Related Applications

The present application claims priority to U.S. Provisional Patent Application No. 61/544,390 filed on October 7, 2011. The U.S. Provisional Patent Application is incorporated herein by reference in its entirety.

Background of the Invention

In all living organisms, gene expression is modulated by proteins and small regulatory RNAs (sRNAs). sRNAs have been identified in many bacteria and a few of them act by binding to proteins and modulating their activity. Most sRNAs function by pairing with target mRNAs, modifying mRNA transcription, stability or translation (Waters et al, Cell, 2009, 136: 615-628). These base pairing sRNAs fall into two categories, the trans- and cz ^' s-encoded. The trans sRNAs are encoded at genomic locations distant from the mRNAs that they regulate and share only limited complementarity with their targets. The czs-encoded antisense RNAs (asRNAs) are transcribed from the DNA strand opposite another gene and have perfect complementarity with their target. Recently, a considerable number of natural asRNAs has been detected in various organisms, including human cells (He et al, Science, 2008, 322: 1855-1857).

In bacteria, antisense transcription was first demonstrated almost 50 years ago and was considered to be the exception rather than the rule. Accumulating evidence from transcriptome studies suggests that extensive antisense transcription occurs in bacteria, producing many cz ^' s-sRNAs of various sizes (Georg et al, Microbiol Mol Biol Rev, 2011, 75: 286-300). In the human pathogen Helicobacter pylori, asRNAs were detected for 46% of all annoted open reading frames (Sharma et al, Nature, 2010, 464: 250- 255), indicating that bacterial transcriptomes are unexpectedly complex. The functional relevance of this massive antisense transcription, however, is poorly understood but these asRNAs are predicted to have significant, as yet largely unexplored, impacts on gene expression. Also, information on the molecular mechanisms of individual asRNAs is only slowly growing. In bacteria, mechanisms of asRNAs action include alteration of target RNA stability, transcription interference or termination, as well as translation modulation (Georg et al, Microbiol Mol Biol Rev, 2011, 75: 286-300). The Gram-positive bacterium Staphylococcus aureus (S. aureus) is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and sRNAs. S. aureus expresses at least 91 sRNAs (Felden et al, PLoS Pathog, 2011, 7: el002006) including asRNAS. Recent high-resolution transcriptome analysis has detected a large proportion of these asRNAs among the inventoried S. aureus sRNAs (Pichon et al., Proc Natl Acad Sci U S A, 2005, 102: 14249-14254; Abu-Qatouseh et al., J Mol Med,

2010, 88: 565-575; Geissmann et al., Nucleic Acids Res, 2009, 37: 7239-7257; Bohn et al., Nucleic Acids Res, 2010, 38: 6620-6636). In S. aureus, many asRNAs are expressed from Pathogenicity Islands (Pis) and mobile elements including plasmids and transposons. Since the SaPIs are the repository of toxins, adherence and invasion factors, superantigens and secretion systems, the location of sRNAs in the Pis suggests that they play important roles during S. aureus infections. In addition to all the Small pathogenicity island mas (Pichon et al., Proc Natl Acad Sci U S A, 2005, 102: 14249- 14254), at least four asRNAs are expressed from the Pis (Felden et al., PLoS Pathog,

2011, 7: el002006). One of these asRNAs, Tegl52, was detected in strains N315 by high throughput sequencing (Beaume et al., PLoS One, 2010, 5: el 0725) and predicted, based on its overall genomic location, to be complementary with SprAl 3 '-end. Because as shown herein, Tegl52 acts as a functional asRNA against SprAl, it was renamed SprAl _A s (wherein AS: antisence). Sequence comparison suggests that the 'SprAl /SprAl AS' pair forms a type I "toxin-antitoxin" (TA) module (Fozo et al., Nucleic Acids Res, 2010, 38: 3743-3759). SprAl was identified by computer searches combined with transcriptome analysis and its expression verified by Nothern blots (Pichon et al, Proc Natl Acad Sci U S A, 2005, 102: 14249-14254).

Because of the emergence of antibiotic-resistant pathogens worldwide, a number of infectious diseases have become difficult to treat. This threatening situation is worsened by the fact that very limited progress has been made in developing new and potent antibiotics in recent years. Some of the promising candidates are antimicrobial peptides produced by bacteria (bacteriocins), which hold a great potential in controlling antibiotic-resistant pathogens. In the invention to be presented in the following sections, novel antimicrobial peptides derived from an SprAl -encoded bacteriocin isolated from S. aureus are described. Summary of the Invention

The present invention encompasses the recognition by the inventors that the SprAl -encoded bacteriocin isolated from S. aureus has antimicrobial and cytolytic properties. Accordingly, in one aspect, the present invention provides an isolated SprAl antimicrobial peptide having the amino acid sequence set forth in SEQ ID NO: 1, a biologically active fragment thereof, or an antimicrobial variant thereof.

In certain embodiments, the biologically active fragment has the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof. For example, the biologically active fragment can have an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

In certain embodiments, the antimicrobial variant is a variant of SEQ ID NO: 2. For example, the antimicrobial variant can have an amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. For example, the antimicrobial variant can have the amino acid sequence set forth in SEQ ID NO: 15.

The present invention also provides a fusion protein comprising an isolated SprAl antimicrobial peptide as described herein.

In another aspect, the present invention relates to an isolated SprAl antimicrobial peptide or fusion protein as described herein for use as a therapeutic agent, in particular as an antimicrobial agent and/or as a cytolytic or pore-forming agent.

In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of at least one isolated SprAl antimicrobial peptide or fusion protein as described herein, and a pharmaceutically acceptable carrier or excipient.

In a related aspect, the present invention relates to the use of an isolated SprAl antimicrobial peptide or fusion protein as described herein for the manufacture of a medicament, in particular a medicament intended to be used as antimicrobial agent or as a cytolytic or pore-forming agent. The invention also provides a product comprising at least one isolated SprAl antimicrobial peptide or fusion protein as described herein or a pharmaceutical composition thereof, wherein the product is selected from the group consisting of bandages, plasters, sutures, adhesives, wound dressings, implants, contact lenses, cleaning solutions, storage solutions, cleaning products, personal care products, and cosmetics.

In still another aspect, the present invention provides a method for preventing or treating a microbial infection in a subject, the method comprising a step of: administering to said subject an effective amount of at least one isolated SprAl antimicrobial peptide or fusion protein described herein or a pharmaceutical composition thereof.

In certain embodiments, the microbial infection is caused by a gram-negative bacterium or by a gram-positive bacterium.

The gram-negative bacterium may be a bacterium selected from the group consisting of bacteria of the genus Salmonella, bacteria of the genus Shigella, and bacteria of the genus Escherichia. The gram-positive bacterium may be a bacterium of the genus Staphylococcus. The therapeutic methods according to the present invention may be applied to humans or other mammals. In certain preferred embodiments, the subject is human.

In yet another aspect, the present invention provides a method for preventing or eliminating microbial contamination of the surface of an object, said method comprising a step of: contacting the surface of said object with an effective amount of at least one isolated SprAl antimicrobial peptide or fusion protein described herein or a pharmaceutical composition thereof.

In certain embodiments, the microbial contamination is caused by a gram-negative bacterium or by a gram-positive bacterium.

The gram-negative bacterium may be a bacterium selected from the group consisting of bacteria of the genus Salmonella, bacteria of the genus Shigella, and bacteria of the genus Escherichia. The gram-positive bacterium may be a bacterium of the genus Staphylococcus.

In certain embodiments, the object to which the decontamination method is applied is selected from sutures, implants, contact lenses, catheters, syringes, and gloves. In yet another aspect, the present invention relates to a SprAl AS antisense R A molecule having the sequence set forth in SEQ ID No: 12:

TATAATTGAGACAACGAAAAUAAGUAUUUACUUAUACACCAAUCCCCUCA CUAUUUGCGGUAGUGAGGGGAUUUUUAUU

for use as an inhibitor of the internal translation of an SprAl RNA molecule having the sequence set forth in SEQ ID NO: 13:

TATAATAGTAGAGUCGCCUAUCUCUCAGGCGUCAAUUUAGACGCAGAGAG GAGGUGUAUAAGGUGAUGCUUAUUUUCGUUCACAUCAUAGCACCAGUCA UCAGUGGCUGUGCCAUUGCGUUUUUUUCUUAUUGGCUAAGUAGACGCAA UAC AAAAUAGGUGACAUAUAGCCGCACCAAUAAAAAUCCCCUC ACUACCG CAAAUAGUGAGGGGAUUGGUGU.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments. Brief Description of the Drawing

Figure 1. Genomic location of sprAl and sprAl _A s, monitoring their lengths, boundaries and expression profiles in vivo during S. aureus growth. (A) Location of sprAl /sprAl AS in Pathogenicity Island (PI, SaPIn3) of S. aureus strain Newman genome. (B) Right panels: Northern blots detection of sprAl and SprAl _A s in a wt and a double deletion mutant. Left panels: Lengths evaluation of SprAl and SprAl AS adjoining synthetic labelled RNAs of known lengths combined to 5 '-end determinations by RACE mapping. The nucleotide numberings of SprAl and SprAl _A s ends are indicated onto the S. aureus Newman genomic sequence. (C-D) Monitoring SprAl and SprAl AS expression profiles during S. aureus growth. The expression levels of SprAl and SprAl _A s during a 10-hour growth of S. aureus Newman strain detected by Northern blots. As loading controls, the blots were also probed for tmRNA. The growth curves of the Newman strain is presented, with the quantification of SprAl (black triangles) and SprAl AS (grey diamonds) levels relative to the amount of tmRNA from the same RNA extraction. AU =arbitrary units. (E) Determination of the in vivo concentrations of SprAl and SprAl _A s in a wild-type S. aureus Newman strain during growth detected by Northern blots. The quantification of SprAl and SprAl _A s in vivo levels (left panels) were performed relative to increasing amounts of synthetic, gel purified SprAl and SprAl AS RNAs (right panels). Figure 2. Detection of the interaction between SprAl and SprAl _A s in vivo and assessing their binding constants. (A) Demonstration of the expression of strepto- tagged SprAl in vivo. Northern blot analysis of SprAl (WT or tagged) expression at mid-exponential (OD ₆ oonm = 3) and stationary (OD ₆ oonm = H) phases in wild-type Newman (WT), deletion mutant Newman AsprAl/sprAl s and Newman AsprAl- AsprAl _A s pCN35Q. STsprAllsprAl _A s- (B) Northern blot analysis of the affinity purification fractions extracted from either Newman AsprAl-AsprAlAs pCN35 .STsprAl/sprAl _A s, or from Newman pCN35 .sprAl _AS as a negative control. Labelled DNA probes were used for SprAl (wt and tagged), for SprAl _A s and for tmRNA, used as an internal negative control. (C) Complex formation between labeled SprAl and SprAl _A s and (D) between labeled SprAl and SprAl _A s detected by native gel retardation assays. Purified labeled SprAl _AS (C) or SprAl (D) with increasing amounts of unlabeled SprAl (C) or unlabeled SprAl _AS (D). The two diamonds indicate the ' SprAl _A s SprAl ' (C) or the 'SprAl/SprAl _AS '. (D) molar ratios used to perform the competition assays with a 1000-fold molar excess of yeast total tRNAs or with a 20-fold molar excess of unlabeled SprAl _AS (C) or SprAl (D), respectively. The apparent binding constant between SprAl _AS and SprAl was inferred from these data.

