WO1996010579A1

WO1996010579A1 - Blocking expression of virulence factors in s. aureus

Info

Publication number: WO1996010579A1
Application number: PCT/US1995/012708
Authority: WO
Inventors: Richard P. Novick; Naomi Balaban; Guangyong Ji
Original assignee: New York University
Priority date: 1994-10-04
Filing date: 1995-10-02
Publication date: 1996-04-11
Also published as: AU3825995A

Abstract

This invention provides a peptide which inhibits agr transcription in S. aureus RN6390B and thereby blocks the expression of virulence factors in S. aureus RN6390B, as well as the use of the peptide to control S. aureus infection. This invention also provides a purified and isolated protein and a peptide which activates agr transcription, and the use of antibodies generated from the activator protein and peptide to control S. aureus infection.

Description

BLOCKING EXPRESSION OF VIRULENCE FACTORS IN S. AUREUS

This invention was made under NIH Grant No. Al- R01-30138. As such, the Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Staphylococcus aureus (S. aureus) is Gram- positive, aerobic bacterial pathogen, distinguished from other staphylococcal species by the production of the enzyme coagulase. Sa is a normal inhabitant of the skin and mucous membranes of man and other animals and under certain circumstances invades the body, causing a wide variety of disease conditions ranging from superficial abscesses (boils and furuncles) to disfiguring and life- threatening deep infections such as endocarditis, pneumonia, osteomyelitis, septic arthritis, meningitis, post-operative wound infections, and septicemia. iSa also causes diseases such as toxic shock syndrome.

Like other Gram-positive pathogens, £>a causes disease chiefly through the production and secretion of injurious proteins. These injurious extracellular proteins, or virulence factors (VF) , include toxins that damage or dissolve host cells, toxins that interfere with the immune system, and enzymes that degrade tissue components such as proteins, nucleic acids, lipids and polysaccharides.

In laboratory cultures, VF are produced and secreted at the end of the standard exponential growth phase, during a segment of the growth cycle known as the post-exponential phase. The production of VF is coordinately regulated and is thought to represent an attempt by the bacteria to generate new sources of nutrition at a time of rapidly diminishing resources. In the infected individual, this may include an attack on the host defenses that have been mobilized to ward off and contain the infection. Sa infections are presently treated with antibiotics, which are natural or semisynthetic chemicals that kill or inhibit the growth of bacterial cells. Unfortunately, antibiotics have become less and less effective in treating Sa infections due to the acquired resistance of Sa to these antibiotics. Major nosocomial epidemics are now caused worldwide by strains of S_a that are resistant to most antibiotics. The antibiotic vancomycin is still effective in treating various strains of Sa, although there is a grave danger that those strains will soon acquire resistance to vancomycin from a closely related Gram-positive pathogen, Enterococcus faecalis.

Since there is little reason to expect the introduction of major new classes of antibiotics, there is an urgent need to develop new methods to control Sa infections, such as interference with the expression of VF. If the bacteria could be disarmed, it is believed that host defenses would do the rest.

In S. aureus. expression of virulence factors is controlled by a global regulator known as aσr (Peng, H. , et al. J. Bacteriol. 179: 4365-4372 (1988); Regassa, L.B., et al. Infect. Immun. 60: 3381-3383 (1992)). Aσr is a genetic locus that contains several genes. Two of these, aσrA and aσrC are thought to constitute a signal transduction (STR) pathway that responds to one or more external signalling molecules by activating the transcription of a third gene, aσr-rnalll (Kornblum, J. , et al., in Molecular Biology of the Staphylococci, R.P. Novick, ed. (VCH Publishers, New York, 1990) ; Bourret, R.B., et al. Annu. Rev. Biochem. 60: 401-441 (1991)). The primary transcript of aσr-rnalll. known as RNAIII, induces transcription of the 20 or more independent genes encoding virulence factors, thereby resulting in the synthesis of VF (Novick, R.P., et al. EMBO Journal 12(10): 3967-3975 (1993)). It has been shown that laboratory-generated mutant strains of Sa, unable to express VF, exhibit greatly reduced virulence (Foster, et al. Molecular Biology of the Staphylococci. Editor: R.P. Novick, VCH Publishers, New York, pp. 403-420 (1990)). Interference with activation of the a r system would therefore afford a simple means of blocking the expression of VF, and thus interfere with the infective process.

Raychoudhury, S. et al. PNAS 90:965-969 (1993) recently described the identification of synthetic chemical compounds that block the expression of alginate, a VF for the cystic fibrosis pathogen, Pseudomonas aeruσinosa. It has not been shown, however, whether these chemicals would have any effect on Sa, or offer any potential clinical utility.

