WO2015075166A1

WO2015075166A1 - Methods and pharmaceutical compositions for treatment of a bacterial infection

Info

Publication number: WO2015075166A1
Application number: PCT/EP2014/075231
Authority: WO
Inventors: Svetlana CHABELSKAIA; Brice Felden; Alex EYRAUD
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université De Rennes 1
Priority date: 2013-11-22
Filing date: 2014-11-21
Publication date: 2015-05-28

Abstract

The present invention relates to methods and compositions for the treatment of a bacterial infection. The present invention relates to oligonucleotides and their use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR TREATMENT

OF A BACTERIAL INFECTION

FIELD OF THE INVENTION:

The present invention relates to methods and compositions for the treatment of a bacterial infection.

BACKGROUND OF THE INVENTION:

Staphylococcus aureus (S. aureus) is a human and animal pathogen that has spectacular adaptive capacities to various antimicrobial agents. S. aureus rapidly acquires multiple antibiotic resistances, causing a wide spectrum of nosocomial and community- associated infections, and is thus a major worldwide health problem [1]. Since the spread of staphylococcal resistance to β-lactams (such as Methicillin), glycopeptide antibiotics have been the most efficient weapons against gram-positive infections, including the problematic methicillin-resistant S. aureus (MRSA). Vancomycin and Teicoplanin glycopeptides antibiotics have a similar mode of action on cell wall synthesis [2,3]. Unlike β-lactams, glycopeptides do not block enzymes involved in cell wall synthesis, but instead sequester the substrates required for peptidoglycan formation. Resistance to glycopeptides appeared through selective pressure as a result of a horizontal gene transfer from Enterococcus faecalis to MRSA clinical isolates [4]. In 1997, the first MRSA with reduced susceptibility to glycopeptides was reported in Japan [1,5]. In 2002, MRSA with Vancomycin-resistance was already isolated from two patients [1]. Therefore, there is an urgent need to increase our knowledge of the molecular events involved in antibiotic resistance of this serious pathogen, with a necessary focus on the precious glycopeptides used for MRSA infections. In this way, it has been suggested that characterization of new therapeutic compounds against bacterial infection and against bacterial antibiotic resistances may be highly desirable.

In S. aureus, methicillin and glycopeptide resistance is adjusted by the yabJ-spoVG operon, which is in turn controlled by a consensus nucleotide sequence of the alternative sigma factor σΒ [6,7]. The yabJ-spoVG mRNA codes for two proteins: YabJ, which has unknown functions; and stage V sporulation protein G (SpoVG). SpoVG was initially identified in Bacillus subtilis and shown to be involved in spore formation [8]. However, in nonsporulating bacteria, its mode of action and the molecular mechanisms involved are not known even though, it was shown recently that SpoVG is a site-specific DNA-binding protein [9]. SpoVG (but not YabJ) was shown to be the major regulator of the yabJ-spoVG operon [10]. This yabJ-spoVG operon has been proposed as the σΒ-dependent secondary regulator [6]. In addition to the control of methicillin and glycopeptide resistance, deletion of the yabJ- spoVG operon was shown to be involved in the control of extracellular nuclease, lipase, and protease expressions [10]. It is also involved in capsule formation and in the transcriptional control of cap and esxA [6,1 1]. Micro-arrays performed on yabJ-spoVG deletion in the Newman strain showed that yabJ-spoVG antagonizes the effects of sigma B on the expression levels of several proteins [10].

The coordinated expression of pathogenicity determinants is tightly-controlled by a complex network of elements, including two component systems, transcription factors, small metabolites, and small regulatory RNAs (sRNAs). Recently, bacterial sRNAs were shown to play a major role in a variety of regulatory processes [12]. S. aureus expresses around 250 sRNAs, most of which have unknown biological functions [13-18]. sRNAs control gene expression through various mechanisms, generally at the post-transcriptional level by direct pairing with mRNA targets [19]. These interactions positively or negatively regulate translation and/or the stability of the mRNAs. Several S. aureus sRNAs have been shown to be involved in pathogenicity, e.g., RNAIII, which regulates the expression of numerous virulence factors [14,20-22]. Moreover, the sRNAs SprD and Ssr42 have been shown to be involved in S. aureus virulence in animal models of infection [23,24]. In a large-scale analysis of S. aureus, the inventors identified the small RNA SprX (alias RsaOR) [18]. SprX is expressed from the genome of a converting phage containing virulence factors [18].

SUMMARY OF THE INVENTION:

The present invention relates to methods and compositions for the treatment of a S. aureus infection. The present invention relates to oligonucleotides and their use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION: In the present invention, the inventors investigated the development of multiple antibiotic resistances by Staphylococccus aureus bacteria, a major cause of opportunistic and nosocomial infections. The inventors reported the first case of an S. aureus sRNA involved in resistance to Vancomycin and Teicoplanin glycopeptides, the invaluable treatments for methicillin-resistant staphylococcal infections. The inventors demonstrated that modifying SprX expression levels in vivo influences Vancomycin and Teicoplanin glycopeptide resistance. The inventors also demonstrated that the SprX sRNA shapes bacterial resistance to glycopeptides resistance by regulating the expression of stage V sporulation protein G (SpoVG), a DNA-binding protein involved in glycopeptide resistance. The inventors then explored the regulatory mechanism at a molecular level, and uncover the functional SprX domain involved. The inventors demonstrated that SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mR A expression levels or affecting YabJ translation. In fact, SprX inhibits SpoVG expression through the direct interaction between the SprX C-rich loop L3 and spoVG ribosomal binding site ofyabJ-spoVG mRNA. This complex prevents ribosomal loading onto spoVG, and specifically inhibits translation of the second downstream gene within the yabJ-spoVG operon without altering the stability of the mRNA operon. The inventors also demonstrated that a mutated SprX that is unable to regulate SpoVG expression loses its influence on antibiotic resistance in vivo, demonstrating that the regulation of glycopeptides sensitivity by SprX involves SpoVG. This emphasizes the importance of sRNA control in S. aureus pathogenesis and is the first time that regulatory RNA has been shown to be involved in antibiotic resistance in a major human pathogen.

Oligonucleotides of the invention:

The present invention relates to an isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 111 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3.

The term "oligonucleotide" as used in the present invention refers to isolated, synthetic or recombinant oligonucleotides recognizing or targeting SpoVG mRNA. The term "oligonucleotide" also relates to the small RNA "sRNA" recognizing or targeting SpoVG mRNA. The term "oligonucleotide" also refers to antisense oligonucletotide (ASO) recognizing or targeting spoVG ribosomal binding site of yabJ-spoVG mRNA. The term "oligonuclotide" also refers to SprX sRNA. The term "SprX" has its general meaning in the art and refers to small pathogenicity island RNA X [18], an S aureus Small regulatory RNAs (sRNA). The term "SprX" also refers to RsaOR.

The term "SpoVG" has its general meaning in the art and refers to stage V sporulation protein G [8,9,10].

The term "Staphylococcus aureus" has its general meaning in the art and refers to a Gram-positive bacterium, a human and animal pathogen [18]. Staphylococcus aureus is a human commensal of skin and nares and an opportunistic pathogen responsible for a wide variety of human infections including superficial skin and wound infections to deep abscesses (endocarditis and meningitis), septicemia or toxin-associated syndromes. S. aureus is a leading cause of nosocomial and community acquired diseases and any stains acquire antibiotic resistance. The term "glycopeptide antibiotic" has its general meaning in the art and refers to a class of antibiotic drugs composed of glycosylated cyclic or polycyclic nonribosomal peptides. Glycopeptide antibiotics include but are not limited to vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, and decaplanin (Pootoolal et al, 2002; Van Bambeke et al, 2004).