Figure 3. SprAl and SprAl _AS both interact by their 5' non-overlapping domains. Complex formation between labelled SprAl _A s with either 5 'SprAl (A) or 3'SprAl (B) and between labelled SprAl with either 5'SprAl _AS (C) or 3'SprAl _AS (D), detected by native gel retardation assays. Purified labeled SprAl _AS with increasing amounts of unlabeled 5 'SprAl (A) or 3 'SprAl (B) Purified labeled SprAl with increasing amounts of unlabeled 5'SprAl _AS (C) or 3'SprAl _AS (D) The apparent binding constants between the RNAs were inferred from these data. The black diamonds indicate the SprAl _AS */5 'SprAl (A) and the SprAl */5 'SprAl _AS (C) molar ratios used to perform the competition assays with a 2000-fold molar excess of polyU RNAs or with a 20-fold molar excess of unlabeled 5 'SprAl (A) or SprAl (C).

Figure 4. SprAl recruits S. aureus ribosomes, is translated in vitro and SprAl _AS hinders SprAl translation by its 5' non-overlapping domain. (A) S. aureus ribosome toeprints onto the SprAl RNA (WT SprAl), and the disappearance of the toeprints onto SD-mutated SprAl . In the presence of SprAl _AS at a two-to-one molar ratio, the toeprints onto SprAl disappear. The experimentally-determined toeprints are indicated with a black line and the RT stop onto SprAl, in the presence of SprAl _A s, is indicated by an arrowhead. 'U', Ά', 'G' and 'C refer to the SprAl sequencing ladders. (B) Detection of the formation of a ~3kDa SprAl -encoded polypeptide by in vitro translation of SprAl R A, but not when in complex with sprAl _A s at a one to one molar ratio or when the predicted internal SD sequence is mutated. Autoradiogram of in vitro translation reactions using [ ³⁵ S]-methionine detected on a 16% tricine SDS PAGE. The 'SprAl -encoded' polypeptide is indicated by an arrowhead. 30 pmol of purified wt SprAl or SD-mutated SprAl were used in the presence or absence of 60 pmol of SprAl _A s or 300 pmol of either 5'SprAl _A s or 3 'SprAl AS, as indicated. (C) Northern blot analysis of SprAl and SprAl _A s in Newman pCN35 (empty plasmid) and Newman pCN35Q.sprAl _A s during growth. 5S rRNAs are the loading controls.

Figure 5. The cis-encoded SprAl _A s RNA operates in trans to downregulate the SprAl-encoded peptide expression in vivo. (A) Detection of the ~5kDa SprAl - encoded flagged peptide at early and mid-exponential phases of growth in strains Newman ' AsprAl-AsprAl _A s pCN34 sprAltag pCN35' and 'AsprAl-AsprAlAs pCN34 sprAltag pCN35Q.sprAl _AS ' by immunoblots using anti- FLAG antibodies. (B) Northern blot analysis monitoring SprAl -tagged (upper panel) and SprAl _A s (lower panel) RNA expression levels at identical phases of growth. 5S rRNAs are the internal loading controls. Figure 6. The SprAl-encoded polypeptide is a hemolysin against human cells and outline of its translational control. (A) Hemolytic activity of the SprAl-encoded polypeptide. ½ serial dilution of peptide in ΙΟΟμί PBS (SprAl peptide/upper row or control non-hemo lytic peptide/lower row) is added to all wells except for the positive control in which PBS is replaced by ΙΟΟμί of water. 3% washed RBC are added to each well and incubated 1 hour at room temperature. The negative control corresponds to 100 μΙ_, washed RBCs incubated with 100 μΙ_, of PBS. RBCs sedimentation indicates the absence of hemolysis, whereas a red supernatant implies hemolysis. (B) Hemolytic activity of the SprAl-encoded polypeptide on human RBCs versus sheep RBCs. Human or sheep RBCs are incubated at 37°C with either 5 μΙ_, of 50% isopropanol (negative control, 'minus' sign) or with 5 μΐ _^ of 50% isopropanol containing 3nmol of the chemically synthesized SprAl-encoded peptide. The SprAl-encoded peptide induces high and very little hemolysis of human and sheep RBCs, respectively; suggesting SprAl possesses a δ-like hemolytic activity. (C) Outline of the trans-regulation of SprAl internal translation by the cz ^' s-SprAl _A s, with emphasis to their contiguous genomic locations. SprAl internal ORF is green and the SprAl and SprAl AS 5' non- overlapping interacting domains are red. Their 3 '-overlapping domains are yellow. Upon duplex formation, SprAl AS 5 'domain pairs at and around the SprAl internal translation initiation signals (SD sequence and start codon, red) by unfolding pseudoknot PK1. During S. aureus growth, translation of the SprAl -encoded peptide is repressed by base pairings in Trans with the cz ^' s-SprAl _A s R A.

Figure 7 shows a picture of petri dishes of S. aureus and of Shigella flexneri prepared as described above for assessing the antimicrobial activity of the SprAl peptide. For comparison purposes, the petri dish obtained in the presence of the SprAl peptide and in the presence of cecropin PI (standard antimicrobial peptide) are presented side-by-side.

Figure 8 shows graphs used for the CMI calculation of SprAl peptide and of standard peptide cecropin PI in the case of S. aureus and of Shigella flexneri. Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

As used herein, the term "subject" refers to a human or another mammal {e.g., primate, mouse, rat, rabbit, dog, cat, horse, cow, pig, camel, and the like). In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an "individual" or a "patient". The terms "individual" and "patient" do not denote a particular age.

The term "treatment" is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about amelioration of the symptoms of the disease or condition; or (4) curing the disease or condition. A treatment may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively or additionally, a treatment may be administered after initiation of the disease or condition, for a therapeutic action. A "pharmaceutical composition" is defined herein as comprising an effective amount of at least one SprAl antimicrobial peptide according to the invention, and at least one pharmaceutically acceptable carrier or excipient.

As used herein, the term "effective amount" refers to any amount of a compound, agent, antibody, or composition that is sufficient to fulfil its intended purpose(s), e.g., a desired biological or medicinal response in a cell, tissue, system or subject.

The term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion, media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example "Remington 's Pharmaceutical Sciences", E.W. Martin, 18 ^th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety). In certain embodiments, the pharmaceutically acceptable carrier or excipient is a veterinary acceptable carrier or excipient.

The term "isolated?', as used herein in reference to a protein or polypeptide, means a protein or polypeptide, which by virtue of its origin or manipulation is separated from at least some of the components with which it is naturally associated or with which it is associated when initially obtained. By "isolated", it is alternatively or additionally meant that the protein or polypeptide of interest is produced or synthesized by the hand of man.

The terms "protein", "polypeptide" , and "peptide" are used herein interchangeably, and refer to amino acid sequences of a variety of lengths, either in their neutral (uncharged) forms or as salts, and either unmodified or modified by glycosylation, side-chain oxidation, or phosphorylation. In certain embodiments, the amino acid sequence is a full-length native protein. In other embodiments, the amino acid sequence is a smaller fragment of the full-length protein. In still other embodiments, the amino acid sequence is modified by additional substituents attached to the amino acid side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains such as oxidation of sulfydryl groups. Thus, the term "protein" (or its equivalent terms) is intended to include the amino acid sequence of the full-length native protein, or a fragment thereof, subject to those modifications that do not significantly change its specific properties. In particular, the term "protein" encompasses protein iso forms, i.e., analogs that are encoded by the same gene, but that differ in their pi or MW, or both. Such iso forms can differ in their amino acid sequence (e.g., as a result of allelic variation, alternative splicing or limited proteolysis), or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation) .

The term "analog", as used herein in reference to a protein or protein portion, refers to a polypeptide that possesses a function similar or identical to that of the protein or protein portion but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the protein or protein portion or a structure that is similar or identical to that of the protein or protein portion. Preferably, in the context of the present invention, a protein analog has an amino acid sequence that is at least 30%, more preferably, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of the protein or protein portion.

The term "homologous" (or "homology"), as used herein, is synonymous with the term "identity" and refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both compared sequences is occupied by the same base or same amino acid residue, the respective molecules are then homologous at that position. The percentage of homology between two sequences corresponds to the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when two sequences are aligned to give maximum homology. Homologous amino acid sequences share identical or similar amino acid sequences. Similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in a reference sequence. "Conservative substitutions" of a residue in a reference sequence are substitutions that are physically or functionally similar to the corresponding reference residue, e.g. that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" as described by Dayhoff et al. ("Atlas of Protein Sequence and Structure", 1978, Nat. Biomed. Res. Foundation, Washington, DC, Suppl. 3, 22: 354-352).

The term "protein fragment" refers to a polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues of the amino acid sequence of a protein. In the context of the present invention a fragment of the SprAl peptide comprises a sequence of 5 consecutive amino acid residues of the sequence of the SprAl peptide, preferably, at least about: 9, 10, 11, 12, 13, 14 or 15 consecutive amino acid residues of the SprAl peptide. The fragment of a protein may or may not possess a functional activity of the protein.

The term "biologically active", as used herein to characterize a fragment, a variant or a derivative of a protein or protein portion, refers to a molecule that retains at least one biological activity of the protein or protein portion. In the context of the present invention, the biological activity may be antimicrobial activity and/or hemolytic activity. For example, in many embodiments of the present invention, a biologically active fragment of the SprQl antimicrobial peptide is a fragment that retains the ability to prevent, inhibit or reduce the growth or function of a microorganism or to kill a microorganism. However, the fragment may or may not exhibit cytolytic activity.