The present invention is based upon the discovery of a peptide which interferes with the activation of rnalll transcription and thus prevents expression of VF. The present invention also is based upon the discovery of a protein and a peptide which activates the expression of mallI and thus induces the production of VF. Antibodies generated against this activator protein and peptides would interfere with the transcription of rnalll. thereby preventing expression of VF.

Prevention of the expression of VF by S. aureus using the peptide which inhibits activation of rnalll transcription, and antibodies directed to the peptide and protein which activate expression of rnalll is expected to prevent or treat diseases caused by Staphylococcal infections.

SUMMARY OF THE INVENTION

This invention provides a peptide capable of inhibiting rnalll transcription in S. aureus and blocking the expression of virulence factors in S. aureus. as well as a method of blocking the expression of virulence factors in S. aureus utilizing the peptide. This invention also provides a peptide and a purified protein which activates rnalll transcription and the use of antibodies directed against this protein and peptide to inhibit rnalll transcription in S. aureus, and thereby block expression of virulence factors in S. aureus.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A represents the autoradiogram of the Northern blot hybridization analysis of RNA electropherograms of whole cell lysates prepared from culture samples, taken at various growth phases of a standard virulent S_a strain, RN6390B, over a 240 minute period. The blot was probed with a radiolabeled sample of DNA specific for RNAIII.

Figure IB is a graphic representation of the levels of RNAIII from Figure 1A during growth of the bacteria. The level of RNAIII is in PhosphorImager units (PIU) . (o) indicates RNAIII level and (I) indicates log cell number.

Figure 2 represents the autoradiogram of the Northern blot hybridization analysis of whole cell lysates separated by agarose gel electrophoresis, blotted to nitrocellulose and probed with a radiolabeled DNA sample specific for RNAIII. The blots indicate levels of RNAIII present in S. aureus cells grown under different conditions. S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0⁷ cells/ml. When the cells reached a density of about 10⁸ cells/ml (time 0) , the culture was divided in four parts. For part A, growth was allowed to continue. For part B, one-tenth the volume of 10-times concentrated post-exponential phase culture supernatant was added and growth was allowed to continue. For part C, the culture was centrifuged, the supernatant discarded, and the cells resuspended in fresh CY broth. For part D, the culture was centrifuged and the cells resuspended in fresh CY broth plus concentrated post-exponential phase supernatant as in part B. Culture samples were taken every 20 minutes from each of the four cultures. Parts A-D are represented in the figure as panels A-D, respectively. Figure 3 represents levels of rnalll transcription when concentrated supernatants of S. aureus RN6390B were collected at various time points during growth and added to S. aureus growth culture. T2 (π) , T3 (0) and T5 (D) represent times (2, 3, and 5 hours, respectively) during growth when supernatants were collected. CY (♦) represents culture broth in which bacteria was not grown.

Figure 4 represents the results of a genetic analysis showing that the activator is either encoded or up-regulated by aσrB and aσrD. At left is a diagrammatic representation of the cloned a r locus showing deletions affecting different genes within agr.. As can be seen, activator activity, measured by the β-lactamase method, is unaffected by deletions affecting main. aσrA or agrC, but is eliminated by deletions affecting aσrB or agrD. All of the tests with the exception of those in the top line, using RN6390B, were performed with an aσr-null strain, RN6778, in which the agr locus was replaced by the tetM gene (bottom line) . Plasmid derivatives containing various agr subclones, in which agr transcription is controlled by the β-lactamase promoter (P-bla) , were introduced into the agr-null strain and the resulting composite strains tested for production of the activator following induction of the β-lactamase promoter. Figure 5A represents the autoradiogram of the

Northern blot analysis of fractions of concentrated late- phase supernatant of RN6390B (T7) collected from an HPLC gel filtration column (BioRad, Bio-Sil SEC 250-5) , lyophilized to 10X, and added to early-exponential Sj_ aureus cultures of RN6390B (to a IX dilution) . Lane 1 represents boiled T7 before being applied to the column, as a positive control. Lane 2 represents Cy broth, as a negative control. Lanes 3-7 represent column fractions corresponding to elution volumes 5-7, 7-10, 10-13, 13- 13.5, and 14-15 ml, respectively. Lane 6 represents the 13-13.5 ml fraction which activates rnalll transcription. Figure 5B represents SDS PAGE analysis of the 7-

10 ml post injection fraction having no activity (lane 1) and the 13-13.5 ml fraction which activates rnalll transcription (lane 2) . As shown in the figure, the purified material which activates rnalll transcription has a molecular weight of about 35 kD.