The present invention also relates to an isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence that has at least about 80%, or at least about 85%o, or at least about 90%>, or at least about 95%>, or at least about 96%>, or at least about 97%o, or at least about 98%>, or at least about 99%> nucleic acid sequence identity with a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 11 1 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3. Nucleic acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST N (Karlin and Altschul, Proc. Natl Acad. Sci. USA 87(6):2264-2268 (1990)). The present invention also relates to an isolated, synthetic or recombinant oligonucleotide consisting of a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 111 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3.

The present invention also relates to an isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence as set forth by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

The present invention also relates to an isolated, synthetic or recombinant oligonucleotide consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

These oligonucleotides may be obtained by conventional methods well known to those skilled in the art.

The oligonucleotide of the invention may be of any suitable type. The one skilled in the art can easily provide some modifications that will improve the clinical efficacy of the oligonucleotide (C. Frank Bennett and Eric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic PlatformAnnu. Rev. Pharmacol. Toxicol. 2010.50:259-293.). Typically, chemical modifications include backbone modifications, heterocycle modifications, sugar modifications, and conjugations strategies. For example the oligonucleotide may be selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, LNA, oligonucleotide, morpholinos, small regulatory RNAs (sRNAs), tricyclo-DNA-antisense oligonucleotides (ASOs), U7- or Ul- mediated ASOs or conjugate products thereof such as peptide-conjugated or nanoparticle- complexed ASOs. Indeed, for use in vivo, the oligonucleotide may be stabilized. A "stabilized" oligonucleotide refers to an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo -nuclease). Stabilization can be a function of length or secondary structure. In particular, oligonucleotide stabilization can be accomplished via phosphate backbone modifications.

In a particular embodiment, the oligonucleotide according to the invention is a LNA oligonucleotide. As used herein, the term "LNA" (Locked Nucleic Acid) (or "LNA oligonucleotide") refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogues also referred to as LNA nucleotides and LNA analogue nucleotides. LNA oligonucleotides, LNA nucleotides and LNA analogue nucleotides are generally described in International Publication No. WO 99/14226 and subsequent applications; International Publication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO 03/006475; U.S. Patent Nos. 6,043,060, 6268490, 6770748, 6639051, and U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241, 2002/0147332, 2004/0244840 and 2005/0203042, all of which are incorporated herein by reference. LNA oligonucleotides and LNA analogue oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA.

Other possible stabilizing modifications include phosphodiester modifications, combinations of phosphodiester and phosphorothioate modifications, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. Chemically stabilized, modified versions of the oligonucleotide also include "Morpho linos" (phosphorodiamidate morpholino oligomers, PMOs), 2'-0-Met oligomers, tricyclo (tc)- DNAs, U7 short nuclear (sn) RNAs, or tricyclo-DNA-oligoantisense molecules (U.S. Provisional Patent Application Serial No. 61/212,384 For: Tricyclo-DNA Antisense Oligonucleotides, Compositions and Methods for the Treatment of Disease, filed April 10, 2009, the complete contents of which is hereby incorporated by reference).

Other forms of oligonucleotides of the present invention are oligonucleotide sequences coupled to small nuclear RNA molecules such as Ul or U7 in combination with a viral transfer method based on, but not limited to, lentivirus or adeno-associated virus (Denti, MA, et al, 2008; Goyenvalle, A, et al, 2004). The oligonucleotide of the invention can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage et al, 1981); nucleoside H-phosphonate method (Garegg et al, 1986; Froehler et al, 1986, Garegg et al, 1986, Gaffney et al, 1988). These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These nucleic acids may be referred to as synthetic nucleic acids. Alternatively, oligonucleotide can be produced on a large scale in plasmids (see Sambrook, et al, 1989). Oligonucleotide can be prepared from existing nucleic acid sequences using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Oligonucleotide prepared in this manner may be referred to as isolated nucleic acids.

In a particular embodiment, the oligonucleotide of the present invention is conjugated to a second molecule. Typically said second molecule is selected from the group consisting of aptamers, antibodies or polypeptides. For example, the oligonucleotide of the present invention may be conjugated to a cell or bacterial penetrating peptide. Cell penetrating peptides are well known in the art and include for example the TAT peptide (Bechara C, Sagan S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013 Jun 19;587(12): 1693-702; Malhi SS, Murthy RS. Delivery to mitochondria: a narrower approach for broader therapeutics. Expert Opin Drug Deliv. 2012 Aug;9(8):909-35; Wesolowski D, Alonso D, Altman S. Combined effect of a peptide-morpholino oligonucleotide conjugate and a cell-penetrating peptide as an antibiotic. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8686-9). The oligonucleotide of the present invention may be conjugated to cell penetrating peptide such as YARVRRRGPRGYARVRRRGPRRC (SEQ ID NO: 4) described in Wesolowski D, Tae HS, Gandotra N, Llopis P, Shen N, Altman S. Basic peptide- morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes. Proc Natl Acad Sci U S A. 2011 Oct 4;108(40): 16582-7. In a particular embodiment, the oligonucleotide-second molecule conjugate is able to target the S. aureus infected cells. In a particular embodiment, the oligonucleotide-second molecule conjugate targets SpoVG. In a particular embodiment, the oligonucleotide-second molecule conjugate is able to target the S. aureus strain.

Therapeutic methods and uses of the invention:

The oligonucleotide of the invention may be used in reducing or eliminating glycopeptide antibiotic resistances in a S. aureus population.

Therefore, a further aspect of the invention relates to the oligonucleotide of the invention for use as a medicament. The present invention also relates to the oligonucleotide according to the invention for use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof. As used herein, the term "subject" denotes a mammal. In one embodiment of the invention, a subject according to the invention refers to any subject (preferably human) afflicted with S. aureus infections.

The term "antibiotic resistance" has its general meaning in the art and refers to bacteria able to survive after exposure to one or more antibiotics. The term "antibiotic resistance" refers to resistance of a bacterium to an antibiotic treatment to which it was originally sensitive.

The method of the invention may be performed for any type of Staphylococcus aureus bacterial infection such as superficial skin and wound infections to deep abscesses (endocarditis and meningitis), septicemia or toxin-associated syndromes, nosocomial and community acquired diseases, any stains acquire antibiotic resistance and methicillm-resistant S. aureus (MRSA) infections, and Sepsis of newborn due to Staphylococcus aureus. In a particular embodiment, oligonucleotides of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the oligonucleotide of the invention to the cells and S. aureus population. Preferably, the vector transports the nucleic acid to cells and S. aureus population with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, naked plasmids, non viral delivery systems (cationic transfection agents, liposomes, etc .), phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the oligonucleotide sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: RNA viruses such as a retrovirus (as for example moloney murine leukemia virus and lentiviral derived vectors), harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus. One can readily employ other vectors not named but known to the art. In a preferred embodiment, the oligonucleotide sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters. In a particular embodiment, two or even more oligonucleotides can also be used at the same time; this may be particularly interesting when the oligonucleotides are vectorized within an expression cassette (as for example by U7 or Ul cassettes).

The present invention also relates to a combination of a least one oligonculeotide according to the invention with at least one glycopeptide antibiotic for use in the treatment of S. aureus infection in a subject in need thereof.

In one embodiment, the glycopeptide antibiotic agent is selected from the group consisting of but not limited to teicoplanin, vancomycin, telavancin, bleomycin, ramoplanin, and decaplanin.

The present invention also relates to a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof, comprising the step of administering to said subject the oligonucleotide according to the invention.