The terms "approximately" and "about", as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context {e.g., where such number would exceed 100% of a possible value).

Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention provides SprAl antimicrobial peptides that can be used in a variety of applications.

I - SprAl Antimicrobial Peptides

The present invention provides several SprAl antimicrobial peptides. As used herein, the term "antimicrobial peptide" refers to a peptide which prevents, inhibits or reduces the growth or function of a microorganism or which kills a microorganism. The antimicrobial activity can be determined by any conventional method known in the art. In certain preferred embodiments of the present invention, an antimicrobial peptide has antibacterial activity. As used herein, the term "antibacterial activity" refers to the ability to kill bacteria (bactericidal activity) or to prevent, inhibit or reduce bacterial growth or function (bacteriostatic activity).

The term "SprAl antimicrobial peptide" more specifically refers to an antimicrobial peptide that is produced by the gram-positive bacterium Staphylococcus aureus, and that is encoded by a small regulatory RNA located in a pathogenicity island of the S. aureus genome. The term "SprAl antimicrobial peptide" also encompasses any antimicrobial peptide that can be derived from the SprAl -encoded antimicrobial peptide.

In particular, the present invention provides an isolated SprAl antimicrobial peptide having the following amino acid sequence:

MMLIF VHII AP VI SGC AI AFF S Y WL SRRNTK (SEQ ID NO: 1), or an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

The present inventors have shown that in addition to an antimicrobial activity, the peptide of SEQ ID NO: 1 also exhibits cytolytic activity against erythrocytes {i.e., hemolytic activity). The term "hemolytic activity" refers to the ability to lyse erythrocytes (i.e., red blood cells). They have also found that it is the C-terminal region of this peptide, and not its N-terminal region, that confers the biological activity to the peptide. In particular, they have determined that the N-terminal portion consisting of the first 16 amino acid residues of SEQ ID NO: 1 is neither antimicrobial nor hemolytic. Accordingly, the present invention also provides an isolated SprAl antimicrobial peptide having an amino acid sequence that is a biologically active fragment of SEQ ID NO: 1. In certain embodiments, the biologically active fragment is both antimicrobial and cytolytic. In other embodiments, the biologically active fragment is antimicrobial but exhibits a lower cytolytic activity than the SprAl antimicrobial peptide of SEQ ID NO: 1.

In certain preferred embodiments, the biologically active fragment has the following amino acid sequence: AI AFFSYWLSRRNTK (SEQ ID NO: 2), or a fragment thereof, such as lAFFSYWLSRRNTK (SEQ ID NO: 3), AFFSYWLSRRNTK (SEQ ID NO: 4), FFSYWLSRRNTK (SEQ ID NO: 5), FSYWLSRRNTK (SEQ ID NO: 6), SYWLSRRNTK (SEQ ID NO: 7), or YWL SRRNTK (SEQ ID NO: 8).

In certain applications (for example in antimicrobial therapy applied to human or other mammals), it is desirable for an antimicrobial peptide to efficiently prevent, inhibit or reduce microbial growth or function or to kill microorganisms without being significantly toxic to mammalian cells. It may also be desirable to increase the stability of an antimicrobial peptide to proteases. In order to obtain such antimicrobial peptides, the inventors have optimized biologically active fragments of SEQ ID NO: 1 by deletion of amino acid residues and/or substitution of amino acid residues.

Accordingly, the present invention provides an isolated SprAl antimicrobial peptide having an amino acid sequence that is an antimicrobial variant of a biologically active fragment of the SprAl antimicrobial peptide of SEQ ID NO: 1. The term "variant" used herein interchangeably in relation to a given peptide refers to a second peptide obtained by deletion of at least one amino acid residue in the first peptide, for example by deletion of 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues in the first peptide. The term "variant" also refers to a second peptide obtained by substitution of at least one amino acid residue of the first peptide (i.e., the replacement of one amino acid residue with another amino acid residue), for example substitution of 1, 2 or 3 amino acid residues of the first peptide. In certain embodiments, the at least one amino acid residue is replaced by an unnatural amino acid residue. As used herein, the term "unnatural amino acids" refers to non-proteinogenic amino acid acids. Examples of unnatural amino acid include, but are not limited to, β-amino acids, homo-amino acids (the prefixing of "homo" to the name of an amino acid indicates the addition of a methylene (CH ₂ ) group on the cc-carbon of an amino acid), proline and pyruvic acid derivatives, 3 -substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, Diamino acids, D- amino acids, and N-methyl amino acids. A variant according to the invention may be obtained by any combination of deletions and substitutions. In certain preferred embodiments, the SprAl antimicrobial peptide has an amino acid sequence that is an antimicrobial variant of the fragment of SEQ ID NO: 2. In particular, the antimicrobial variant may have an amino acid sequence selected from the group consisting of FFSYWLSRRTK (SEQ ID NO: 9), FFSWLSRRTK (SEQ ID NO: 10) and FFWLSRRTK (SEQ ID NO: 11). Alternatively, the antimicrobial variant may have the amino acid sequence FFWLRRT*K (SEQ ID NO: 15), wherein T* is a homohydroxy threonine.

The invention also relates to fusion proteins comprising at least one SprAl antimicrobial peptide (as described above) and a fusion partner. The term "fusion partner" refers to an amino acid sequence that confers to the fusion protein one or more desirable properties. Thus, a fusion partner may be an amino acid sequence that improves the expression of the SprAl antimicrobial peptide in host cells during preparation of the fusion protein, and/or an amino acid sequence that facilitates purification of the fusion protein, and/or an amino acid sequence that increases the stability of the fusion protein compared to the stability of the non- fused protein (e.g., to obtain a fusion protein with increased stability to proteases) and/or an amino acid sequence exhibiting a desirable biological activity (e.g., a targeting property).

Preparation of the SprAl Antimicrobial Peptides

The SprAl antimicrobial peptides according to the present invention may be prepared using any of a variety of suitable methods known in the art, including chemical synthesis and recombinant methods.

For example, the SprAl antimicrobial peptides of the invention may be prepared using standard chemical methods. Solid-phase peptide synthesis, which was initially described by R.B. Merrifield (J. Am. Chem. Soc. 1963, 85: 2149-2154) is a quick and easy approach to synthesizing peptides and peptidic molecules of known sequences. A compilation of such solid-state techniques may be found, for example, in "Solid Phase Peptide Synthesis" (Methods in Enzymology, G.B. Fields (Ed.), 1997, Academic Press: San Diego, CA, which is incorporated herein by reference in its entirety). Most of these synthetic procedures involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain. For example, the carboxy group of the first amino acid is attached to a solid support via a labile bond, and reacted with the second amino acid, whose amino group has, beforehand, been chemically protected to avoid self-condensation. After coupling, the amino acid group is deprotected, and the process is repeated with the following amino acid. Once the desired peptide is assembled, it is cleaved off from the solid support, precipitated, and the resulting free peptide may be analyzed and/or purified as desired. Solution methods, as described, for example, in "The Proteins ^' " (Vol. II, 3 ^rd Ed., H. Neurath et al. (Eds.), 1976, Academic Press: New York, NY, pp. 105-237), may also be used to synthesize the SprAl antimicrobioal peptides of the invention.

Crude synthesized antimicrobial peptides may be purified using any suitable preparative technique such as reversed-phase chromatography, partition chromatography, gel filtration, gel electrophoresis, and ion-exchange chromatography. Alternatively, the SprAl antimicrobial peptides provided herein can be produced by recombinant DNA methods. These methods generally involve isolation of the gene encoding the desired peptide, transfer of the gene into a suitable vector, and bulk expression in a cell culture system. The DNA coding sequences for the antimicrobial peptides of the invention may be readily prepared synthetically using methods known in the art (see, for example, M.P. Edge et ah, Nature, 1981, 292: 756-762).

After synthesis, the DNA encoding the desired peptide is inserted into a recombinant expression vector, which may be a plasmid, phage, viral particle, or other nucleic acid molecule-containing vectors or nucleic acid molecule-containing vehicles which, when introduced into an appropriate host cell, contains the necessary genetic elements to direct expression of the coding sequence of interest. Standard techniques well known in the art can be used to insert the nucleic acid molecule into the expression vector. The insertion results in the coding sequence being operatively linked to the necessary regulatory sequences. Host cells for use in the production of proteins are well known and readily available. Examples of host cells include bacteria cells such as Escherichia coli, Bacillus subtilis, attenuated strains of Salmonella typhimurium, and the like; yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins; insect cells such as Spodoptera frugiperda; non-human mammalian tissue culture cells such as Chinese Hamster Ovary (CHO) cells, monkey COS cells, and mouse 3T3 cells; and human tissue culture cells such as HeLa cells, HL-60 cells, kidney 293 cells and epidermal S431 cells.

Several expression vectors to produce polypeptides in well-known expression systems are commercially available. For example, the plasmids pSE420 (available from Invitrogen, San Diego, CA) and pBR322 (available from New England Biolabs, Beverly, MA) may be used for the production of the inventive peptides in E. coli. Similarly, the plasmid pYES2 (Invitrogen) may be used for peptide production in S. cerevisiae strains of yeast. The commercially available MacBacR™ kit (Invitrogen) for baculovirus expression system or the BaculoGold™ Transfection Kit available from PharMingen (San Diego, CA) may be used for production in insect cells, while the plasmids pcDNA I, pcDNA 3, and pRc/RSV, commercially available from Invitrogen, may be used for the production of the peptides of the invention in mammalian cells such as Chinese Hamster Ovary (CHO) cells.

Other expression vectors and systems can be obtained or produced using methods well known to those skilled in the art. Expression systems containing the requisite control sequences, such as promoters and polyadenylation signals, and preferably enhancers are readily available for a variety of hosts (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ^nd Ed., 1989, Cold Spring Harbor Press: Cold Spring, NY; and R. Kaufman, Methods in Enzymology, 1990, 185: 537-566).

The expression vector including DNA that encodes the desired peptide is used to transform the compatible host cell. The host cell is then cultured and maintained under conditions favoring expression of the desired peptide. The peptide thus produced is recovered and isolated, either directly from the culture medium or by lysis of the cells. The expressed antimicrobial peptide may be isolated by conventional isolation techniques such as affinity, size exclusion, or ion exchange chromatography, HPLC and the like.