Figure 6A represents the autoradiogram of the Northern blot analysis of fractions of concentrated late- phase supernatant T6 of RN6390B separated on a reverse phase C18 HPLC column which were collected, lyophilized, and added to early-exponential S. aureus cultures of RN6390B. Cy represents Cy broth, as a negative control. Lanes 1-8 represent fractions collected from the column with increasing amounts of acetonitrile. Lane 6 represents the fraction which activates rnalll transcription, and eluted from the column at 35% acetonitrile. T6 represents the boiled T6 before being applied to the column, as a positive control.

Figure 6B represents an SDS PAGE of the fractions corresponding to lanes 5-8 shown in Figure 6A. The protein which activates rnalll transcription has a molecular weight of about 40 kDa.

Figure 6C represents a spectrophotometer tracing (at low OD (0-0.12)) of the HPLC fractionation of the material eluted from the C18 reverse phase HPLC column at 35% acetonitrile, and corresponding to the fraction shown in lane 6 of Figure 6A.

Figure 7 represents a chromatogram of the purification of the peptide which activates rnalll transcription. The peptide was fractionated on an HPLC C18 column, using a 20-32% acetonitrile gradient at 0.2% acetonitrile per min. The peptide eluted at an acetonitrile concentration of about 28.5%. Figure 8A represents an autoradiogram of RNAIII Northern blots resulting from the addition of culture supernatants from different S. aureus strains to a standard S. aureus RN6390B culture. Duplicate numbers represent duplicate experiments. Lane 1, no supernatant; Lane 2, S♦ aureus RN7111; lane 3, RN7112; lane 4, RN8470; lane 5, RN8471; (6) lane 6, RN831; lane 7, RN833; and lane 8, RN6390B.

Figure 8B represents a densitometric analysis of the autoradiogram of Figure 8A.

Figure 9 represents the level of RNAIII in S. aureus RN6390B cells when various dilutions of concentrated supernatant (none (♦) ; 6 hr supernatant (T6) , diluted 1:10 (D) ; T6, diluted 1:20 (0); and T6, diluted 1:40 (D) ) from a post-exponential phase culture of mutant S. aureus strain RN833, were added to S. aureus RN6390B growth culture.

Figure 10A represents an autoradiogram of a Northern blot analysis of RN6390B cells treated with RN833 concentrated supernatant fractionated on a reverse phase C18 HPLC column, and eluted with increasing amounts of acetonitrile in 0.1% TFA. Lanes 1-12 represent fractions collected from the column with increasing amounts of acetonitrile. Lane 13 represent cells collected before addition of any fractionated material at time 0. Lane 14 represents no addition (control) at 40 minutes. Lane 9 represents the fraction eluted at 35% acetonitrile which retained its inhibitory activity.

Figure 10B represents the absorbance at 214nm of the fraction from lane 9 of Figure 10A which was reapplied to the C18 reverse phase column.

Figure 11 represents levels of RNAIII in S. aureus RN6390B cells when various concentrations of S. aureus mutant strain RN833 supernatant, either alone or in combination with various concentrations of S. aureus RN6390B supernatant, were added to RN6390B growth culture. The culture supernatants that were added were as follows: (1) CY; (2) lOOμl RN833; (3) 50μl RN833; (4) lOμl RN833; (5) lμl RN833; (6) 50μl RN6390B; (7) lOOμl RN833 + 50μl RN6390B; (8) 50μl RN833 + 50μl RN6390B; (9) lOμl RN833 + 50μl RN6390B; and (10) lμl RN833 + 50μl RN6390B.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a peptide which inhibits agr-rnalll transcription in S. aureus, and thereby blocks expression of virulence factors in S. aureus. Preferably, the peptide of the present invention is produced by S. aureus mutant strain RN833 deposited with the American Type Culture Collection, Rockville, Maryland on October 5, 1994 under ATCC Accession No. 55619. In the more preferred embodiment, the peptide is purified from a post-phase supernatant of S. aureus mutant strain RN833 and elutes as a single peak at about 35% acetonitrile on reverse phase HPLC, and has a molecular weight of less than 3 kDa. In the most preferred embodiment, the peptide comprises the amino acid sequence Pro-X-Thr-Asn-Phe, wherein X is Cys, Trp or a modified amino acid selected from the group consisting of modified amino acids known in the art.

The present invention also provides a method for blocking expression of virulence factors in S♦ aureus which comprises contacting S. aureus with the peptide above which inhibits agr-rnalll transcription in S. aureus. The peptide will block expression of virulence factors, thereby preventing or treating diseases caused by S. aureus infections.