The present invention also relates to a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof, comprising the step of administering to said subject at least one oligonucleotide according to the invention in combination with at least one glycopeptides antibiotic.

The present invention also relates to a method of treating S. aureus infection in a subject in need thereof, comprising the step of administering to said subject the oligonucleotide according to the invention in combination with at least one glycopeptides antibiotic.

In the present invention, the inventors also demonstrated that SprX negatively regulates the expression of the general transcriptional factor SpoVG. Accordingly, the present invention also relates to the oligonucleotide according to the invention for use in the treatment of S. aureus infection in a subject in need thereof.

Pharmaceutical compositions and kits of the invention:

The oligonucleotide of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the oligonucleotide of the invention and a pharmaceutical acceptable carrier for use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.

Typically, the oligonucleotide of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, inhalation, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration of the oligonucleotide, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, inhalation administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and nasal or intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being administered by nasal administration or by inhalation. Nasal administration may be under the form of liquid solution, suspension or emulsion. Solutions and suspensions are administered as drops. Solutions can also be administered as a fine mist from a nasal spray bottle or from a nasal inhaler. Inhalation may be accomplished under the form of solutions, suspensions, and powders; these formulations are administered via an aerosol, droplets or a dry powder inhaler. The powders may be administered with insufflators or puffers.

Pharmaceutical compositions of the present invention include a pharmaceutically or physiologically acceptable carrier such as saline, sodium phosphate, etc. The compositions will generally be in the form of a liquid, although this need not always be the case. Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, celluose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, mineral oil, etc. The formulations can also include lubricating agents, wetting agents, emulsifying agents, preservatives, buffering agents, etc. Those of skill in the art will also recognize that nucleic acids are often delivered in conjunction with lipids (e.g. cationic lipids or neutral lipids, or mixtures of these), frequently in the form of liposomes or other suitable micro- or nano-structured material (e.g. micelles, lipocomplexes, dendrimers, emulsions, cubic phases, etc.).

One skilled in the art will recognize that the amount of an oligonucleotide to be administered will be an amount that is sufficient to induce amelioration of unwanted disease symptoms. Such an amount may vary inter alia depending on such factors as the gender, age, weight, overall physical condition, of the subject, etc. and may be determined on a case by case basis. The amount may also vary according to the type of condition being treated, and the other components of a treatment protocol (e.g. administration of other medicaments such as steroids, etc.). If a viral-based delivery of oligonucleotides is chosen, suitable doses will depend on different factors such as the viral strain that is employed, the route of delivery (intramuscular, intravenous, intra-arterial, oral, inhalation or other). Those of skill in the art will recognize that such parameters are normally worked out during clinical trials. In addition, treatment of the subject is usually not a single event. Rather, the oligonucleotides of the invention will likely be administered on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart.

Pharmaceutical compositions of the invention may include any further glycopeptide antibiotic agent which is used in the treatment of S. aureus infection. For example, pharmaceutical compositions of the invention can be co-administered with glycopeptide antibiotic agent selected from the group consisting of teicoplanin, vancomycin, telavancin, bleomycin, ramoplanin, and decaplanin. The invention also provides kits comprising at least one oligonucleotide of the invention. Kits containing oligonucleotide of the invention find use in therapeutic methods.

Diagnostics methods A further aspect of the invention relates to a method of identifying a S. aureus infected subject having or at risk of having or developing a glycopeptide antibiotic resistance which comprises the steps of:

i) measuring the expression level of SprX in a biological sample obtained from said subject,

(ii) comparing the expression level measured at step i) with a reference value,

(iii) detecting differential in the SprX expression level between the biological sample and the reference value is indicative that said subject is having or at risk of having or developing a glycopeptide antibiotic resistance. The term "biological sample" refers to sample obtained from S. aureus infected subject such as blood and urine. The term "biological sample" also refers to S. aureus strain isolated from S. aureus infected subject.

A "reference value" can be a "threshold value" or a "cut-off value". Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the SprX expression levels obtained according to the method of the invention with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the SprX expression level (or ratio, or score) determined in a biological sample derived from one or more subjects having a glycopeptide antibiotic resistance. In one embodiment of the present invention, the threshold value may also be derived from SprX expression level (or ratio, or score) determined in a biological sample derived from one or more subjects not having glycopeptide antibiotic resistance. Furthermore, retrospective measurement of the SprX expression levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

In one embodiment of the invention, the reference value may consist in expression level measured in a biological sample associated with a subject having glycopeptide antibiotic resistance or in a biological sample associated with a subject not having glycopeptide antibiotic resistance.

Typically, when the expression level determined for Spr-X is lower than the corresponding reference value it is concluded that the S. aureus infected subject is having or at risk of having or developing a glycopeptide antibiotic resistance.

A further aspect of the invention relates to a method for determining whether a S. aureus infected subject is a responder or is a non-responder to a glycopeptide antibiotics treatment which comprises the steps of:

(ii) comparing the expression level measured at step i) with a reference value,

(iii) detecting differential in the SprX expression level between the biological sample and the reference value is indicative that said subject is a responder or a non-responder.

The term "responder" refers to S. aureus infected subject that will respond to glycopeptide antibiotics treatment. The term "non-responder" refers to a subject that will not respond to glycopeptide antibiotics treatment. The term "non-responder" also refers to a subject infected with S. aureus strain resistante to glycopeptide antibiotics treatment.

Methods for measuring the expression level of SprX in a biological sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a sRNA. Typically the prepared nucleic acid can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays (AFFYMETRIX). Advantageously, the analysis of the expression level of SprX involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (BARANY, Proc. Natl. Acad. Sci. USA, vol.88, p: 189-193, 1991), self sustained sequence replication (GUATELLI et al, Proc. Natl. Acad. Sci. USA, vol.57, p: 1874-1878, 1990), transcriptional amplification system (KWOH et al, 1989, Proc. Natl. Acad. Sci. USA, vol.86, p: 1173-1177, 1989), Q-Beta Replicase (LIZARDI et al, Biol. Technology, vol.6, p: 1197, 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Real-time quantitative or semi-quantitative RT-PCR is preferred. In a particular embodiment, the determination comprises hybridizing the sample with selective reagents such as probes or primers and thereby detecting the presence, or measuring the amount of SprX. Hybridization may be performed by any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth. Nucleic acids exhibiting sequence complementarity or homology to the nucleic acid of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e.g. avidin/biotin). The probes and primers are "specific" to SprX they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature -Tm-, e.g., 50 %> formamide, 5x or 6x SCC. lx SCC is a 0.15 M NaCl, 0.015 M Na-citrate). Many quantification assays are commercially available from Qiagen (S.A. Courtaboeuf, France) or Applied Biosystems (Foster City, USA). Expression level of SprX may be expressed as absolute expression profile or normalized expression profile. Typically, expression profiles are normalized by correcting the absolute expression profile of SprX by comparing its expression to the expression of a nucleic acid that is not a relevant, e.g., a housekeeping nucleic acid that is constitutively expressed. This normalization allows the comparison of the expression profile in one sample, e.g., a patient sample, to another sample, or between samples from different sources. Probe and or primers are typically labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. The term "labelled" is intended to encompass direct labelling of the probe and primers by coupling (i.e., physically linking) a detectable substance as well as indirect labeling by reactivity with another reagent that is directly labeled. Examples of detectable substances include but are not limited to radioactive agents or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)). The invention also relates to a kit for performing the methods as above described, wherein said kit comprises means for measuring the expression level of SprX that is indicative of subject having or at risk of having or developing a glycopeptide antibiotic resistance. Typically the kit may include primers or probes as above described. The kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.