As understood by one skilled in the art, an antimicrobial peptide of the invention may be produced as a fusion protein {i.e., a molecule in which the antimicrobial peptide sequence is linked to a polypeptide entity). Such a polypeptide entity may be selected to confer any of a number of advantageous properties to the resulting fusion protein. For example, the polypeptide entity may be selected to provide increased expression of the recombinant fusion protein. Alternatively or additionally, the polypeptide entity may facilitate purification of the SprAl antimicrobial peptide for example, by acting as a ligand in affinity purification. A proteolytic cleavage site may be added to the recombinant protein so that the desired antimicrobial sequence can ultimately be separated from the polypeptide entity after purification. The polypeptide entity may also be selected to confer an improved stability to the fusion protein, when stability is a goal. Examples of suitable polypeptide entities include, for example, polyhistidine tags, that allow for the easy purification of the resulting fusion protein on a nickel chelating column. Glutathione-S-transferase (GST), maltose B binding protein, or protein A are other examples of suitable polypeptide entities that can be fused to a SrpAl antimicrobial peptide of the invention using commercial fusion expression vectors. II - Uses of the Spr Al Antimicrobial Peptides

Due to their biological activity, the SprAl antimicrobial peptides of the invention (including fusion proteins thereof) may be used in a variety of applications, including therapeutic applications. Indeed, the peptides disclosed have been found to exhibit antimicrobial activity against gram-positive and gram-negative bacteria. Detailed description of the microorganisms belonging to gram-positive and gram-negative bacteria can be found in "Medical Microbiology" 3 ^rd Ed., 1991, Churchill Livingstone, NY).

Examples of gram-positive bacteria against which the SprAl antimicrobial peptides of the invention may be used include, but are not limited to, bacteria belonging to the Staphylococcus, Micrococcus, Lactococcus, Lactobacillus, Clostridium, Bacillus, Streptococcus, Enter ococcus, or Listeria genus. In particular, the SprAl antimicrobial peptides of the invention may be used against gram-positive bacteria that are potentially pathogenic to humans or other mammals. Such gram-positive bacteria include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus hyicus, Staphylococcus intermedius, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Bacillus cereus, Bacillus anthracis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, and Listeria ivanovii.

Examples of gram-negative bacteria against which the SprAl antimicrobial peptides of the invention may be used include, but are not limited to, bacteria belonging to the Bordetella, Salmonella, Enterobacter, Klebsiella, Shigella, Yersinia, Escherichia coli, Vibrio, Pseudomonas, Neisseria, Haemophilus, or Agrobacterium genus. In particular, the SprAl antimicrobial peptides of the invention may be used against gram- negative bacteria that are potentially pathogenic to humans or other mammals. Such gram-negative bacteria include, but are not limited to, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter sakazakii, Klebsiella pneumoniae, Yersinia pestis, Yersina enter ocolitica, Yersina pseudotuberculosis, Salmonella enterica, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio fluvialis, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Haemophilus aegypticus, and Haemophilus ducreyi. The antimicrobial peptides of the present invention (and the fusion proteins thereof) are therefore useful as bactericides and/or bacteriostats for modification of infectivity, killing microorganisms, or inhibiting microbial growth or function, and thus are useful for the treatment of an infection or contamination caused by such microorganisms.

1 - Therapeutic Applications

A. Indications

The present invention concerns both humans and other mammals such as horses, dogs, cats, cows, pigs, camels, among others, and is applicable in human medicine and veterinary therapy.

Thus, an antimicrobial peptide according to the present invention may be used for the treatment of any disease or condition caused by or due to a gram-positive or gram- negative bacterium. Bacterial infections include, but are not limited to, urinary infections, skin infections, intestinal infections, lung infections, ocular infections, otitis, sinusitis, pharyngitis, osteo-articular infections, genital infections, dental infections, oral infections, septicemia, nocosomial infections, bacterial meningitis, gastroenteritis, gastritis, diarrhea, ulcers, endocarditis, sexually transmitted diseases, tetanus, diphtheria, leprosy, cholera, listeriosis, tuberculosis, salmonellosis, dysentery, and the like. Bacterial diseases are contagious and can result in many serious or life-threatening complications, such as blood poisoning, kidney failure and toxic shock syndrome.

Methods of treatment of the present invention may be accomplished using an inventive SprAl antimicrobial peptide, a fusion protein thereof or a pharmaceutical composition thereof. These methods generally comprise administering an effective amount of at least one SprAl antimicrobial peptide or fusion protein (as defined above), or a pharmaceutical composition thereof, to a subject in need thereof. Administration may be performed using any of the methods known to one skilled in the art. In particular, an antimicrobial peptide or composition thereof may be administered by any of various routes including, but not limited to, aerosol, parenteral, oral or topical route.

In general, an inventive SprAl antimicrobial peptide or a composition thereof will be administered in an effective amount, i.e., an amount that is sufficient to fulfill its intended purpose. The exact amount of SprAl antimicrobial peptide or pharmaceutical composition to be administered will vary from subject to subject, depending on the age, sex, weight and general health condition of the subject to be treated, the desired biological or medical response and the like. In certain embodiments, an effective amount is one that prevents bacterial infection. In other embodiments, an efficient amount is one that treats bacterial infection by killing microorganisms and/or by inhibiting bacterial growth or function. In most embodiments, an effective amount of an SprAl antimicrobial peptide or of a pharmaceutical composition thereof is one that results in treatment of the disorder for which it is administered, e.g. slowing down or stopping the progression, aggravation or deterioration of the symptoms of the disorder and/or bringing about amelioration of the symptoms of the disorder, and/or curing the disorder. The effects of a treatment according to the invention may be monitored using any of the assays known in the art for the diagnosis of the disease being treated.

In certain embodiments, an inventive SprAl antimicrobial peptide or a composition thereof is administered alone according to a method of treatment of the present invention. In other embodiments, an inventive SprAl antimicrobial peptide or a composition thereof is administered in combination with at least one additional therapeutic agent or therapeutic procedure. The SprAl antimicrobial peptide or composition may be administered prior to administration of the therapeutic agent or therapeutic procedure, concurrently with the therapeutic agent or procedure, and/or following administration of the therapeutic agent or procedure.

Therapeutic agents that may be administered in combination with an inventive SprAl antimicrobial peptide or composition may be selected among a large variety of biologically active compounds including compounds that are known to have a beneficial effect in the treatment of the infection for which the SprAl antimicrobial peptide is administered; compounds that are known to be active against a condition or symptom associated with the infection treated; and compounds that increase the availability and/or activity of the SprAl antimicrobial peptide. Examples of such biologically active compounds include, but are not limited to, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and the like.

Therapeutic procedures that may be performed in combination with administration of an inventive SprAl antimicrobial peptide or composition thereof include, but are not limited to, surgery, catheterization and other invasive therapeutic procedures. Indeed, an inventive SprAl antimicrobial peptide may be used to prevent bacterial infection in association with urinary catheter use or use of central venous catheters. An inventive SprAl antimicrobial peptide, in plasters, adhesives, sutures or wound dressings, may also be used for prevention of infection postsurgery.

B. Administration

An inventive SprAl antimicrobial peptide or fusion protein thereof (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), in a desired dosage can be administered to a subject in need thereof by any suitable route. Various delivery systems are known and can be used to administer SprAl antimicrobial peptides of the present invention, including tablets, capsules, injectable solutions, encapsulation in liposomes, microp articles, microcapsules, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. An inventive SprAl antimicrobial peptide or composition thereof may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by adsorption through epithelial or mucocutaneous linings {e.g., oral, mucosa, rectal and intestinal mucosa, etc). Administration can be systemic or local. Parenteral administration may be directed to a given tissue of the patient, such as by catheterization. As will be appreciated by those of ordinary skill in the art, in embodiments where an inventive SprAl antimicrobial peptide is administered along with an additional therapeutic agent, the SprAl antimicrobial peptide and therapeutic agent may be administered by the same route {e.g., orally) or by different routes {e.g., orally and intravenously).

As mentioned above, an inventive SprAl antimicrobial peptide (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) may alternatively be administered incorporated in or coating bandages, plasters, sutures, catheters, needles, adhesives, wound dressings or implants.

C. Dosage

Administration of an inventive SprAl antimicrobial peptide (or a composition thereof) of the present invention will be in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend upon the therapeutic effect desired, the severity of the disease being treated, the age, sex, weight and general health condition of the patient as well as upon the potency, bioavailability and in vivo half-life of the SprAl antimicrobial peptide used, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively or additionally, the dosage to be administered can be determined from studies using animal models. Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using SprAl antimicrobial peptides of the invention, further information will emerge regarding the appropriate dosage levels and duration of treatment.

A treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of an inventive SprAl antimicrobial peptide, or composition thereof, may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval); monthly, yearly {e.g., in a time release form). Alternatively, the delivery may occur at multiple times during a given time period, e.g., two or more times per week, two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g., intravenous delivery.

2 - Cleaning, Disinfection, Decontamination Applications

The SprAl antimicrobial peptides of the present invention or fusion protein thereof may also be used in any application where the absence of bacterial contamination on inanimate (non-living) objects is desired. Examples of such inanimate objects include, but are not limited to, medical devices (e.g., instruments, apparatus, implants, contact lenses, laboratory coats, scrubs, gloves, and the like); surfaces {e.g., floors, furniture, and the like) of operating rooms, hospitals, laboratories, industrial installations, public places or private housing. Accordingly, the present invention provides for the use of an inventive SprAl antimicrobial peptide or fusion protein thereof as disinfectant. The invention also provides a method for cleaning or disinfecting the surface of an object or for preventing bacterial contamination of the surface of an object comprising a step of contacting the surface of the object with an effective amount of an inventive SprAl antimicrobial peptide or fusion protein thereof. The SprAl antimicrobial peptide or fusion protein thereof may be comprised in a cleaning solution or product or a cleaning pad or wipe. Other examples of inanimate objects include products that come in contact with the human body including personal care products (e.g., soap, shampoos, tooth paste, deodorant, sunscreens, tampons, diapers, and the like) and cosmetics. The SprAl antimicrobial peptide may be comprised in the products or the products may be soaked, sprayed or coated with the SprAl antimicrobial peptide.

3 - Other Applications

The SprAl antimicrobial peptides of the present invention have been shown to exhibit cytolytic activity, and more specifically hemolytic activity, with different degrees of efficiency. Cytolytic pore-forming peptides have been proposed as potential therapeutics for the treatment of cancer.

Accordingly, the present invention relates to the use of a SprAl antimicrobial peptide, or fusion protein thereof, as a therapeutic agent for the treatment of diseases or disorders in which cytolytic or pore-forming activity is beneficial to the patient.