The method of the present invention may be used in any human or animal which may be suspectable to S. aureus infection, and preferably those with compromised immune systems such as patients with AIDS, or other immunodeficiencies, and transplant patients. For purposes of treatment or prevention, the peptide may be administered as in conjunction with pharmaceutically acceptable carriers well known in the art such as aqueous solutions containing sodium chloride, glycine, and the like. In addition, the peptide may be administered in combination with traditional antibiotics which are used to treat diseases caused by S. aureus infections.

Accordingly, the present invention also provides a peptide composition comprising the peptide hereinabove and a pharmaceutically or physiologically acceptable carrier. Such formulations may be prepared by adding the peptide to water containing physiologically compatible substances such as sodium chloride, glycine, and the like, having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The peptide or peptide composition is administered in vaccine form using procedures known in the art such as intravenous, intramuscular, subcutaneous, or intraperitoneal routes of administration.

The present invention also provides a peptide and a purified and isolated protein which activates agr- rnalll transcription in S. aureus. Preferably, the peptide and protein of the present invention are produced by S. aureus strain RN6390B deposited with the American Type Culture Collection, Rockville, Maryland on October 5, 1994 under ATCC Accession No. 55620. In the more preferred embodiment, the peptide is purified from a post-exponential phase supernatant of S. aureus strain RN6390B and elutes as a single peak at about 28.5% acetonitrile on reverse phase HPLC, and has a molecular weight of less than 3 kDa. The protein also is purified from a post-exponential phase supernatant of S. aureus strain RN6390B and elutes as a single peak at about 35% acetonitrile on reverse phase HPLC, and has a molecular weight of about 35-40 kDa on SDS PAGE.

The present invention also provides a peptide or protein composition comprising the activator peptide or protein, respectively, conjugated to a carrier known in the art to enhance immunogenicity such as keyhole limpet hemocyanin (KLH) , BSA, and the like. This peptide or protein composition may be utilized as a vaccine to treat or prevent diseases caused by S. aureus infections.

In addition, the present invention provides an antibody which specifically binds and is immunoreactive with the activator peptide and protein hereinabove. The antibody binds to the site on the peptide or protein which activates agr-rnalll transcription in S. aureus. The antibody therefore inhibits agr-rnalll transcription in S. aureus. and thus is useful for blocking expression of virulence factors in S. aureus.

The antibody of the present invention may be polyclonal or monoclonal, and is prepared using methods well known in the art. In particular, the polyclonal antibody may be produced by immunizing a rabbit, mouse or rat with the protein or the peptide as an immunogen and collecting the serum produced thereby. The protein or peptide may be coupled to a protein carrier to enhance immunogenicity such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) . An adjuvant also may be used. A booster injection should be given 4-6 weeks after the primary injection. Additional booster injections may be given at later periods if necessary. The presence of antibody in the serum may be tested by radioimmunoassay (RIA) , enzyme-linked immunosorbent assay (ELISA) , or immunoprecipitation.

To produce monoclonal antibodies, the spleen cells from the rabbit, mouse or rat are removed and fused with a myeloma cell to form a hybridoma, which is grown in culture to produce the desired monoclonal antibody by standard procedures.

Lastly, the present invention provides a method for blocking expression of virulence factors in S. aureus which comprises contacting the S. aureus with the antibody above so that the antibody inhibits agr-rnalll transcription. Again, the method of the present invention may be used in any human or animal which may be suspectable to S. aureus infection, and preferably those with compromised immune systems such as patients with AIDS, or other immuno-deficiencies, and transplant patients. For purposes of treatment or prevention, the antibody may be administered using procedures known in the art such as intravenous, intramuscular, subcutaneous, or intraperitoneal routes of administration.

The present invention is described in the following Experimental Details section, which sets forth specific examples to aid in an understanding of the invention, and should not be construed to limit in any way the invention as defined in the claims which follow thereafter.

Experimental Details section

I. Characterization of Material Which Activates rnalll Transcription in S. aureus

A. RNA III Synthesis in S. aureus RN6390

S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting in the early exponential phase at approximately 10⁸ cells/ml. Samples containing an equal number of cells were removed at various intervals over a 240 minute period. The whole-cell lysates were analyzed by Northern blotting. The level of RNAIII in the cells was determined using a radiolabeled rnalll-specific DNA as a probe (Kornblum, J., et al. Gene 63: 75-85 (1988)). The blot was exposed to a storage phosphor screen (Molecular Dynamics) , which was scanned in a Phosphorlmager 425 (Molecular Dynamics) . The relative levels of RNAIII were measured using Image Quant software.

