEQ ID No. 7) of sprD folds up as a stem-loop structure flanked by single RNA strands (FIG. 1C) and causes a direct repression of the translation of sbi mRNA by extensive pairing with its target mRNA encoding the sbi protein (FIG. 5A), forming a long duplex. Of the 142 nucleotides in the SprD sequence, 96 are involved in pairing interactions (Figure 1C), indicating that about 68% of its sequence is not accessible for exonuclease digestion, which accounts for its stability in vivo (Figure 2). The molecular interaction between SprD and mRNA encoding the sbi protein is supported by mutational analysis of the two RNAs (Figure 5B) as well as the use of structural probes in solution (Figure 6). The 5 'leader sequence of sbi mRNA, 41 nucleotides in length (nucleotides 1-41 of sequence SEQ ID No. 3), is crucial for regulation because sprD is unable to interact with the mRNA deletion mutant, sbiΔ61 (Figure 5B). The H2-L2 hairpin pattern from sprD could facilitate the initial "sprD-sbi mRNA" contact that would then further extend upstream and downstream, in a manner similar to the "RNAIII-mRNA" (Boisset) regulations. et al 2007). In fact, effective pairings between interacting RNAs initially occur between limited "loop-to-loop" or "unpaired-loop sequence" interactions (Wagner et al., 2002). At the same time, the 5 'leader sequence of sbi mRNA and the central domain of sbi mRNA were subjected to strong evolutionary constraints in order to maintain the pairing interaction (the leader nucleotide sequence sbi mRNA is conserved among S. aureus sequences). When this "antisense-target RNA" complex is formed, ribosomes are unable to bind to the translation initiation region of sbi mRNA (Figure 7). Formation of a complex between SprD and target mRNA increases the amount of sbi mRNA detected in vivo, as evidenced by Northern blots (data not shown). This result could be due either to a direct stimulation of sprD for sbi mRNA transcription or to an indirect consequence of an increase in the stability of the target mRNA due to extensive pairing with its regulatory RNA. Experiments performed by the inventors are under way to try to make a choice between these hypotheses.

The formation of a complex between SprD and the mRNA encoding the sbi protein in vitro does not require the presence of the Sm protein-related Hfq protein (Figure 5B), and the in vivo expression levels of SprD are not affected. by the presence or absence of the Hfq protein (data not shown). These results are consistent with previous studies since the Hfq protein is expressed at low levels in S. aureus, with no detectable effect on virulence gene expression (Geisinger et al 2006, Bohn et al., 2007). ) and since Hfq has no major effect on the stability of RNAIII and its in vzvo mRNA targets and on the formation of an "RNAIII-mRNA" complex in vitro (Boisset et al, 2007). The inventors have thus demonstrated another example of sophisticated regulation of genes coding for virulence factors in bacteria of the genus Staphylococcus, in particular S. aureus, suggesting that the chronology and the amount of synthesis of the virulence factors are precisely controlled during the infectious process.