In addition, the SprAl-encoded peptide (of SEQ ID NO: 1) shares physico- chemical properties with S. aureus phenol- soluble modulins (PSMs) that are small, amphipathic and a-helical peptides with significant cytolytic activity against human neutrophils and erythrocytes (Otto et ah, Annu Rev Microbiol, 2010, 64: 143-162). a- type PSMs also cause chemotaxis and cytokines release.

Antimicrobial peptides have also been reported to promote wound healing. Accordingly, the present invention also relates to the use of a SprAl antimicrobial peptide, or fusion protein thereof, as a therapeutic agent for the treatment of diseases or disorders in which any one of wound healing activity, chemotaxis activity and cytokine release is beneficial to the patient.

Ill - Products and Pharmaceutical Compositions

As mentioned above, the SprAl antimicrobial peptides of the invention, or fusion proteins thereof, may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising an effective amount of at least one SprAl antimicrobial peptide or fusion protein (as defined herein) and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biologically active agents. The SprAl antimicrobial peptides and pharmaceutical compositions thereof may be administered in any amount and using any route of administration effective for achieving the desired prophylactic and/or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.

The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "unit dosage form", as used herein, refers to a physically discrete unit of a SprAl antimicrobial peptide, or fusion protein thereof, for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

A. Formulation

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection. In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to the SprAl antimicrobial peptide, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizes or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils {e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, an inventive SprAl antimicrobial peptide may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannital, and silicic acid; (b) binders such as, for example, carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as non-ionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition such that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally, in a delaying manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

In certain embodiments, it may be desirable to administer an inventive composition locally to an area in need of treatment. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, by injection, by means of a catheter, by means of suppository, or by means of a skin patch or stent or other implant. For topical administration, the composition is preferably formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingredient by either passive or active release mechanisms. Transdermal administrations include all administration across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of a transdermal patch containing an active ingredient (i.e., the SprAl antimicrobial peptide) and a carrier that is non-toxic to the skin, and allows the delivery of the ingredient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil- in- water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerine. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations for the delivery of antibodies can be found, for example, in "Remington 's Pharmaceutical Sciences ^' ", E.W. Martin, 18 ^th Ed., 1990, Mack Publishing Co.: Easton, PA.

B. Additional Biologically Active Agents

In certain embodiments, an inventive SprAl antimicrobial peptide, or fusion protein thereof, is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, but are not limited to, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof.

In such pharmaceutical compositions, the SprAl antimicrobial peptide and the at least one additional therapeutic agent may be combined in one or more preparations for simultaneous, separate or sequential administration of the SprAl antimicrobial peptide and therapeutic agent(s). More specifically, an inventive composition may be formulated in such a way that the SprAl antimicrobial peptide and therapeutic agent(s) can be administered together or independently from each other. For example, the SprAl antimicrobial peptide and therapeutic agent can be formulated together in a single composition. Alternatively, they may be maintained {e.g., in different compositions and/or containers) and administered separately. C. Products Comprising an Inventive SprAl Antimicrobial Peptide

The invention also relates to a product comprising an inventive SprAl antimicrobial peptide, a fusion protein thereof, or a pharmaceutical composition thereof, as defined above. The product may be selected from bandages, plasters, sutures, adhesives, wound dressings, implants, contact lenses, cleaning solutions, storage solutions (e.g., for contact lenses or medical devices), cleaning products (e.g., cleaning pads or wipes), personal care products (e.g., soap, shampoos, tooth paste, sunscreens, tampons, diapers, and the like), and cosmetics.

The products comprising an inventive SprAl antimicrobial peptide may be prepared by any suitable method. Generally, the method of preparation will depend on the nature of the object. For example, an inventive SprAl antimicrobial peptide, or a pharmaceutical composition thereof, may be added to the product by mixing, or may be incorporated or applied to a product by soaking the product into a solution of the SprAl antimicrobial peptide, by coating the product with the SprAl antimicrobial peptide, or by spraying the product with a solution of the SprAl antimicrobial peptide.

D. Pharmaceutical Packs or Kits

In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers {e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of an SprAl antimicrobial peptide of the present invention or a fusion protein thereof.

Different ingredients of a pharmaceutical pack or kit may be supplied in a solid {e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Packs or kits according to the invention may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale. In certain embodiments, a pack or kit includes one or more additional therapeutic agent(s). Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.

An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained. Some of the results presented below have been described in a scientific article (Sayed et al, Nat. Struct. Mol. Biol, December 2011, 19: 105-112). The entire content of the scientific paper is incorporated herein by reference in its entirety.

Example 1: Biological Properties of the SprAl Peptide Materials and Methods

Strains and Plasmids. Several strains and different plasmids were used in this study. S. aureus strains were grown at 37°C in brain heart infusion broth (BHI, Oxoid). When necessary, chloramphenicol and erythromycin were added at 10 μg/mL, and kanamycin was added at 200 μg/mL. Genetic Manipulations. Primers were used for all the constructions. In all constructions, RNAs are expressed from their endogenous promoters. In pCN35QsprAl _A s, SprAl _A s sequence with 113 nts upstream and 26 nts downstream was amplified from Newman genomic DNA as a 215-bp fragment, with flanking Pstl/EcoRI sites. The fragment was inserted in pCN35. In pCN35QSTsprAl/sprAl _A s, the sprAl /sprAl AS locus was amplified from Newman genomic DNA (with 48 upstream and 45 nt downstream) with streptotag (Windbichler et al, Nat Protoc, 2006, 1 : 637-640) (46b aptamer with affinity to streptomycin) incorporated between sprAl and its promoter. For the truncated- flagged SprAl construct, sprAl gene sequence, from positions -171 to +110, was amplified from Newman genomic as a 368-bp fragment flanked by Pstl and EcoRI restriction sites. In frame with the first 20 amino acids encoded within SprAl, the reverse primer contains 3XFlag (66-bp, 20 amino acids) followed by two UAG termination codons. The PCR fragment was inserted into pCN48 (Charpentier et al., Appl Environ Microbiol, 2004, 70: 6076-85) and then digested by Pstl/Narl (the insert followed by the blaZ transcription terminator). The resulting 684- bp fragment (truncated sprAl ending by 3XFlag followed by the blaZ transcription terminator) was inserted into pCN34 (Charpentier et al., Appl Environ Microbiol, 2004, 70: 6076-85). The resulting amino acid sequence of the SprAl fusion peptide is:

MLIFVHIIAPVISGCAIAFDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 14). The construction of the deletion mutant S. aureus strain Newman ASprAl/SprAl _A s was performed as previously described (Chabelskaya et al., PLoS Pathog, 2010, 6: el000927). Briefly, chromosomal gene disruption of SprAl/ SprAl _A s locus was constructed by deletion of targeted locus and insertion of erythromycin resistance gene by using the temperature-sensitive vector pBT2 (Bruckner et al., FEMS Microbiol Lett, 1997, 151 : 1-8).

In vitro Transcription and RNA Labelling. All the RNAs used herein were transcribed from PCR-amplified templates using Newman genomic DNA. Forward primers contained a T7 promoter sequence. PCR generated DNA was used as template for transcription using the 'Ambion T7Megascript' kit. For synthesis of short RNAs (SprAl _A s, 5'SprAl _A s, 3'SprAl _A s), template was produced by annealing the primers. RNAs were gel purified, eluted passively and ethanol precipitated. 5' and 3' end labelling of the RNAs were performed as previously described (Antal et al., J Biol Chem, 2005, 280: 7901-7908).

Primer Extension, RACE Mapping. For SprAl _A s 5' end mapping, RNA extracts from Newman pCN35QsprAlAs were used. Primer extension carried out as previously described (Antal et al., J Biol Chem, 2005, 280: 7901-7908) using Superscript III reverse transcriptase. 5 '-RACE of SprAl were carried out as previous described (Antal et al., J Biol Chem, 2005, 280: 7901-7908).

RNA Extraction and Northern Blots. Isolated colonies were suspended in 5 mL of BHI and incubated at 37°C overnight. Culture was diluted 1 : 100 then incubated at 37°C with agitation and stopped at various phases of growth. RNA extraction was performed as previously described (Cheung et al., Anal Biochem, 1994, 222: 511-514). Total RNA (10 μg) was separated on denaturing PAGE and transferred onto a Zeta probe GT membrane (Bio-Rad). Specific ³² P-labelled probes were hybridized with ExpressHyb solution (Clontech) for 90 minutes, washed, exposed and scanned with a Phosphorlmager (Molecular Dynamics).

Streptomycin-streptotag Purification. RNA extracts of Newman AsprAl/sprAl _A s pCN35QSTsprAl/sprAl _A s were used for affinity purification. Streptomycin sepharose preparation and the affinity purification were performed as previously described (Windbichler et al., Nat Protoc, 2006, 1 : 637-640). Eluted RNAs were ethanol-precipitated.

Gel-Mobility Assays, Structural Probing and Toeprints Gel-mobility assays were performed as previously described (Antal et al., J Biol Chem, 2005, 280: 7901- 7908). RNAs mix were incubated in binding buffer (80 mM K-HEPES pH 7.5, 4 mM MgCl ₂ , 330 mM KC1) 20 minutes at 30°C before native gel separation. Structural assays were performed as previously described (Antal et al, J Biol Chem, 2005, 280: 7901-7908). Structural analysis of duplexes between SprAl and SprAl _A s were prepared by incubating 0.5 pmol of labeled RNA with 1 pmol of unlabeled RNA in binding buffer 20 minutes at 30°C. Digestions with the various ribonucleases were performed for 15 minutes 30°C in the presence of 1 μg of total tRNA from yeast (VI at 5.10 ^"5 units; SI at 5 units, lead acetate at 0.75 mM). The toeprints were done as previously described (Chabelskaya et al., PLoS Pathog, 2010, 6: el 000927) with some modifications. S. aureus purified 70S were used and reverse transcription was performed using labeled primer 'sprAlToep'. For the assays in the presence of SprAl _A s, 2-fold excess of sprAl _A s was added and incubated 15 minutes at 30°C. The reactions were precipitated and the pellets dissolved in loading buffer (Ambion). The samples were separated on denaturing 8% PAGE. The gels were dried and visualized with Phosphorlmager.