The autoradiogram of the Northern blot is shown in Figure 1A. A graphic representation of the levels of

RNAIII in Phosphorlmager units (PIU) is shown in Figure

IB. (o) indicates RNAIII level and (■) indicates log cell number. Figures 1A and IB demonstrate that the RNAIII level (as represented by rnalll transcription) is low during the early exponential growth phase and increases sharply during the mid- and late-exponential phases. B. Effect of Exponential Growth Supernatant on RNAIII Synthesis in S. aureus RN6390B

S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0⁷ cells/ml. When the cells reached a density of about 10⁸ cells/ml (time O) , the culture was divided in four parts. For part A, growth was allowed to continue. For part B, one-tenth the volume of

10-times concentrated post-exponential phase culture supernatant was added and growth was allowed to continue. For part C, the culture was centrifuged, the supernatant discarded, and the cells resuspended in fresh CY broth. For part D, the culture was centrifuged and the cells resuspended in fresh CY broth plus concentrated post- exponential phase supernatant as in part B. Culture samples were taken every 20 minutes from each of the four cultures. The whole-cell lysates were analyzed by Northern blotting using a radiolabeled rnalll-specific DNA as described above.

The results are presented in Figure 2. As shown in panels B and D, the addition of concentrated post- exponential phase supernatant caused the immediate synthesis of RNAIII, at higher levels than seen in untreated cultures.

In another experiment, early-, mid- and post exponential phase supernatants were added to the culture. In particular, S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0⁷ cells/ml. When the cells reached a density of about 10⁸ cells/ml, 10- fold concentrated supernatants collected at various time points from the culture of S. aureus RN6390B were added, and growth continued. Samples containing an equal number of cells were removed at various intervals over a 120 minute period. The whole-cell lysates were analyzed by Northern blotting using a radiolabeled rnalll-specific DNA as described above.

The results are presented in Figure 3. The concentrated RN6390B supernatants added were: none (♦) (CY) ; a 2hr old culture, i.e. of an early exponential phase culture (□) (T2) ; a 3hr culture, i.e. of a mid- exponential phase culture (0) (T3) ; and a 5hr old culture, i.e. of a post-exponential phase culture (D) (T5) . Agr activating activity was demonstrated in supernatants from mid-exponential phase, RN6390B cultures, when agr is normally activated, as well as from post-exponential phase supernatants. The activity increased with the age of the culture suggesting that the activity is continuously produced.

C. Effect of Proteinase K, pH and Boiling on Agr-Activation

S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0⁷ cells/ml. When the cells reached a density of about 10⁸ cells/ml, concentrated supernatants were collected from a late post-exponential phase culture of S. aureus RN6390B and subjected to various treatments. The post-exponential culture supernatant was adjusted to a pH of 7.4 (pH 5.5). This change in pH did not effect the activity. The supernatant also was treated with proteinase K. The proteinase K treatment eliminated the agr-activating effect. The effect of heat also was tested. Boiling the supernatant for 10 minutes caused some reduction in agr-activity.

D. Genetic Analysis of Activator Material The results of a genetic analysis showing that the activator is either encoded or up-regulated by agrB and agrD is presented in Figure 4. At left is a diagrammatic representation of the cloned agr locus showing deletions affecting different genes within agr. As can be seen, activator activity, measured by the β- lacta ase method, is unaffected by deletions affecting main. agrA or agrC, but is eliminated by deletions affecting agrB or agrD. All of the tests with the exception of those in the top line, using RN6390B, were performed with an agr-null strain, RN6778, in which the agr locus was replaced by the tetM gene (bottom line) . Plasmid derivatives containing various agr subclones, in which agr transcription is controlled by the β-lactamase promoter (P-bla) , were introduced into the agr-null strain and the resulting composite strains tested for production of the activator following induction of the β-lactamase promoter.

E. Purification of Activator Material

From Late Phase Culture Supernatants 1) Purification of Protein Using

HPLC Gel Filtration

The concentrated late-phase supernatant of

RN6390B (T7) (7 hour) was boiled for 10 minutes, centrifuged for 10 minutes at 14K, and applied to an HPLC gel filtration column (BioRad, Bio-Sil SEC 250-5) . Fractions were collected, lyophilized to 10X, and added to early-exponential S. aureus cultures of RN6390B (to a IX dilution) . Samples were analyzed for RNAIII after 20 minutes using Northern blot analysis as described above. The results are presented in Figure 5A. Lane 1 represents boiled T7 before being appled to the column, as a positive control. Lane 2 represents Cy broth, as a negative control. Lanes 3-7 represent column fractions corresponding to elution volumes 5-7, 7-10, 10-13, 13- 13.5, and 14-15 ml, respectively. Lane 6 represents the

13-13.5 ml fraction which activates rnalll transcription.