The method of the invention allows to define a subgroup of subjects who are responder or non responder to the glycopeptide antibiotic treatment. A further object of the invention relates to a method for the treatment of S. aureus infection in a subject in need thereof comprising the steps of:

a) determining whether a S. aureus infected subject is a responder or a non-responder to glycopeptide antibiotic treatment by performing the method according to the invention, b) administering the glycopeptide antibiotic, if said subject has been considered as a responder, or,

c) administering the oligonucleotide according to the invention in combination with at least one glycopeptide antibiotic, if said subject has been considered as a non responder,

A further object of the invention relates to a glycopeptide antibiotic for use in the treatment of S. aureus infection in a subject in need thereof, wherein the subject was being classified as responder by the method as above described.

A further object of the invention relates to the oligonucleotide according to the invention in combination with at least one glycopeptide antibiotic for use in the treatment of S. aureus infection in a subject in need thereof, wherein the subject was being classified as non responder by the method as above described.

Oligonucleotide sequences

SEQ ID NO: 1 for SprX-N315:

ACACAUGCAUCAACUAUUUACAUCCUUGUUCACCCAAGCAUGUCACUGGGUGUUUU 56

UUCUUAUGAUAGAGAGCAUAGUUUUCAUACUACUCCCCCGUAGUAUAUAUGACUUUAGCA 1 1 6

UUCCCGUAUAAUAGUUUACGGGGUGCUUUUU 147

SEQ ID NO: 2 for SprXl-HGOOl :

ACACAUGCAUCAACUAUUUACAUCUAUCCUUGUUCACCCAAGCAUGUCACUGGGUGUUUU 60

UUCUUAUGAUAGAGAGCAUAGUUUUCAUACUACUCCCUCGUAGUAUAUAUGACUUUAGCA 12 0

UUCCCGUAUAAUAGUUUACGGGGUGCUUUUU 151

SEQ ID NO: 3 for SprX2-HG001 :

ACACAUGCAUCAACUAUUUACAUCUAUCCUUGUUCACCCAAGCAUGUCACUGGGUGUUUU 60

UUCUUACGAUAGAGAGCAUAGUUUUCAUACUACUCCCCGUAGUAUAUAUGACUUUAGCA 1 1 9

UUCCCGUAUAACAGUUUACGGGGUGCUUUUU 150

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Alignment of the nucleotide sequences of SprX from the N315 strain and the two SprX copies from the HGOOl strain. Identical nucleotides in the sequences are indicated with asterisks. The nucleotide numbering is based on the SprX2 sequence.

EXAMPLE:

Material & Methods

Bacterial strains, plasmids construction, and culture conditions.

The bacterial strains and plasmids used in this study are listed in Table I. The bacteria were grown at 37°C in Brain-Heart Infusion broth (BHI, Oxoid) or in Tryptic Soy Broth (TSB, Oxoid). When necessary, the media were supplemented with either 10 μg of chloramphenicol or^ erythromycin for S. aureus, or 50 μg ampicillin per mL for E. coli. In pCN38-sprX, sprX2 was expressed from its own promoter. The sprX2 sequence (containing the 207-base pair (bp), upstream and 258-bp downstream from sprX) was amplified from HGOOl genomic DNA as a 615-bp fragment. The pCN38-sprX_mutL3 was produced using the mutagenized oligonucleotides 'mutfor' and 'mutrev'. All fragments were flanked with Pstl and EcoBl restriction sites. The PCR products were cloned in pCN38 [37]. For the spot assays, overnight cultures of the tested strains were diluted to obtain a OD600 of 8. From these cultures, seven consecutive ten-fold dilutions were prepared. 3 jiL of each dilution was dropped on Mueller-Hinton (MH ) or on MH supplemented by 0.75 or 0.85 μg/mL of Teicoplanin or Vancomycin antibiotics, then incubated for 24 hours at 37°C. The first dilution corresponds to 107 bacteria.

IcanaBry in resistant

Table I: Strains and plasmids used in this study.

Strain construction. To inactivate the HG001 [38] sprX genes, DNA fragments upstream and downstream of sprX were amplified by PCR from genomic DNA. For the sprXl, the upstream fragment was 889 bp-long and the downstream fragment was 949 bp-long; for sprX2 the upstream was 988 bp-long and the downstream was 949 bp-long. A second PCR amplification was done by combining these fragments (using primer pairs sprXDl and sprXD6 for sprXl, and primer pairs 2sprXDl and 2sprXD4 for sprXl). These were then cloned using the Pstl-BamHI sites in the temperature-sensitive plasmid pBT2 [39]. To achieve gene disruption in the genome by homologous recombination, the resulting plasmids pBT2AsprXl and pBT2AsprX2 were transformed into S. aureus strain RN4220 and then into S. aureus HG001. Mutants were enriched by cultivation at 42°C. Cells from the stationary phase culture were plated on TSA plates and incubated at 37°C. Colonies were imprinted on plates that were supplemented with 10 μg/mL chloramphenicol. Chloramphenicol-sensitive colonies were tested by PCR for deletion of sprXl and sprX2. The deletions were confirmed by Northern blot assays.

The S. aureus HG001 AyabJ-spoVG: :erm mutant was constructed by transducing the AyabJ-spoVG::erm mutation of strain RN4220 [7] into strain HG001. The deletion of the yabJ-spoVG locus was confirmed by PCR, Northern blot, and Western blot analysis.

SpoVG-His6 expression and antibody preparation.

The pSTM33 vector containing SpoVG-His6 [7] was electroporated into BL21 strain (DE3, Novagen). Cells were grown in LB broth to a 600 nm optical density of 0.5, and SpoVG-His6 expression was induced with 0.3 mM isopropyl-D-thiogalactopyranoside (IPTG, Eurobio). After 3 hours, cells were collected by centrifugation and resuspended in 30 mL phosphatebuffered saline (pH 7.4) supplemented with a cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) and 0.1 mg/mL DNase. The cells were disrupted and the debris separated by centrifugation at 13,000 g for 10 minutes. Purification of the His-tagged protein was performed on nickel Sepharose High Performance columns (HisTrap HP; GE Healthcare) using an AKTA fast-performance liquid chromatography system. The correct molecular weight of the purified protein was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. One milligram of the purified SpoVG-His₆ was used to raise polyclonal mouse antibodies (Eurogentec, Seraing, Belgium).

Protein isolation and western blots.

For the total protein extractions, culture pellets corresponding to 2 mL of culture at a OD600 of 1 were suspended in 0.2 mL of lysis buffer (10 mM Tris-HCl pH 7.5, 20 mM NaCl, 1 mM EDTA, 5 mM MgC12, and cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) containing 0.1 mg/mL lysostaphin. Following incubation at 37°C for 10 minutes, Laemmli sample buffer was added [40]. Samples were then boiled for 5 minutes, separated by SDSPAGE, and transferred onto a hybond-P PVDF membrane (Amersham). To visualize SpoVG, antibodies were used at a dilution of 1 :5,000. The anti-rabbit antibodies were used at a dilution of 1 :20,000. Western blots were revealed using the Amersham ECL Plus detection Kit. Signals were visualized using a Typhoon FLA 9500 and quantified using Image-QuantTL 7.0 (GE Healthcare). Bi-dimensional gel electrophoresis (2D-DiGE).