Protein Extractions, Western Blots and in vitro Translation Assays. For protein extractions during growth, the pellets were re-suspended in lysis buffer (50mM Tris-Cl pH7.5, 3mM MgCl ₂ , 0.1 mg/mL lysostaphin and 0.2υ/μΤ of Benzonase) incubated 15 minutes at 37°C then transferred into ice. Bradford assays were performed on the samples and equal amounts of total proteins were used for the Western Blots. The samples were separated on Tricine-SDS-PAGE 16% gel (Schagger et al, Nat Protoc, 2006, 1 : 16-22), transferred on Hybond™-P PVDF membrane (Amersham) and revealed using the Amersham™ ECL™ Plus detection Kit. In vitro translation was performed using E. coli S30 extract system for linear templates (Promega) following the manufacturer's instruction. For the assays in the presence of SprAl _A s or other RNAs, the RNA mix was incubated in binding buffer 20 minutes at 30°C. Samples were separated by Tricine-SDS-PAGE 16% gel (Schagger et al, Nat Protoc, 2006, 1 : 16-22). The gel was fixed, exposed and visualized with Phosphorlmager.

Hemolytic Assays. Human or sheep RBC (Elsevier) were washed 3 times diluted to 3% in PBS. For the titration of hemolytic activity of SprAl peptide, 100 of serial ½ dilutions of PBS containing peptides were pipetted in a V Bottom 96 Well Plate (Sigma). 100 μΐ, of 3% RBC were pipetted in each well and mixed gently by pipetting. Hemolysis positive control is done by adding 100 μΐ, of water to 100 μΐ, of RBC. Hemolysis negative control was done by adding in a well 100 μΐ, of PBS to 100 μΐ, of RBC. Ribonucleotides, Oligonucleotides and Proteins. SprAl peptide was synthesized by PROTEOGENIX (Oberhausbergen, France). Its sequence is set forth in SEQ ID NO: 1 (MMLIF VHII AP VI S GC AI AFF S Y WLSRRNTK) . Superscript III reverse transcriptases, lysostaphin, Benzonase were purchased from Invitrogen. Restriciton enzymes were from New England Biolabs (Berverly, MA). [γ ³² Ρ]ΑΤΡ, [cc ³² P]pCp (3000 mCi/mmol) and [ ³⁵ S] methionine at (1150 mmol/ at 10 mci^L) were from Perkin-Elmer (Courtaboeuf, France).

Antimicrobial Activity. The antimicrobial activity of the SprAl peptide was assessed using a radial diffusion assay (RDA), using a method adapted from that described by Lehrer et al. (J. Immunol. Methods, 1991, 137: 167-173). Briefly, one bacterial colony of each organism (Staphylococcus aureus Newman, Staphylococcus aureus N315, Salmonella enterica, Shigella flexneri, Escherichia coli 0157:H7) was inoculated in 5 mL TSB (ON/37°C/220 rpm). 75 of the ON culture were inoculated in 15 mL of fresh TSB medium for 2 hours at 37°C/220 rpm. After centrifugation at 5000 rpm for 5 minutes, the bacteria were washed with 10 mL of sterile, ice cold PBS, and centrifuged at 5000 rpm for 5 minutes. The bacteria were resuspended with 5 mL of sterile, ice cold PBS. OD(600 nm) was measured using 1 mL of the bacteria suspension and CFU (=OD(600 nm) x 2.5xl0 ⁸ ) was calculated (CFU/mL).

10 mL of Underlay gel (50 mL 100 mM phosphate buffer pH 7.4, 5 mL TSB, 5 g low melting agarose, qsp 500 mL ddH ₂ 0 - autoclaved) maintained at 42°C were mixed with 4xl0 ⁶ CFU, and immediately poured into sterile plates (diameter of 80 mm). After solidification of the underlay, the wells (diameter of 2 mm) were dug into the agar. 4 of SprAl peptide (in 50% isopropanol at 5 mg/mL), of a negative control (isopropanol 50%), of a positive control (5% acetic acid), and of cecropin PI were pipetted into the wells. The agar plates were incubated for 3 hours at 37°C. 10 mL of Overlay gel (30 g TSB, 5 g low melting agarose, qsp 500 mL ddH ₂ 0 - autoclaved) maintained at 42 were poured over the underlay layer. After solidification, the plates were incubated ON/37°C.

CMI measurement: Diameters of growth inhibition were photographed for CMI (minimal inhibitory concentration) calculation. A ruler was placed beside the petri dish to help accurate diameter measurement. The diameter of the well (2 mm) was subtracted from each diameter of inhibition. The resulting value was converted into arbitrary units, where 1 OU = 1 mm. Semi-logarithmic curves were drawn for each organism using diameter of standard peptide cecropin PI .

Results

Monitoring SprAl and SprAl _A s Expression. Up to five copies of sprA were detected in the S. aureus strains (Pichon et al, Proc Natl Acad Sci U S A, 2005, 102: 14249-14254). In strain Newman, a human clinical isolate, only two copies, sprAl and sprA2, were identified. Therefore, that strain is a simplified model for studying the multicopy sprA gene. In Newman, sprAl was located in the ν8αβ pathogenicity island (Baba et al, J Bacteriol, 2008, 190: 300-310), between a transposase and a hypothetical protein (Figure 1A). An antisense RNA (asRNA) to SprAl was detected in strain N315 by high-throughput sequencing (Beaume et al, PLoS One, 2010, 5: el 0725). The sprAl and sprAl _A s genes read in opposite directions with a predicted sequence overlap at their 3 '-ends (Figure 1A). A second copy, sprA2, is detected in the core genome at position '2560389-2560600', between a HP and a protein from the GtrA family (not shown). SprAl and sprA2 share 74% nucleotide identity. Using a DNA probe specific of SprAl, the RNA was found to be expressed in Newman, as well as its asRNA, SprAl _A s, (Figure IB). To distinguish between the expression of the two copies of sprA, as well as to assess their functional implications independently one another, a sprAl- sprAl _A s" deletion mutant (AsprAl-sprAl _A s) was constructed in Newman by homologous recombination. SprAl- and SprAl _A s-specific DNA probes confirmed the absence of expression of both SprAl and SprAl _A s in the deletion strain (Figure IB). Quantification of the expression levels of SprAl and of SprA2 indicate that SprAl is expressed two- to three-fold more than SprA2 in the Newman strain (not shown). The present study focuses on the functional investigation of the SprAl and SprAl _A s RNAs. Determination of the Nucleotide Overlaps Between SprAl and SprAl _AS .

Determining SprAl and SprAl _AS 5' and 3' boundaries was required for subsequent functional and structural analysis. Since they are predicted to overlap, the extent of sequence overlay between the two RNAs was assessed experimentally in Newman. For SprAl, it was resolved using RACE (rapid amplification of cDNA ends), as previously described (Antal et al, J Biol Chem, 2005, 280: 7901-7908), combined with direct size assessment using Northern blots on polyacrylamide gels (Figure IB).

SprAl 5 '-end maps at position G188 5 7 in strain Newman (at Gi856485 in strain N315, data not shown), twelve nucleotides downstream from a _i ₂ TATAAT_ ₇ box that is the predicted promoter. SprAl 3 '-end forms an intrinsic terminator characterized by a stem loop (H6) followed by an imperfect U-tract (UUGGUGU). In E. coli, about half of the Rho-independent terminators possess imperfect U-tract (Peters et ah, J Mol Biol, 2011, doi: 10.1016/j.jmb.2011.03.036).

SprAl AS ends were very difficult to assess experimentally because its small size precludes from using RACE on circularized RNAs (Redko et ah, Mol Microbiol, 2008, 68: 1096-1106). Therefore, SprAl _A s transcriptional start site was resolved by primer extension analysis on total RNAs from wt Newman cells containing a pCN35QsprAl _A s plasmid expressing SprAl _A s from its endogenous promoter, to bypass the obstacle of its small size. SprAl _A s expressed in vivo from the plasmid was verified to have a similar length than wt SprAl _A s (see Figure ID). SprAl _AS 5 '-end was assigned at position Gi88 77o- Therefore, SprAl _AS 5 '-end is positioned ten nucleotides downstream from a _ ioTATAAT_5 box that is its predicted promoter. SprAl _A s 3 '-end forms an intrinsic transcription terminator characterized by a stem loop (H2 _A s) followed by a near-perfect U-tract (UUUUUAUU). SprAl _AS is a -60 nt-long asRNA. The experimental determination of the two RNA boundaries allowed producing them as synthetic transcripts.

Phylogenetic Distribution of the sprAl and sprAl _A s Genes. The phylogenetic distribution of sprAl and sprAl _AS was studied in all sequenced bacterial genomes. The genes encoding SprAl and SprAl _AS were identified in two orders of the bacilli class, the bacillales (staphylococcaceae, genus staphylococcus) and the lactobacillales (enterococcaceae, genus enterococcus). They were distinguished from sprA2 that is in the core genome by their locations within the Pis as well as by their systematic association with a sprAl _AS gene on the opposite DNA strand. Sequence alignments of SprAl _AS indicate sequence conservation in the non-overlapping region of the RNA pair, corresponding to -20 nucleotides at the 5 'side of the RNA. Within all the aureus species in which the RNA pair was detected, there is conservation of the two genes at the nucleotide level, suggesting selective pressure. Within the Staphylococcus genus, however, there are differences scattered through the RNA sequence. An in-depth experimental analysis was performed onto the 'SprAl-SprAl _AS ' duplex.