The 7-10 ml post injection fraction having no activity but maximum protein concentration and the 13-13.5 ml fraction which activates rnalll transcription were separated on SDS 12% PAGE. The gel was silver stained (BioRad) . The results of the SDS PAGE are shown in Figure 5B. Lane 1 contains the 7-10 ml fraction, and lane 2 contains the 13-13.5 ml fraction. As shown in Figure 5B, the purified material which activates rnalll transcription has a molecular weight of about 35 kD. 2) Purification of Protein Using C18 Reverse Phase HPLC

Concentrated wild type late phase RN6390B supernatant (T6) (6 hour) was fractionated on a C18 reverse phase HPLC column, and eluted with increasing amounts of acetonitrile in 0.1% TFA. Fractions were collected, lyophilized, and added to early exponential S. aureus RN6390B cultures in CY culture broth with shaking at 37°C. Growth was allowed to continue for another 20 minutes. Samples containing equal numbers of cells were then removed and whole-cell lysates were analyzed by Northern blotting. The ability of the fractions to activate the agr response was analyzed using radiolabeled rnalll-specific DNA as a probe. The blot was exposed to a storage phosphor screen (Molecular Dynamics) , which screen was then scanned in a Phosphorlmager 425 (Molecular Dynamics) .

The results of the Northern blot analysis is presented in Figure 6A. Cy represents Cy broth, as a negative control. Lanes 1-8 represent fractions collected from the column with increasing amounts of acetonitrile. Lane 6 is the fraction which activates rnalll transcription, and eluted from the column at 35% acetonitrile. T6 represents the boiled T6 before being appled to the column, as a positive control.

The fractions corresponding to lanes 5-8 from Figure 6A were separated on SDS 12.5% PAGE, and the gel was silver stained (BioRad) . The results of the SDS PAGE are shown in Figure 6B. The numbers on the left side of Figure 6B represent the sizes of molecular weight markers, in kilodaltons (kDa) . The most active fraction was shown to be enriched for a protein of about 40 kDa. The material eluted from the C18 reverse phase HPLC column at 35% acetonitrile, and corresponding to the fraction shown in lane 6 of Figure 6A, was rerun on HPLC. The results are presented in the spectrophotometer tracing shown in Figure 6C. 3) Purification of Peptide Using C18 Reverse Phase HPLC

Strain RN6390B was grown in a predialyzed synthetic medium (Difco tryptophan assay medium) . The supernatant was filtered through a Millipore 0.45μ filter, boiled 10 min., concentrated 10-fold by lyophilization, then applied to an HPLC C18 column in 2.5% acetonitrile and eluted with an acetonitrile gradient (16-48%) at 0.27% acetonitrile per min. Fractions with activator activity were pooled, concentrated 10-fold by lyophilization, and filtered through an Amicon ultrafilter (Centricon 3) which allows only material of less than 3 kDa molecular weight to pass through. The activator was found to pass through this filter indicating it had a molecular weight less than 3 kDa. The flow-through was then refractionated on the HPLC C18 column, using a 20-32% acetonitrile gradient at 0.2% acetonitrile per min. The peptide which activates rnalll transcription eluted at an acetonitrile concentration of about 28.5% as shown in Figure 7.

II. Characterization of Material Which Inhibits rnalll Transcription in S. aureus

A. Effect of Exponential Growth Supernatant from S. aureus Strain RN833 on RNAIII Synthesis in S. aureus RN6390B

S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0⁷ cells/ml. When the cells reached a density of about 10⁸ cells/ml, concentrated supernatants collected from a late post-exponential phase culture from various S. aureus strains and concentrated 10 times by lyophilization were added, and growth continued. Samples containing equal numbers of cells were removed 20 minutes and 40 minutes later, and whole-cell lysates were analyzed for RNAIII by Northern blotting. The expression of agr-rnalll was determined using radiolabeled main- specific DNA as a probe. The concentrated 6 hr (T6) supernatants that were added to the culture broth were taken from the following cultures: (1) no supernatant; (2) S. aureus RN7111; (3) RN7112; (4) RN8470; (5) RN8471; (6) RN831; (7) RN833; and (8) RN6390B. These strains were grown in CY culture broth with shaking at 37°C, starting at 5x10⁸ cells/ml, to late post exponential phase 6 hours later (T6).

The results are presented in the autoradiogram shown in Figure 8A, and the densitometric analysis of the autoradiogram shown in Figure 8B. As can be seen in both

Figures 8A and 8B, a product of S. aureus strain RN833 inhibits rnalll activation in growth culture of RN6390B.