Overnight cultures of bacteria were diluted 1 : 100 in BHI and grown at 37°C to the exponential phase, then the cells were pelleted for 10 minutes at 4°C (8,000 g). Pellets of 2 mL culture were suspended in the same lysis buffer already described with the addition of 2 UI of DNase amp grade and 2 UI of RNase A. Following incubation at 37°C for 30 minutes, 1 mL of Tri-Reagent was added, then the samples were sonicated (3x30s 20% active cycle). 100 chloroform was added to each sample and incubated for 5 minutes at room temperature (RT). Next, 300 ethanol was added and samples were incubated for 3 minutes at RT and precipitated overnight at -20°C using acetone. The proteins were centrifuged for 15 minutes at 4°C (4,000 g) and washed with 1 mL 80% acetone, then pelleted for 5 minutes at 4°C (4,000 g). The pellets were dried at room temperature. Duplicate pellets were dissolved in 200 urea 8M. 2D-DIGE and mass spectrometry identification of proteins of interest were performed by the Cochin Institute (Paris). Spots corresponding to proteins with expression modified between HG001 wt and AsprX were subjected to in-gel trypsin digestion, peptide extraction, and desalting, followed by MALDI-ToF/ToF analysis. Peptides were analyzed using MASCOT and the NCBlnr database to identify the selected protein spots. The average ratio of protein levels was calculated using DeCyder 2D software (GE Healthcare) to determine the change in normalized spot volume between HG001 wt and HG001 AsprX samples. RNA isolation, Northern blots, transcription and RNA labelling.

Total RNAs were prepared as previously described [18]. For SprX, Northern blots were performed with 10 μg of total RNA [13]. Specific ³²P-labeled probes were hybridized with ExpressHyb solution (Clontech) for 90 minutes, washed, exposed, then scanned with a Typhoon FLA 9500 scanner (GE Healthcare). For the yabJ-spoVG mRNA, Northern blots were performed [6]. The primer pair oSTM29/oSTM30 was used to generate adCTP-labeled spoVG-specific probes by PCR labeling. For in vitro experiments, RNAs were transcribed from PCR fragments generated from genomic DNA. To produce the template-encoding SprX_mutL3, mutagenized oligonucleotides were used. RNA was produced by in vitro transcription using MEGAscript (Ambion). 5 '-RNA γΑΤΡ labeling was performed [32]. The RNA was purified by 8% PAGE, eluted, ethanol precipitated, then stored at -80°C.

RNA structural probing.

Structural assays were performed as previously described [32]. SprX was prepared by incubating 14 pmol of unlabeled RNA in a buffer (10 mM Tris-HCl pH 7.5, 60 mM NaCl, 1 mM EDTA) for 10 minutes at 25°C. I e yabJ-spo

mRNA was prepared by incubating 1 pmol of labeled RNA in the aforementioned buffer. MgCl₂ was added to obtain a final concentration of 2.5 mM and this was then incubated for 10 minutes at 25°C. Preparation for structural analysis of the duplexes between SprX and yabJ-spoVG^-] mRNA was done by incubating 0.2 pmol of labeled RNA with 10 pmol of unlabeled RNA for 15 minutes at 25°C. Cleavages with SI nuclease (0.063 U^L),V1 RNase (6.25x10-7 υ/μί), and 1.25 mM lead acetate were carried out for 10 minutes at 25°C in the presence of 1 g of total yeast tRNA. For sequencing, Tl at 0.038 \JI\xL and U2 at 0.0015 Ό/μΙ, were used. The reactions were precipitated, and the pellets dissolved in loading buffer II (Ambion). The samples were denatured for 10 minutes at 65°C prior to separation on 8% polyacrylamide/8M urea gels. Gels were dried and visualized (Typhoon FLA 9500).

Toeprint assays.

The toeprint assays were performed as previously described [18], but with modifications. Annealing mixtures containing 13 nM of yabJ-spoVG mRNA with either 67 nM of labeled Toeprint spoVG or Toeprint yabJ primer were incubated with a buffer (20 mM Tris-HCl pH 7.5, 60 mM NH4C1) for 2 minutes at 90°C followed by 1 minute at RT. Renaturation was realized in the presence of 10 mM MgC12 at 25 °C for 20 minutes. For the assays in the presence of SprX, various concentrations of SprX were added prior to the purified E. coli 70S ribosomes. The ribosomes were reactivated for 15 minutes at 37°C and diluted in the reaction buffer in the presence of 1 mM MgC12. 33 nM 70S were added in each assay, incubated for 5 minutes and the MgC12 was adjusted to 10 mM. After 5 minutes, 0.83 μΜ of uncharged tRNAfMet was added and this was incubated for 15 minutes. cDNA was synthesized with 4 UI of AMV RT (Biolabs) for 15 minutes. Reactions were ended by the addition of 15 μΐ of loading buffer II (Ambion). The cDNAs were loaded and separated onto 8% polyacrylamide/8M urea gels. Sequencing ladders were generated with the same 5'- endlabeled primer. Results

SprX regulates S. aureus resistance to glycopeptide antibiotics.

In a large-scale analysis of S. aureus, the inventors identified the small R A SprX (alias RsaOR) [18]. SprX is expressed from the genome of a converting phage containing virulence factors [18]. To elucidate the functions of SprX, the inventors analyzed the phenotypes of strains with different sRNA expression levels. For this purpose, the inventors used HG001 agr+ S. aureus strain carrying two copies of sprX. Alignments of SprXl and SprX2 showed that the RNA sequences are identical in each, except for three nucleotides (Figure 1). The inventors disrupted the two copies of sprX in strain HG001 (AsprX) by homologous recombination, thus abolishing SprX expression. Overexpression of SprX was achieved using a multicopy plasmid expressing SprX from its endogenous promoter (pCN38- sprX). Among the phenotypes tested of strains possessing different SprX expression levels, the inventors measured their resistance to Teicoplanin and Vancomycin, two antibiotics from the glycopeptide family. SprX deletion and overexpression had no effect on bacterial growth in Mueller-Hinton (MH) medium. Interestingly, the inventors observed and reproduced a 2- log 10 diminution of Teicoplanin and Vancomycin resistance in the strain overexpressing SprX when compared to wild-type (wt) strain. The inventors also observed that the sprX deletion strain was more resistant than the wt strain. These results indicate that SprX is involved in S. aureus resistance to Teicoplanin and Vancomycin. This interesting observation prompted us to identify the SprX targets involved in this antibiotic resistance, and to elucidate the mechanism of their regulation by SprX.

SprX reduces the expression of a protein involved in bacterial resistance to numerous antibiotics.

To identify SprX targets, we analyzed whether SprX modulates the expression of S. aureus proteins. We thus compared the protein profiles of HG001 wt and AsprX strains by bidimensional gel electrophoresis (2D-DiGE). In total, five proteins were identified as being regulated by SprX, and all of these were upregulated in the AsprX strain. Interestingly, mass spectrometry analysis of these proteins identified stage V sporulation protein G (SpoVG), which is involved in bacterial resistance to methicillin and other glycopeptide antibiotics [7]. To confirm the regulation of SpoVG expression by SprX, SpoVG protein levels were monitored by Western blots using polyclonal antibodies raised against the SpoVG protein. In agreement with the 2D-DiGE data, we observed an increase of SpoVG protein levels in the AsprX strain. To confirm that the enhanced expression of SpoVG was linked to SprX inactivation, we performed complementation experiments. Introducing pCN38-s/?r into the AsprX strain decreased SpoVG protein levels as compared to the AsprX strain and also compared to those in the wt strain. These results are explained by the overexpression of SprX from pCN38 compared to its endogenous expression levels in the wt strain. Since SpoVG protein expression is downregulated by SprX, we monitored mRNA levels in order to determine whether this occurs at transcriptional and/or translational levels. SpoVG is translated from a -1200 nt-long bicistronic yabJ-spoVG mRNA that is cleaved by an unknown mechanism into two transcripts of -600 and -500 nts, fragments which correspond respectively to yabJ and spoVG mRNA. The inventors used a labeled DNA probe of 300 bp encompassing SpoVG open reading frame (ORF) to detect both the spoVG and yabJ-spoVG mRNA. No detectable changes were seen in levels of either mRNA in the AsprX strain when compared to the wt parental strain. Also, no SprX-induced changes were observed for the yabJ mRNA using a ja^J-specific probe. Collectively, these results show that SprX lowers SpoVG expression, and that regulation occurs at the posttranscriptional level. Structural changes of the yabJ-spo VG mRNA induced by SprX.