SprAl and SprAl _A s Expression Profiles During Growth. SprAl and SprAl _AS expression levels were monitored by northern blots during growth of S. aureus strain Newman (Figure 1, panels C-D). Their expression levels were quantified relative to tmRNA, a ubiquitous eubacterial sRNA expressed at constant levels during growth. SprAl is constitutively expressed, detected early and present at all phases. SprAl _A s is also expressed early and reproducibly exhibits a peak of expression at mid-exponential phase and is also expressed later. Similar expression patterns for the two RNAs were also observed in SHI 000 and N315 S. aureus strains (data not shown). The in vivo concentrations of SprAl and SprAl AS in wild-type Newman strain was determined by including a range of each of the purified synthetic RNAs in the Northern blots, for quantitative estimations (Figure IE). At all times during growth, SprAl _A s is in large excess relative to SprAl, from a 35- to a -90 fold molar excess (Figure IE). In vivo Detection of the "SprAl-SprAlAS" Duplex and Assessment of

Binding Constants. Since the two RNA genes are partially overlapping, the present inventors tested experimentally if the two RNAs, SprAl and SprAl _A s were interacting in vivo. For that purpose, a streptomycin-binding aptamer (Windbichler et ah, Nat Protoc, 2006, 1 : 637-640) was fused at the 5 '-end of the sprAl gene and cloned into a plasmid expressing 5'ST-SprAl (STSprAl) and SprAl AS from their endogenous promoters. The AsprAl-AsprAl s Newman strain was complemented with the 'pCN35Q STsprAl -sprAl AS' plasmid and Northern blots demonstrated that STSprAl is expressed in the complemented strain at both mid- exponential (OD=3) and early stationary (OD=l l) growth phases (Figure 2A, STSprAl is expressed to higher levels than wt SprAl in vivo because of the plasmid copy number). As controls, the expression of SprAl in wt cells, but not in the deletion strain, was monitored and the size difference between STSprAl and wt SprAl corresponds to the 46 nt-long ST. Total RNAs extracted from Newman 'AsprAl-AsprAlAs- pCN35Q. STsprAl -sprAl AS' cells were loaded on a streptomycin affinity matrix. As shown by PAGE, the flow through (FT) contains the non specific RNAs (tRNAs and ribosomal RNAs), the last two washes cause some loss of STSprAl from the column and, interestingly, the elution performed with 100 μΜ streptomycin contains STSprAl and SprAl AS, indicating that SprAl _A s is in complex with immobilized STSprAl . As a negative control, total RNAs were extracted from Newman 'WT-pCNSSQsprAl AS' cells and loaded onto the affinity matrix. Northern blots demonstrate that in the absence of STSprAl in vivo, SprAl _A s cannot bind the column by itself and is only detected in the FT, together with a non specific sRNA, tmRNA (Figure 2B). However, with the ⁱ AsprAl-AsprAl _A s-pCN35Q. STsprAl -sprAl AS' cells, Northern blots demonstrate that the eluted fraction contains both S rAl _A s and STSprAl, but not tniRNA (Figure 2B). Altogether, these experiments indicate that SprAl forms a complex with SprAl _A s in vivo and that its 5 'ST does not hamper recognition.

Duplex formation between SprAl and SprAl _AS was analyzed by gel retardation assays. A 'SprAl-SprAl _A s' duplex was detected at a 1 :0.3 molar ratio and all SprAl was in complex with sprAl _A s at a 1 : 1 molar ratio (Figure 2C). Complex formation between labeled SprAl and SprAl _AS was also analyzed and nearly all labeled SprAl was in complex at a 1 : 1 molar ratio (Figure 2D). In the two experiments, the binding is specific since a 1,000-fold molar excess of total tR As do not displace SprAl _A s or SprAl from preformed 'SprAl _A s- SprAl ' complexes, whereas a twenty-fold excess of unlabeled SprAl _A s or SprAl R As does. SprAl _A s binds SprAl with an apparent ¾ of 15nM ±5 (Figure 2, panels C and D), a value that was mandatory for complex formation between the two RNAs subsequently analyzed by structural probes in solution.

Monitoring of the Interacting between SprAl and SprAl _A s by Structural Probes. The conformations of free SprAl and SprAl _A s, as well as the SprAl-SprAl _A s duplexes were analyzed by structural probes (lead, RNAse VI and nuclease SI). The data are summarized onto SprAl and SprAl _A s models (Figure 3).

SprAl _A S has two folded stems (Hl _A s and H2 _A s) separated by an 8 nt-long junction (Hl-H2 _A s). Sequence alignments provide strong phylogenetic support for stem Hl _A s, but much less for H2 _A s- In SprAl, the presence of Vi cuts with no Si or lead cuts supports the existence of six stems, HI to H6. Probing data support the existence of two loops, LI and L6, capping respectively HI and H6. Internal bulges within HI and H6 are supported by nuclease Si and lead cleavages. A 5 nt junction separates HI from the first pseudoknot, PK1 (H2-L2-H3-L3), followed by PK2 (H4-L4 H5-L5). SprAl is a compact RNA made of two tandem pseudoknots flanked by two stable helices, the second acting as a transcription terminator. Sequence alignments provide strong phylogenetic support for stem H1-H6 but not for H3.

The structural changes induced by duplex formation between the two RNAs were examined. Upon SprAl binding, the most striking reactivity changes involve the single- stranded Hl/H2 _A s junction that is cleaved by RNase VI and protected from lead cuts, suggesting that it becomes double stranded upon duplex formation (data not shown). An overall destabilization of the RNA is evidenced by the disappearance of VI cuts in HI _A S and H2 _A s as well as by the fading of SI and lead cuts in Ll _A s and L2 _A s- Conversely, in the presence of SprAl _A s, all the reactivity changes in SprAl are centred between lower portion of HI and PKl, indicating that it turns double-stranded, as it becomes protected from lead and SI cuts (Figure 3). Interestingly, there are no structural changes at the czs-overlapping region between the two RNAs. Altogether, the probing data collected on the 'SprAl-SprAl _A s' duplex support an interaction between the two RNAs. Comparing sequences from genomes and plasmids provides phylogenetic support of the interaction.

SprAl and SprAl _A s Interact by their Non-overlapping Domains. Between SprAl and SprAl _A s, there is an intuitive binding site that involves a 35-nt overlap at their 3 '-ends (data not shown). Experimental evidence supports a different, unanticipated binding site that involves pairings between nucleotides located at their 5'- domains. To assess the contribution of those binding sites in duplex formation, SprAl and SprAl _A S were cleaved in two halves to retain a single binding domain on each RNA variant ('5'-SprAl \ '3'-SprAl \ '5'-SprAl _AS ' and '3'-SprAl _AS '). Duplex formation between each of these shorter RNAs was analyzed by gel retardation assays (Figure 3). A specific '5'SprAl-SprAl _A s' duplex is detected (Figure 3A) with a Kj similar to that observed with wt SprAl (Figure 2, panels C and D), whereas the 3 'SprAl construct is unable to bind SprAl _AS , even at a 20 fold molar excess (Figure 3B), indicating that SprAl 5'-domain, including Hl-Ll and PKl, is necessary and sufficient to interact with SprAl _AS . Also, a specific '5 ' SprAl _AS -5' SprAl ' duplex is detected, with a weaker affinity than that between the two native RNAs (Figure 2, panels C and D), probably because of two conformations for PKl from 5 'SprAl including an open and closed pseudoknotted structures. In addition, a specific '5'SprAl _AS -SprAl ' duplex is detected (Figure 3C) but its apparent KJ is ~20 fold weaker than that between the two native RNAs, probably because of the reduced stability of 5 'sprAl _AS lacking the H2 _AS terminal helix, and also because of the reduced accessibility of the folded PKl in full-length SprAl . Reciprocally, the 3'SprAl _AS construct is unable to bind SprAl, even at a 200 fold molar excess (Figure 3D). Overall, the non-overlapping 5 '-domains of each RNA are necessary and sufficient for duplex formation, whereas the complementary 35 nucleotides at SprAl and SprAl _AS 3 '-ends are dispensable for duplex formation.

Translation Initiates Internally onto SprAl to Produce a Peptide whose Expression is Repressed by SprAl _A s via its 5' Non-overlapping Domain. In all the sprAl genomic sequences, an internal ORF was identified, predicted to encode a 30-33 amino acid- long peptide, starting at a ₅₁ GUG ₅₃ or ₅₄ AUG ₅₆ initiation codons and ending at a 144UAG146 stop codon. Moreover, an internally conserved '37AGGAGG42' Shine- Dalgarno (SD) sequence was identified 8 to 11 nucleotides upstream the predicted start codons. To test whether or not ribosomes can form translation initiation complexes onto SprAl, toeprint assays were performed on ternary initiation complexes including purified 70S ribosomes from S. aureus, initiator tRNA ^Met and SprAl . In the presence of the purified S. aureus ribosomes, toeprints were detected onto SprAl at C65-C69 within L2 from pseudoknot PKl, 14 to 18 nucleotides downstream from the predicted initiation codons (Figure 4A). The presence of multiple toeprints suggests that ribosome loading onto SprAl takes place at several positions; probably because of two available initiation codons and also since there is a compact pseudoknot structure at the loading site that needs opening by the ribosomes. Within SprAl, the predicted SD sequence was mutated into ₃₇ UCCUCC ₄₂ and, to maintain the conformation of helix HI, ₅ CCUAUCU _{1 1} was also mutated into ₅ GGAAGGA _{1 1} . Ribosomes are unable to load onto this SprAl variant named 'SD-mutated sprAl ' (Figure 5A, right panel), demonstrating that the ₃₇ AGGAGG ₄₂ sequence is required for translation initiation. Since the interaction between SprAl _A s and SprAl coincides with the region covered by the ribosomes during translation initiation, SprAl _A s should prevent ribosome loading onto SprAl . Indeed, in the presence of SprAl _A s, SprAl toeprints disappear (Figure 5 A), indicating that the asRNA prevents ribosome loading onto SprAl . Interestingly, a strong stop was detected at U61 within SprAl structure likely because RNA duplex formation induces a conformational change.

In vitro translation assays were performed to provide direct experimental evidence that SprAl can express a polypeptide predicted to contain 31 amino acids. In the presence of wt SprAl, a 2 to 5 KDa polypeptide is detected (Figure 4B), in agreement with its -3.45 KDa theoretical molecular weight. Remarkably, when SprAl AS was added to the reaction (2-fold molar excess compared to SprAl), SprAl translation was blocked. The inventors concluded that the SprAl _A s down regulates SprAl translation by direct pairing interactions at and around the SprAl translation initiation signals. A ten- fold molar excess of either 5'SprAl _A s or 3'SprAl _A s was added in the translation assays of SprAl (5'SprAl _AS has a ~10-fold weaker binding affinity for SprAl than wt SprAl _AS has, Figures 2 panels C and D, and 4C). Whereas 5'SprAl _AS reduces significantly SprAl translation, 3'SprAl _AS does not (Figure 5B), providing direct evidence that SprAl _AS prevents SprAl translation by its 5 '-domain, but not by its 3'- domain. As a negative control, the 'SD-mutated SprAl ' cannot be translated in vitro (Figure 5B), in agreement with the toeprint data, as it fails to recruit the S. aureus ribosomes (Figure 5 A, right panel). Altogether, these experiments indicate that SprAl AS binds SprAl at its 5' non-overlapping sequence to block internal translation of SprAl . To test whether SprAl _A s also regulates SprAl transcription and/or stability, in addition to its translational control, SprAl _A s was over expressed in vivo and the expression levels of SprAl were monitored by northern blots during bacterial growth. Increasing the expression levels of SprAl _A s was found not to significantly affect SprAl levels in vivo, ruling out a direct regulation at the RNA level (Figure 5C). The S/)f"/4/-encoded Peptide Expression is Down-regulated in trans by the cis-