In another experiment, S♦ aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0⁷ cells/ml. When the cells reached a density of about 10⁸ cells/ml, increasing amounts of supernatant collected from a late post exponential phase culture of S. aureus mutant strain RN833 were added (T6) , and growth continued. Samples containing an equal number of cells were removed at various intervals over a 150 minute period, and whole- cell RNA lysates were analyzed by Northern blotting. The expression of agr-RNAIII was determined using radiolabeled rnalll DNA as a probe. The results are presented in Figure 9. The following RN833 supernatants were added to growth culture; none (♦) ; 6 hr supernatant (T6) , diluted 1:10 (D) ; T6, diluted 1:20 (0); and T6, diluted 1:40 (D) . As shown in Figure 9, increased concentrations of S. aureus strain RN833 supernatant decreased levels of agr RNAIII in growth culture, proportionately.

B. Purification of Inhibitor Peptide

From Late Phase Culture Supernatants The inhibitory material secreted by S. aureus mutant strain RN833 was purified. Post-exponential culture supernatant of RN833 was passed through a 3 kD cutoff membrane (A icon) , and fractionated on a C18 reverse phase HPLC column and eluted with increasing amounts of acetonitrile in 0.1% TFA. Fractions were collected, lyophilized, and added to early exponential phase culture of S. aureus RN6390B cells that were growing in CY culture broth with shaking at 37°C. Growth continued for another 40 minutes. Samples containing an equal number of cells were removed and whole-cell lysates were analyzed by Northern blotting. The ability to inhibit main activation was determined using radiolabeled rnalll-specific DNA as a probe. T h e results of the Northern blot analysis is presented in the Figure 10A. Lanes 1-12 represent the fractions collected from the column with increasing amounts of acetonitrile. Lane 13 represents cells collected before addition of any fractionated material at time 0. Lane 14 represents no addition (control) at 40 minutes. Lane 9 represents the fraction eluted at 35% acetonitrile which retained its inhibitory activity. The fraction from lane 9 was reapplied to the C18 reverse phase column, and the absorbance at 214nm is shown in Figure 10B. The amino acid sequence of the fraction was determined to be Pro-X- Thr-Asn-Phe, wherein X is Cys, Trp or any modified amino acid known in the art, using the Ed an degradation procedure.

III. Effect of agr-Inhibitor on agr-Activator

In order to determine whether the presence of the agr-inhibitor decreases the level of RNAIII activation caused by the agr-activator, culture supernatants containing the agr-activator were added to growing cultures along with increasing amounts of the agr- inhibitor. A constant amount of concentrated activator- containing S. aureus (RN6390B) T6 was added with increasing amounts of mutant S. aureus (RN833) T6 to an early exponential phase S. aureus RN6390B culture growing in CY culture broth with shaking at 37°C. The culture supernatants that were added were as follows: (1) CY; (2) lOOμl RN833; (3) 50μl RN833; (4) lOμl RN833; (5) lμl RN833; (6) 50μl RN6390B; (7) lOOμl RN833 + 50μl RN6390B; (8) 50μl RN833 + 50μl RN6390B; (9) lOμl RN833 + 50μl RN6390B; and (10) lμl RN833 + 50μl RN6390B. Growth continued for another 40 minutes. Samples containing equal numbers of cells were removed and whole-cell lysates were analyzed by Northern blotting. The activity of rnalll was determined using radiolabeled malll-specific DNA as a probe. The results are presented in Figure 11. The resulting RNA level for each of the added supernatant combinations is shown. Lanes 2-5 of Figure 11 confirm that RN833 supernatant alone inhibits RNAIII induction. When mutant RN833 and wild type RN6390B supernatants were added together, RNAIII induction decreased when the concentration of mutant RN833 supernatant was greater than or equal to the concentration of wild type RN6390B supernatant. RNAIII induction was slightly decreased when the concentration of wild type RN6390B was greater than the concentration of mutant RN833. This indicates that the activator and the inhibitor act in a competitive manner, that they both may bind to the same receptor, and the inhibitor is derived from the activator by mutation. It is also possible that the activator and the inhibitor bind to different receptors and activate different signalling pathways which in turn induce or repress rnalll transcription.

All publications mentioned hereinabove are hereby incorporated by reference in their entirety. Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention in the appended claims.

Claims

What is Claimed is:

1. A peptide which inhibits agr-rnalll transcription in S. aureus.

2. The peptide of claim l which is produced by S. aureus mutant strain RN833 deposited under ATCC Accession No. 55619.

3. The peptide of claim 2 which elutes as a single peak at about 35% acetonitrile on reverse phase HPLC, and has a molecular weight of less than 3 kDa.