In vivo results prompted us to explore the interaction between SprX and yabJ-spoVG mRNA. Interestingly, we identified a putative pairing site between SprX and the translational initiation site of spoVG in silico using targetRNA2 [25] and sRNA TarBase [26]. The predicted interaction between the two RNAs includes 26 bp and occurs between the U79- Ul 15 nts from SprX and the A566-A606 nts from the yabJ-spoVG mRNA (a fragment which includes its Shine-Dalgarno (SD) sequence and the initiation codon of spoVG). To determine whether SprX interacts with the yabJ-spoVG mRNA in vitro, we used enzymatic probes to monitor the structural changes of yabJ-spoVG mRNA induced by SprX binding. For this purpose, we designed and produced a 167 nt-long mRNA construct, yabJspoVGiei, which contains the ribosome binding site (RBS) of spoVG and the first 86 nts of the spoVG ORF. The inventors subjected the yabJ-spoVGiei alone or in complex with SprX to statistical nuclease SI (specific for single-stranded RNAs) and to RNase VI cleavages (specific for double-stranded RNAs). The presence of many SI cleavages in the region A558-A630 of yabJ-spoVGiei mRNA supports the existence of unpaired single-strand domains. These data indicate that the proposed yabJ-spoVG mR A region that binds with SprX is in fact accessible for interaction. SprX- induced structural changes on the yabJ-spoVGi ei mRNA are located from A563 to U621. Si cleavages disappeared at positions A563-A578 and A602- A616. Several weak Vi cleavages situated at A593, U605, and G580-G584 disappeared within the mRNA. Moreover, upon duplex formation a strong Vi cut at position G613 and two weak ones at G596 and U599 appeared in the mRNA. Therefore, we can conclude that the binding of SprX induces structural changes encompassing the Shine-Dalgarno and AUG initiation codon of spoVG in yabJ-spoVG mRNA. The inventors investigated the structure of free SprX (nts 1-150) using chemical and enzymatic probes. The inventors used lead to probe the SprX solution structure, as it cleaves to accessible singlestranded RNA, RNase Vi, and nuclease Si . The simultaneous presence of Vi and Si cleavages at the same nucleotide position within Hl-Ll and H2'-L2' may be explained by the coexistence of two alternating structures at the SprX 5 '-end. Hairpins H3-L3 and H4-L4 in the 3 ' region of SprX are however well-supported by the probing patterns. Moreover, a C-rich nucleotide sequence situated within the SprX L3 loop has been proposed to interact through base pairings with the spoVG SD sequence. The inventors tested the importance of the SprX L3 loop in the interaction between SprX and yabJ-spoVG mRNA by mutating the 5 '-CUCCCCG-3 ' sequence (SEQ ID NO: 5) of the SprX L3 loop termed SprX_mutL3. Mutated SprX failed to induce structural changes in the spoVG RBS of the yabJspoVGi₆₇ mRNA, thus emphasizing the importance of that loop in the interaction with the yabJ-spoVG target. Taken together, these mutational data support the proposed model of SprX-yabJ-spoVG mRNA interaction, which involves pairings between the SprX L3 loop and the spoVG RBS of the yabJ-spoVG mRNA.

SprX specifically reduces spoVG translation initiation of the yabJ-spoVG operon mRNA.

Since SprX interacts with the spoVG RBS of yabJ-spoVG mRNA covered by the ribosomes during translation initiation, we conjectured that SprX could prevent ribosome loading on the spoVG RBS. We tested this hypothesis by performing toeprint analysis. We formed a ternary initiation complex consisting of 70S ribosomes, initiator tRNA⁰^¹, and yabJ- spoVG mRNA. The ribosome blocked the elongation of reverse transcription, and produced a toeprint 16 nts downstream from the AUG initiation codon of spoVG. SprX significantly reduced this toeprint in a concentration-dependent manner, indicating that in vitro SprX inhibits ribosome binding onto the spoVG RBS of yabJ-spoVG mRNA. These results are in agreement with our in vivo data, which show that SprX reduces SpoVG protein expression. Interestingly, SprX_mutL3 failed to prevent ribosome loading onto the spoVG ofyabJ-spoVG mRNA, indicating the essential role of the SprX L3 loop in regulating SpoVG expression. Since yabJ-spoVG is a bicistronic mRNA, the inventors next tested whether SprX might also prevent ribosome loading onto the yabJ RBS of the yabJ-spoVG mRNA. One ribosome toeprint was detected 16 nts downstream from the predicted yabJ initiation codon. The addition of increasing concentrations of SprX did not alter the ribosome binding. These results show that in vitro, SprX specifically inhibits SpoVG translation initiation by antisense pairings with the spoVG RBS of the yabJ-spoVG mRNA, but it has no effect on YabJ translation initiation.

To assess the functional importance of the SprX L3 loop in vivo, we tested whether SprX_mutL3 is able to regulate SpoVG protein expression in vivo. To address this, we transformed strain AsprX by pCN3%sprX_mutL3 and monitored SpoVG protein levels using Western blots. Overexpressing SprX strongly reduces SpoVG protein levels. In contrast, overexpressing SprX_mutL3 was unable to inhibit SpoVG protein expression, resulting in SpoVG protein levels identical to those in the strain AsprX transformed with pCN38. SprX_mutL3 thus failed to regulate SpoVG expression, demonstrating the importance of the SprX loop in regulating SpoVG in vivo. Northern blots indicated that the SprX_mutL3 and wt SprX are expressed at similar levels from pCN38, indicating that the absence of SpoVG regulation by SprX_mutL3 is not due to its in vivo instability. Taken altogether, our in vitro and in vivo results show that SprX inhibits SpoVG expression at the translational level by antisense pairings occurring between a C-rich loop from SprX and the spoVG RBS from yabJ-spoVG mRNA.

SprX influences glycopeptide antibiotic resistance by regulating SpoVG.

The deletion of the yabJ-spoVG operon reduces the resistance to glycopeptide antibiotics [7]. Complementation by a vector allowing the expression of only the SpoVG protein was sufficient to restore the antibiotic resistance [7]. We tested whether the deletion of yabJspoVG would affect resistance to Teicoplanin and Vancomycin in the S. aureus strain HG001. In agreement with a previous report [7], deletion of the yabJ-spoVG operon reduces bacterial resistance to both antibiotics. To see if SprX modulates bacterial resistance to glycopeptide antibiotics through control of SpoVG levels, we tested whether SprX_mutL3 (already shown to not influence SpoVG expression) could reduce this resistance. As demonstrated in this report, the overexpression of SprX reduced S. aureus resistance to Teicoplanin and to Vancomycin. In contrast, since the strain transformed by pCN3%sprX_mutL3 exhibits the same resistance levels as the one transformed with pCN38, the over-expression of SprX_mutL3 failed to modify their antibiotic resistance. Collectively, our results emphasize the importance of the C-rich sequence within the third loop of SprX in regulation of SpoVG and the S. aureus resistance to two glycopeptides antibiotics.