SprAl _A s in vivo. For the in vitro data presented above, the sprAl and sprAl _A s genes were linked genetically. Decoupling genetically the location and expression of the two overlapping R As was required to demonstrate in vivo that a cz ^' s-sRNA operates in trans. The SprAl peptide has very low immunogenicity (not shown), and its overexpression in vivo inhibits S. aureus growth (data not shown). A reporter peptide construct was designed by combining the 5'- sequence of SprAl including 20 amino acids at N-ter from its internal coding sequence, merged to a 22 amino acid 3XFlag, for detection. SprAl peptide truncation of its 11 amino acids at C-ter was required to lower its toxicity in vivo. Also, the transcription terminator sequence of SprAl that overlaps with sprAl _AS was replaced by another unrelated terminator sequence (blaZ). A low- copy (-20 copies per bacterium) vector was used (pCN34) and the pCN34ΩsprAltag was transformed into Newman AsprAl-AsprAl AS- Immunoblots using anti-FLAG antibodies demonstrate that the SprAl fusion peptide is expressed in vivo (Figure 5 A). To monitor the impact of SprAl _AS on the SprAl-encoded fusion peptide in vivo, strain AsprAl-AsprAl _A s pCN34ΩsprAltag was transformed with either pCN35 or pCN35QSprAlAs- In the strain containing the two RNAs in trans, each expressed from a different plasmid, SprAl peptide levels are drastically reduced (Figure 5 A), demonstrating the down regulation of the expression of the SprAl peptide by SprAl _AS in vivo. Northern blots validate the presence of the SprAl-flagged and SprAl _AS RNAs in the 'AsprAl-AsprAl AS' double deletion strain containing the two plasmids whereas, as expected, there is only the SprAl-flagged RNA in the AsprAl-AsprAl _A s pCN34ΩsprAltag strain transformed with the empty pCN35 plasmid (Figure 5B). Therefore, SprAl _A s prevents sprAl -encoded peptide expression in trans in vivo and its 3 '-overlapping region with SprAl is dispensable for the regulation.

The S/)f"/4/-encoded Peptide is Cytolytic in Human Cells. Sequence alignments of the SprAl -encoded peptide indicate a-helicity and amphipathy, which are typical features of pore-forming peptides (Mellor et al, Biochim Biophys Acta, 1988, 942: 280-294). The lytic activity of the SprAl peptide was demonstrated by adding increasing concentrations of the chemically synthesized peptide on human erythrocytes (Figure 6A). The SprAl peptide lyses human erythrocytes at a concentration of 1 μΜ and above, but is less active towards sheep erythrocytes (Figure 6B), suggesting a narrow hemolytic range. Some microbial hemolysins display antibacterial activity (Verdon et al, Peptides, 2009, 30: 817-823). The cytolytic activity of the SprAl peptide against S. aureus cells gives a rationale as to why SprAl _A s is continuously expressed during bacterial growth, preventing SprAl translation and toxicity against S. aureus cells.

The S/)f"/4/-encoded Peptide Exhibits Antimicrobial Activity. The SprAl peptide was found to exhibit antimicrobial activity. Using a radial diffusion assay (Figure 7), CMI values were calculated for the SprAl peptide and for the standard antimicrobial peptide, Cecropin PI in Staphylococcus aureus Newman, Staphylococcus aureus N315, Salmonella enterica, Shigella flexneri, and Escherichia coli 0157:H7 (Figure 8). The results obtained are presented in the following table.

Table 1. CMI values for the SprAl peptide and for Cecropin PI in different micro- organisms.

Discussion

Many asRNAs were recently inventoried in bacteria and eukaryotes but information on the molecular mechanisms underlying their functions is, for the most part, unknown. By definition, cz ^' s-asRNAs regulate the genes encoded opposite them (Thomason et al., Annu Rev Genet, 2010, 44: 167-188). It is soundly accepted that cis- asR A functions are linked to their sequence overlap with genes transcribed from the opposite DNA chain.

The present study reports an unusual case of an asR A that acts as a trans regulator in S. aureus. It demonstrates that asRNAs can interact in trans with complementary target genes and possibly also with other genes at remote genetic loci. Base complementarities between overlapping RNAs do not necessarily imply that they are the functional unit of the pair. Because czs-RNAs can work in trans, the distinction between cis- and trans- RNAs should be revised. The present findings also suggest that the mechanisms of gene regulations of the identified asRNAs should be re-evaluated, as some could operate in trans on targets. It may be advantageous for trans sRNAs to be located in cis on the chromosome because, during genomic rearrangements, they will move with their target genes. In many bacteria, trans-acting sRNAs require the Hfq RNA chaperone protein for pairings with target RNAs (Chao et αί, Curr Opin Microbiol, 2010, 13: 24-33). In vivo, SprAl steady state levels are unaffected by the presence or absence of Hfq, suggesting that the protein is dispensable for the interaction between SprAl _A s and SprAl . The cz ^' s-overlap between SprAl and SprAl _A s 3 '-ends operates as a bi-directional transcription terminator, presumably for genome compaction and tightness. Their 5' non-overlapping domains, however, interact in trans to repress SprAl encoded toxic peptide synthesis (Figure 6C). SprAl has a compact secondary structure made of RNA pseudoknots flanked by stable stem-loops, hindering internal translation initiation signals from the ribosomes. Despite such structural lock, the S. aureus ribosomes can load onto SprAl in vitro to produce a 31 amino acid peptide. SprAl _A s is therefore required to block internal translation onto SprAl, inducing a conformation rearrangement by pairing interactions. These observations are consistent with a continuous and tight repression of peptide synthesis that is detrimental for S. aureus growth (data not shown). The SprAl peptide is probably only expressed under restricted, currently unknown, physiological conditions by specific environmental clue(s) reducing SprAl _A s levels. The peak of SprAl _AS expression at mid-E phase suggests that translation repression of SprAl is optimized during the E phase, probably to insure that the toxic peptide is not produced when the S. aureus cells are actively spreading. Alternatively, SprAl _AS could have other functions; it might act in trans on other RNAs. Indeed, in Listeria monocytogenes, riboswitches that are cz ^' s-RNA elements can also function in trans, therefore acting as regulatory RNAs (Loh et al, Cell, 2009, 139: 770-779). SprAl could have other functions, at the RNA level, in addition to expressing a peptide. As a recent example of such a dual function for an RNA, both the transcription and translation products of a cytolysin (phenol-soluble modulin, PSM) regulate virulence in S. aureus (Kaito et al, PLoS Pathog, 2011, 7, el 001267).

The SprAl peptide is a novel virulence factor that lyses erythrocytes and probably other cell types and organelles. Its function is consistent with its genomic location within a PI containing virulence genes. Another S. aureus cytolytic peptide, the δ- hemolysin (Janzon et al, Mol Gen Genet, 1989, 219: 480-485), is internally encoded within a sRNA, the RNAIII. δ-hemolysins are a-helical and amphipatic PSMs, as the SprAl peptide and active against a wide range of cells and organelles, δ-hemolysin enables the phagosomal escape of staphylococci in human cells (Giese et al, Cell Microbiol, 2011, 13: 316-329). Therefore, the SprAl peptide could also be involved in evading the human phago-endosomes, the acidic pH could lower SprAl _A s levels, leading to SprAl peptide expression.

Based on sequence comparisons, SprAl and SprAl _A s were proposed to form a type I TA module resembling those of the LDR/Fts family (Fozo et al. , Nucleic Acids Res, 2010, 38: 3743-3759). Type-I TA modules kill cells usually by forming pores (Fozo et al, Nucleic Acids Res, 2010, 38: 3743-3759), some are involved in the general stress response (Wang et al, Nat Med, 2007, 13: 1510-1514) or in specific functions including controlling production of multidrug tolerant cells (Dorr et al, PLoS Biol, 2010, 8: el000317). Here, the toxin is internally encoded by a sRNA and not by an mRNA and protein synthesis is prevented by the sRNA antitoxin in trans. Bacterial Type-I TA systems are usually found in multiple copies and there is up to five copies of sprA in some S. aureus strains (Pichon et al, Proc Natl Acad Sci U S A, 2005, 102: 14249-14254), but the systematic expression of an associated asRNA with each copy is unknown. However, one difference between the TA systems and ' SprAl -SprA l _A s' is that in vivo, SprAl levels are unaffected by increasing SprAl _A s expression. The SprAl- encoded peptide shares physico-chemical properties with S. aureus PSMs that are small, amphipathic and α-helical peptides with significant cytolytic activity against human neutrophils and erythrocytes (Otto et al, Annu Rev Microbiol, 2010, 64: 143-162). a- type PSMs also cause chemotaxis and cytokines release. Therefore, similar biological activities are anticipated for the SprAl peptide. PSMs are regulated by the accessory gene regulator A through its direct interaction with the PSM promoter (Queck et al., Mol Cell, 2008, 32: 150-158). In contrast, SprAl translation is down regulated by an asRNA, as for the TA systems.

In conclusion, cis antisense R As can officiate in trans, implying mechanistic re- evaluations of the identified antisense R A-mediated target gene regulations. Also, a novel virulence factor expressed by S. aureus is reported and its expression is locked in vivo by an inhibitory sR A pair. The next challenges are to understand when this novel cytolysin is expressed and its biological functions during staphylococcal infections.

Example 2: Optimization of the SprAl Peptide

The CMI determinations using a radial diffusion assay (RDA) and haemo lytic assays were performed as described in Example 1. However, hemolytic activity was assessed by determining the concentration of haemoglobin released measured at 414 nm. A positive control (1% triton in water) for which all the red blood cells present in a well are lyzed (100% hemolysis) and a negative control (PBS) for which none of the red blood cells are lyzed (0% hemolysis) were used.

Table 2. CMI values and haemolytic activity for 5 SprAl antimicrobial peptides derived from the SprAl peptide.

In addition, CMI values were measured under microdilution conditions. 10 ⁴ to 10 ⁵ CFU of S. aureus or E. coli in Muller Hinton medium were contacted with decreasing quantities of the peptide to be tested diluted in PBS. The plates were incubated at 37°C for 20 hours. The last concentration at which there was no bacterial growth was determined as the CMI (minimal inhibitory concentration). CMI values, which were found to be identical for S. aureus or E. coli, were of 200 μΜ for FFWLSRRTK (SEQ ID NO: 11), and of 100 μΜ for FFWLRRT*K (SEQ ID NO: 15), wherein T* is a homohydroxy threonine.