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Claims

An isolated polynucleotide characterized in that it comprises a sequence chosen from the group comprising: the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and the fragments of these; and the sequence SEQ ID No. 2, the derived sequences having at least 70% identity with the sequence SEQ ID No. 2 and the fragments thereof; and the sequence complementary to the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with said complementary sequence and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 3 and further having the ability to negatively regulate the expression of the Sbi protein. 2. isolated polynucleotide according to claim 1, characterized in that it comprises a sequence selected from the group comprising: the sequence SEQ ID No. 6, the derived sequences having at least 70% identity with the sequence SEQ ID No. 6 and the fragments thereof; and the sequence SEQ ID No. 7, the derived sequences having at least 70% identity with the sequence SEQ ID No. 7 and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 3 and further having the ability to negatively regulate the expression of the Sbi protein. 3. isolated polynucleotide according to one of claims 1 or 2, characterized in that it is an RNA. 4. isolated polynucleotide according to claim 3, characterized in that it is an RNA comprising a sequence selected from the group comprising: the sequence SEQ ID No. 2, in which the nucleotides in positions 23, 24 and 36 are u; the sequence SEQ ID No. 2, in which the nucleotides in positions 23 and 24 are c and the nucleotide in position 36 is u; and the sequence SEQ ID No. 2, in which the nucleotides at positions 23, 24 are c and the nucleotide at position 36 is a; and the sequence SEQ ID No. 7, in which the nucleotides at positions 44, 45 and 57 are u; the sequence SEQ ID No. 7, in which the nucleotides at positions 44 and 45 are c and the nucleotide at position 57 is u; and the sequence SEQ ID No. 7, in which the nucleotides at positions 44, 45 are c and the nucleotide at position 57 is a. 5. Expression cassette comprising a DNA sequence chosen from the group comprising: the sequence SEQ ID No. 1, the derived sequences having at least 70% identity with the sequence SEQ ID No. 1 and the fragments of those -this ; and the sequence SEQ ID No. 6, the derived sequences having at least 70% identity with the sequence SEQ ID No. 6 and the fragments thereof; and a DNA sequence that can be transcribed into an RNA according to one of claims 3 or 4; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 3 and further having the ability to negatively regulate the expression of the Sbi protein. A recombinant vector comprising an isolated polynucleotide according to any one of claims 1 to 4 or an expression cassette according to claim 5. 7. Vector according to claim 6, characterized in that it is a bacteriophage. 8. Host cell transformed by an expression cassette according to claim 5 or a vector according to one of claims 6 or 7. A composition comprising at least one component selected from the group consisting of an isolated polynucleotide according to any one of claims 1 to 4, an expression cassette according to claim 5, a vector according to one of claims 6 or 7 and a host cell according to claim 8. 10. Composition according to claim 9, characterized in that it is a pharmaceutical composition. An isolated polynucleotide according to any one of claims 1 to 4, or an expression cassette according to claim 5, or a vector according to one of claims 6 or 7 or a host cell according to claim 8, for its application as a medicament for the prevention and / or treatment of a staphylococcal infection. 12. Use of a component selected from the group comprising an isolated polynucleotide according to any one of claims 1 to 4, an expression cassette according to claim 5, a vector according to one of claims 6 or 7, a cell Host according to claim 8 for the preparation of a medicament for the prevention and / or treatment of a staphylococcal infection. 13. Isolated polynucleotide characterized in that it consists of a sequence selected from the group consisting of: the sequence SEQ ID No. 3, the derived sequences having at least 70% identity with the sequence SEQ ID No. 3 and the fragments of these; and the sequence SEQ ID No. 4, the derived sequences having at least 70% identity with the sequence SEQ ID No. 4 and the fragments thereof; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2; and said derived sequences and said fragments comprising nucleotides gaaaggg located at position 28 to 34 and nucleotides aug located at position 42 to 44 in the sequence SEQ ID No. 3 or SEQ ID No. 4. 14. Isolated polynucleotide, characterized in that it consists of: a sequence consisting of nucleotides 1 to 41 of one of the sequences SEQ ID No. 3 or SEQ ID No. 4, coupled at its 3 'end to a coding sequence of a gene of interest. 15. Isolated polynucleotide according to one of claims 13 or 14, characterized in that it is an RNA. An expression cassette comprising a DNA sequence chosen from the group comprising: the sequence SEQ ID No. 4, the derived sequences having at least 70% identity with the sequence SEQ ID No. 4 and the fragments of those -this ; and a DNA sequence transcribable to a polynucleotide according to claim 15; said derived sequences and said fragments having the ability to hybridize specifically with the sequence SEQ ID No. 1 and / or with the sequence SEQ ID No. 2; and said derived sequences and said fragments comprising gaaaggg nucleotides located in position 28 to 34 and the nucleotides aug located in position 42 to 44 in the sequence SEQ ID No. 3 or SEQ ID No. 4. A recombinant vector comprising an expression cassette according to claim 16. 18. Host cell transformed with an expression cassette according to claim 16 or a vector according to claim 17. 19. Product containing: at least one component selected from the group comprising an isolated polynucleotide according to any one of claims 1 to 4, an expression cassette according to claim 5, a vector according to one of claims 6 or 7, a host cell according to claim 8; and at least one other antistaphylococcal agent, as a combination product for simultaneous, separate and / or spreading use for the prevention and / or treatment of a staphylococcal infection. 20. Pharmaceutical composition according to claim 10, characterized in that it further comprises: an isolated polynucleotide according to claim 14 wherein said coding sequence is a sequence encoding at least one domain of the Sbi protein, and / or an expression vector comprising an isolated polynucleotide according to claim 14 wherein said coding sequence is a sequence coding for at least one domain of the Sbi protein, said domain of the Sbi protein being capable of binding specifically to the complement C3 protein and / or IgG immunoglobulins. 21. Method for detecting, in vitro, the presence of bacteria of the genus Staphylococcus in a biological sample, characterized in that it comprises the detection of at least one sequence chosen from the group comprising: the sequences as defined according to any one of claims 1 to 4; and the sequences as defined according to claim 13; with at least one appropriate detection means of said sequence. 22. A method for detecting, in vitro, mRNA of the gene encoding the Sbi protein present in a biological sample, characterized in that it comprises the following steps: contacting said biological sample with at least one probe comprising a sequence chosen from the sequences defined according to claim 1; and detecting the hybridization of said probe with said mRNA of the gene encoding the Sbi protein with at least one means for detecting said appropriate hybridization. 23. mRNA detection kit of the gene encoding the Sbi protein, characterized in that it comprises: at least one probe as defined according to claim 22; and at least one means for detecting the hybridization of said probe with said mRNA of the gene encoding the Sbi protein. 24. A kit for regulating the expression of a gene of interest characterized in that it comprises: at least one first component selected from the group comprising a polynucleotide according to any one of claims 13 to 15, a cassette; expression system according to claim 16, a vector according to claim 17, a host cell according to claim 18; and at least one second component selected from the group comprising an isolated polynucleotide according to any one of claims 1 to 4, an expression cassette according to claim 5, a vector according to one of claims 6 or 7, a host cell according to claim 8. 25. Non-therapeutic use of an isolated polynucleotide according to any one of claims 1 to 4 for inhibiting the expression of the gene encoding the Sbi protein. The non-therapeutic use of an isolated polynucleotide according to any one of claims 13 to 15 in combination with an isolated polynucleotide according to any one of claims 1 to 4 for inhibiting the expression of a gene of interest.