Discussion

Here, we report on the function of SprX (alias RsaOR), a recently-identified small

RNA expressed from an S. aureus pathogenicity island. We provide evidence that SprX shapes S. aureus resistance to Vancomycin and Teicoplanin glycopeptides, two invaluable antibiotics for treatment of methicillin-resistant staphylococcal infections. This is the first time that a regulatory RNA has been shown to be involved in antibiotic resistance in a major human bacterial pathogen.

To investigate the mechanism underlying this modification in antibiotic resistance, we searched for SprX targets. 2D-DiGE analysis of wild-type (wt) and isogenic sprX deletion strains allowed us to identify the SpoVG protein, whose expression is reduced by SprX. SpoVG is translated from bicistronic yabJ-spoVG mRNA transcribed from a sigma Independent promoter that is responsible for yabJ-spoVG mRNA accumulation during bacterial growth [6]. This operon is involved in capsule formation; controls the expression of extracellular lipase, nuclease, and protease; and is also involved in bacterial resistance to methicillin and glycopeptides [6,7,10]. In fact, SpoVG (not YabJ) is the major regulator of the yabJ-spoVG operon [7,10] and on its own it can rescue the phenotypes related to the deletion of the yabJ-spoVG operon. However, the molecular mechanisms underlying SpoVG action in these biological pathways remain unknown. Recently, it was reported that SpoVG is a DNA- binding protein supporting the hypothesis that it could act as a transcriptional factor [9]. In this report, we found that by reducing SpoVG expression levels, SprX affects S. aureus resistance to two glycopeptide antibiotics. Further studies on the regulation of the additional phenotypes related to yabJ-spoVG will be necessary to fully understand the importance of SprX in the regulation of other bacterial processes. We have uncovered the mechanism by which SprX lowers SpoVG expression. SprX interacts by antisense pairings with the spoVG ribosomal binding site (including its SD and AUG initiation codon) of the yabJ-spoVG mRNA. By mutational analysis, we identified the functional sequence within SprX that interacts with spoVG RBS: a C-rich sequence situated in an accessible loop within the SprX structure. This functional sequence contains an UCCC motif, a specific conserved signature that has been detected in several previously-studied S. aureus mRNAs [14]. This reinforces the notion that gene expression regulation by these sRNAs in S. aureus occurs through a shared mechanism. In vitro, SprX-mediated downregulation of SpoVG translation does not require any additional factors. This result is in agreement with previously-reported S. aureus sRNAs that overcome the Hfq protein requirement for sRNA-mRNA duplex stabilization in enterobacteria [27].

The SprX-yabJ-spoVG mRNA interaction inhibits SpoVG translation initiation by preventing the binding of ribosomes to the spoVG RBS. The translation initiation inhibition is a strategy commonly used by bacterial sRNAs to control gene expression. sRNA pairing with mRNA targets can enhance or repress targeted gene translation [19,28,29]. Generally, duplex formation between the mRNA target and sRNAs induces mRNA target degradation through the recruitment of bacterial ribonucleases [30]. Here, we show that SprX inhibits SpoVG translational initiation without inducing yabJ-spoVG mRNA degradation. Such strategy was shown to be sufficient for gene silencing in both Gram-negative and -positive bacteria [23,31]. Furthermore, the bicistronic yabJ-spoVG mRNA is cleaved by an unknown mechanism into two separate transcripts that correspond to the yabJ and the spoVG mRNAs. In vivo, SpoVG cannot be translated from the spoVG mRNA because the mRNA cleavage occurs downstream from its SD nucleotide sequence [7]. The physiological functions of the yabJ-spoVG mRNA processing remain to be identified, but this cleavage could serve to irreversibly inhibit spoVG expression. The full-length yabJ-spoVG mRNA and spoVG mRNA are observed at similar levels in both wt and AsprX strains. The inventors measured the levels of the yabJ transcript and determined that SprX has no effect on its amount. This suggests that the internal processing ofyabJ-spoVG mRNA is SprX-independent.

Our results raised an important question concerning the combination of sRNA- mediated translational inhibition with mRNA degradation in S. aureus. mRNA-sRNA interactions can result in mRNA target degradations, therefore gene silencing becomes irreversible, leading to the elimination of target mRNAs and sRNAs. In contrast, in the absence of sRNA-triggered mRNA degradation, mRNA target translation would rapidly resume once the sRNAs released target repression. Moreover, as SpoVG is translated from bicistronic yabJ-spoVG mRNA, sRNA-triggered operon mRNA degradation would also affect the expression of the operon's first protein, YabJ. We hypothesize that the strategy wherein SprX inhibits translation of SpoVG but without promotion of mRNA degradation, may allow for discoordinate gene expression in the yabJ-spoVG operon. Such a strategy of specific trans lational repression of a targeted gene within an operon while not modifying other operon gene expressions was described in E. coli, where Spot42 specifically inhibits galK of galETKM operon [31]. Since SprX binding on the RBS of spoVG does not affect yabJ-spoVG mRNA levels, specific repression of SpoVG expression can occur without affecting YabJ translation. Indeed, we showed in vitro that SprX does not influence YabJ translation. Since the conditions of YabJ protein expression are not known [10], further studies will be necessary to investigate the conditions of YabJ expression in vivo and to show whether SprX sRNA is involved in its regulation in vivo.

In fact, sRNA-mediated operon control adds an additional layer to gene expression regulation. Diverse sRNA mechanisms for the adjustment of operon expression have been discovered. In addition to the aforementioned mechanism of specific translational inhibition of target operon genes, sRNA could also influence operon mRNA stability. Indeed, sRNAs could trigger degradation of the entire operon mRNA [32], or of just a part of the operon, releasing a translationally-active mRNA fragment [33]. Furthermore, sRNA could also induce operon-mRNA cleavages [34]. We presume that further studies on sRNA-mediated regulation of operon expression will help us to understand the complexity of their regulation, and will reveal new mechanisms of action.

Until now, sRNAs were described as being involved in the regulation of diverse cellular processes, including pathogenicity control. An S. aureus paradigm for this emerging and expanding class of regulatory RNAs is RNAIII, the effector of the global agr regulon that controls the synthesis of multiple virulence factors [14,20-22]. RNAIII is produced at the end of the exponential phase and allows for the transition between synthesis of surface-associated proteins and secreted factors [35]. Others regulatory sRNAs such as SprD and Ssr42 have been shown to be essential for the virulence of S. aureus in an animal model of infection [23,24]. SprD was shown to regulate the expression of an immune evasion molecule (Sbi) secreted by bacteria to impair the host immune responses. Recently, a case of antibiotic resistance regulation by a riboswitch was described [36]. In that report, a riboswitch (a 5 ' leader sequence within mR A) was shown to control the translation of the mRNA-encoding aminoglycoside adenyl-transferase enzymes that confer resistance to aminoglycoside antibiotics. Indeed, drug-binding to the mRNA leader releases the translation repression imposed by the riboswitch and induces bacterial resistance to aminoglycosides. Here, we report the first case other than a riboswitch of a small RNA involved in bacterial resistance to antibiotics. Our study thereby emphasizes the importance of sRNA control in bacterial pathogenesis. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E (2006) Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet 368: 874-885.

2. Boger DL (2001) Vancomycin, teicoplanin, and ramoplanin: synthetic and mechanistic studies. Med Res Rev 21 : 356-381.

3. Courvalin P (2006) Vancomycin resistance in gram-positive cocci. Clin Infect Dis 42 Suppl 1 : S25-34.

4. Arthur M, Reynolds PE, Depardieu F, Evers S, Dutka-Malen S, et al. (1996) Mechanisms of glycopeptide resistance in enterococci. J Infect 32: 11-16.

5. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, et al. (1997) Methicillin- resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 40: 135-136.