4. The peptide of claim 3 comprising the amino acid sequence Pro-X-Thr-Asn-Ph , wherein X is Cys, Trp or a modified amino acid.

5. A peptide composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier.

6. A peptide composition comprising the peptide of claim 2 and a pharmaceutically acceptable carrier.

7. A peptide composition comprising the peptide of claim 3 and a pharmaceutically acceptable carrier.

8. A peptide composition comprising the peptide of claim 4 and a pharmaceutically acceptable carrier.

9. A method for blocking expression of virulence factors in S. aureus which comprises contacting S. aureus with a peptide which inhibits agr-rnalll transcription in S. aureus.

10. The method of claim 9, wherein the peptide is produced by S. aureus mutant strain RN833 deposited under ATCC Accession No. 55619.

11. The method of claim 10, wherein the peptide elutes as a single peak at about 35% acetonitrile on reverse phase HPLC, and has a molecular weight of less than 3 kDa.

12. The method of claim 7, wherein the peptide comprises the amino acid sequence Pro-X-Thr-Asn-Phe, wherein X is Cys, Trp or a modified amino acid.

13. A method for blocking expression of virulence factors in S. aureus which comprises contacting

S. aureus with a peptide composition comprising a peptide which inhibits agr-rnalll transcription in S. aureus and a pharmaceutically acceptable carrier.

14. The method of claim 13, wherein the peptide is produced by S. aureus mutant strain RN833 deposited under ATCC Accession No. 55619.

15. The method of claim 14, wherein the peptide elutes as a single peak at about 35% acetonitrile on reverse phase HPLC, and has a molecular weight of less than 3 kDa.

16. The method of claim 15, wherein the peptide comprises the amino acid sequence Pro-X-Thr-Asn-Phe, wherein X is Cys, Trp or a modified amino acid.

17. A peptide which activates agr-rnalll transcription in S♦ aureus.

18. The peptide of claim 17 which is produced by S. aureus strain RN6390B deposited under ATCC Accession No. 55620.

19. The peptide of claim 18 which elutes as a single peak at about 28.5% acetonitrile on reverse phase HPLC, and has a molecular weight of less than 3 kDa.

20. A peptide composition comprising the peptide of claim 17 conjugated to an immunogenic carrier.

21. A peptide composition comprising the peptide of claim 18 conjugated to an immunogenic carrier.

22. A peptide composition comprising the peptide of claim 19 conjugated to an immunogenic carrier.

23. An antibody which specifically binds to the peptide of claim 17.

24. An antibody which specifically binds to the peptide of claim 18.

25. An antibody which specifically binds to the peptide of claim 19.

26. A method for blocking expression of virulence factors in S♦ aureus which comprises contacting S. aureus with the antibody of claim 23.

27. A method for blocking expression of virulence factors in S. aureus which comprises contacting S. aureus with the antibody of claim 24.

28. A method for blocking expression of virulence factors in S. aureus which comprises contacting S. aureus with the antibody of claim 25.

29. A purified and isolated protein which activates agr-rnalll transcription in S. aureus.

30. The protein of claim 29 which is produced by S. aureus strain RN6390B deposited under ATCC Accession

No. 55620.

31. The protein of claim 30 which elutes as a single peak at about 35% acetonitrile on reverse phase HPLC.

32. The protein of claim 31 which has a molecular weight of about 35-40 kDa on SDS PAGE.

33. A protein composition comprising the protein of claim 29 conjugated to an immunogenic carrier.

34. A protein composition comprising the protein of claim 30 conjugated to an immunogenic carrier.

35. A protein composition comprising the protein of claim 31 conjugated to an immunogenic carrier.

36. A protein composition comprising the protein of claim 32 conjugated to an immunogenic carrier.

37. An antibody immunoreactive with the protein of claim 29.

38. An antibody immunoreactive with the protein of claim 30.

39. An antibody immunoreactive with the protein of claim 31.

40. An antibody immunoreactive with the protein of claim 32.

41. A method for blocking expression of virulence factors in S. aureus which comprises contacting S. aureus with the antibody of claim 37.

42. A method for blocking expression of virulence factors in S. aureus which comprises contacting S. aureus with the antibody of claim 38.

43. A method for blocking expression of virulence factors in S. aureus which comprises contacting

S. aureus with the antibody of claim 39.

44. A method for blocking expression of virulence factors in S. aureus which comprises contacting S. aureus with the antibody of claim 40.