6. Meier S, Goerke C, Wolz C, Seidl K, Homerova D, et al. (2007) sigmaB and the sigmaBdependent arlRS and yabJ-spoVG loci affect capsule formation in Staphylococcus aureus. Infect Immun 75: 4562-4571.

7. Schulthess B, Meier S, Homerova D, Goerke C, Wolz C, et al. (2009) Functional characterization of the sigmaB-dependent yabJ-spoVG operon in Staphylococcus aureus: role in methicillin and glycopeptide resistance. Antimicrob Agents Chemother 53: 1832-1839. 8. Matsuno K, Sonenshein AL (1999) Role of SpoVG in asymmetric septation in Bacillus subtilis. J Bacteriol 181 : 3392-3401.

9. Jutras BL, Chenail AM, Rowland CL, Carroll D, Miller MC, et al. (2013) Eubacterial SpoVG Homologs Constitute a New Family of Site-Specific DNA-Binding Proteins. PLoS One 8: e66683.

10. Schulthess B, Bloes DA, Francois P, Girard M, Schrenzel J, et al. (2011) The sigmaBdependent yabJ-spoVG operon is involved in the regulation of extracellular nuclease, lipase, and protease expression in Staphylococcus aureus. J Bacteriol 193: 4954-4962.

11. Schulthess B, Bloes DA, Berger-Bachi B (2012) Opposing roles of sigmaB and sigmaBcontrolled SpoVG in the global regulation of esxA in Staphylococcus aureus. BMC

Microbiol 12: 17.

12. Beisel CL, Storz G (2010) Base pairing small RNAs and their roles in global regulatory networks. FEMS Microbiol Rev 34: 866-882.

13. Pichon C, Felden B (2005) Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains.

Proc Natl Acad Sci U S A 102: 14249-14254.

14. Geissmann T, Chevalier C, Cros MJ, Boisset S, Fechter P, et al. (2009) A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence mot if for regulation. Nucleic Acids Res 37: 7239-7257.

15. Marchais A, Naville M, Bohn C, Bouloc P, Gautheret D (2009) Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles. Genome Res 19: 1084-1092.

16. Abu-Qatouseh LF, Chinni SV, Seggewiss J, Proctor RA, Brosius J, et al. (2010) Identification of differentially expressed small non-protein-coding RNAs in Staphylococcus aureus displaying both the normal and the small-colony variant phenotype. J Mol Med (Berl) 88: 565-575.

17. Beaume M, Hernandez D, Farinelli L, Deluen C, Linder P, et al. (2010) Cartography of methicillin-resistant S. aureus transcripts: detection, orientation and temporal expression during growth phase and stress conditions. PLoS One 5: el 0725.

18. Bohn C, Rigoulay C, Chabelskaya S, Sharma CM, Marchais A, et al. (2010)

Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism. Nucleic Acids Res 38: 6620-6636.

19. Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136: 615-628. 20. Morfeldt E, Taylor D, von Gabain A, Arvidson S (1995) Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J 14: 4569-4577.

21. Boisset S, Geissmann T, Huntzinger E, Fechter P, Bendridi N, et al. (2007) Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism. Genes Dev 21 : 1353-1366.

22. Chevalier C, Boisset S, Romilly C, Masquida B, Fechter P, et al. (2010) Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation. PLoS Pathog 6: el000809.

23. Chabelskaya S, Gaillot O, Felden B (2010) A Staphylococcus aureus small RNA is required for bacterial virulence and regulates the expression of an immune-evasion molecule. PLoS Pathog 6: el000927.

24. Morrison JM, Miller EW, Benson MA, Alonzo F, 3rd, Yoong P, et al. (2012) Characterization of SSR42, a novel virulence factor regulatory RNA that contributes to the pathogenesis of a Staphylococcus aureus USA300 representative. J Bacteriol 194: 2924-2938.

25. Tjaden B (2012) Computational identification of sRNA targets. Methods Mol Biol 905: 227-234.

26. Cao Y, Wu J, Liu Q, Zhao Y, Ying X, et al. (2010) sRNATarBase: a comprehensive database of bacterial sRNA targets verified by experiments. RNA 16: 2051- 2057.

27. Jousselin A, Metzinger L, Felden B (2009) On the facultative requirement of the bacterial RNA chaperone, Hfq. Trends Microbiol 17: 399-405.

28. Frohlich KS, Vogel J (2009) Activation of gene expression by small RNA. Curr Opin Microbiol 12: 674-682.

29. Storz G, Vogel J, Wassarman KM (2011) Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 43: 880-891.

30. Lalaouna D, Simoneau-Roy M, Lafontaine D, Masse E (2013) Regulatory RNAs and target mRNA decay in prokaryotes. Biochim Biophys Acta 1829: 742-747.

31. Moller T, Franch T, Udesen C, Gerdes K, Valentin-Hansen P (2002) Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon. Genes Dev 16: 1696-1706.

32. Antal M, Bordeau V, Douchin V, Felden B (2005) A small bacterial RNA regulates a putative ABC transporter. J Biol Chem 280: 7901-7908.

33. Desnoyers G, Morissette A, Prevost K, Masse E (2009) Small RNA-induced differential degradation of the polycistronic mRNA iscRSUA. EMBO J 28: 1551-1561. 34. Opdyke JA, Fozo EM, Hemm MR, Storz G (2011) RNase III participates in GadYdependent cleavage of the gadX-gadW mRNA. J Mol Biol 406: 29-43.

35. Novick RP, Ross HF, Projan SJ, Kornblum J, Kreiswirth B, et al. (1993) Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12: 3967-3975.

36. Jia X, Zhang J, Sun W, He W, Jiang H, et al. (2013) Riboswitch control of aminoglycoside antibiotic resistance. Cell 152: 68-81.

37. Charpentier E, Anton Al, Barry P, Alfonso B, Fang Y, et al. (2004) Novel cassette- based shuttle vector system for gram-positive bacteria. Appl Environ Microbiol 70: 6076- 6085.

38. Pohl K, Francois P, Stenz L, Schlink F, Geiger T, et al. (2009) CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression. J Bacteriol 191 : 2953-2963.

39. Bruckner R (1997) Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol Lett 151 : 1-8.

40. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

Claims

CLAIMS:

1. An isolated, synthetic or recombinant oligonucleotide comprising a nucleic acid sequence ranging from nucleic acid at position 75 to nucleic acid at position 111 in SEQ ID NO: 1, a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 115 in SEQ ID NO: 2, or a nucleic acid sequence ranging from nucleic acid at position 79 to nucleic acid at position 114 in SEQ ID NO: 3.

2. A method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof, comprising the step of administering to said subject the oligonucleotide according to claim 1.

3. A method of treating S. aureus infection in a subject in need thereof, comprising the step of administering to said subject the oligonucleotide according to claim 1 in combination with at least one glycopeptides antibiotic.

4. The method according to claim 3 wherein said glycopeptide antibiotic agent is selected from the group consisting of teicoplanin, vancomycin, telavancin, bleomycin, ramoplanin, and decaplanin.

5. A pharmaceutical composition comprising the oligonucleotide of the invention and a pharmaceutical acceptable carrier for use in a method for enhancing the clinical efficacy of glycopeptide antibiotics for the treatment of a S. aureus infection in a subject in need thereof.

6. A method of identifying a S. aureus infected subject having or at risk of having or developing a glycopeptide antibiotic resistance which comprises the steps of: i) measuring the expression level of SprX in a biological sample obtained from said subject,

(ii) comparing the expression level measured at step i) with a reference value,

(iii) detecting differential in the SprX expression level between the biological sample and the reference value is indicative that said subject is having or at risk of having or developing a glycopeptide antibiotic resistance.