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WO1996010579A1 - Blocage de l'expression des facteurs de virulence du staphylocoque dore - Google Patents

Blocage de l'expression des facteurs de virulence du staphylocoque dore Download PDF

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Publication number
WO1996010579A1
WO1996010579A1 PCT/US1995/012708 US9512708W WO9610579A1 WO 1996010579 A1 WO1996010579 A1 WO 1996010579A1 US 9512708 W US9512708 W US 9512708W WO 9610579 A1 WO9610579 A1 WO 9610579A1
Authority
WO
WIPO (PCT)
Prior art keywords
aureus
peptide
protein
antibody
rnalll
Prior art date
Application number
PCT/US1995/012708
Other languages
English (en)
Other versions
WO1996010579A9 (fr
Inventor
Richard P. Novick
Naomi Balaban
Guangyong Ji
Original Assignee
New York University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by New York University filed Critical New York University
Priority to AU38259/95A priority Critical patent/AU3825995A/en
Publication of WO1996010579A1 publication Critical patent/WO1996010579A1/fr
Publication of WO1996010579A9 publication Critical patent/WO1996010579A9/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a ⁇ r is a genetic locus that contains several genes. Two of these, a ⁇ rA and a ⁇ rC 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, a ⁇ r-rnalll (Kornblum, J.
  • 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.
  • 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 5xl0 7 cells/ml. When the cells reached a density of about 10 8 cells/ml (time 0) , the culture was divided in four parts. For part A, growth was allowed to continue.
  • 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.
  • the antibody of the present invention may be polyclonal or monoclonal, and is prepared using methods well known in the art.
  • 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) .
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • 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.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent as
  • S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting in the early exponential phase at approximately 10 8 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.
  • S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0 7 cells/ml. When the cells reached a density of about 10 8 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
  • 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.
  • S. aureus RN6390B was grown in CY culture broth with shaking at 37°C starting at 5xl0 7 cells/ml. When the cells reached a density of about 10 8 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un peptide qui inhibe la transcription d'agr dans RN6390B du staphylocoque doré et, de ce fait, bloque l'expression de facteurs de virulence dans RN6390B du staphylocoque doré, ainsi que l'utilisation de ce peptide dans la lutte contre l'infection par le staphylocoque doré. Elle concerne également une protéine purifiée et isolée et un peptide qui active la transcription d'agr, ainsi que l'utilisation d'anticorps produits à partir de la protéine et du peptide d'activation, afin de lutter contre l'infection par le staphylocoque doré.
PCT/US1995/012708 1994-10-04 1995-10-02 Blocage de l'expression des facteurs de virulence du staphylocoque dore WO1996010579A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU38259/95A AU3825995A (en) 1994-10-04 1995-10-02 Blocking expression of virulence factors in s. aureus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31849994A 1994-10-04 1994-10-04
US08/318,499 1994-10-04

Publications (2)

Publication Number Publication Date
WO1996010579A1 true WO1996010579A1 (fr) 1996-04-11
WO1996010579A9 WO1996010579A9 (fr) 1996-07-18

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/012708 WO1996010579A1 (fr) 1994-10-04 1995-10-02 Blocage de l'expression des facteurs de virulence du staphylocoque dore

Country Status (2)

Country Link
AU (1) AU3825995A (fr)
WO (1) WO1996010579A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040979A1 (fr) * 1995-06-07 1996-12-19 Microcide Pharmaceuticals, Inc. Procedes d'evaluation de cibles antimicrobiennes
WO1997011690A3 (fr) * 1995-09-29 1997-09-12 Microcide Pharmaceuticals Inc Inhibiteurs des voies de regulation
WO1997044349A1 (fr) * 1996-05-22 1997-11-27 New York University BLOCAGE DE L'EXPRESSION DE L'AGGRESSINE DANS $i(S. AUREUS)
WO1999026968A1 (fr) * 1997-11-26 1999-06-03 The University Of Nottingham Oligopeptides et leur utilisation comme agents antibacteriens contre des souches de staphylococcus
US6291431B1 (en) 1997-12-19 2001-09-18 Panorama Research Methods and compositions for the treatment and prevention of Staphylococcal infections
WO2001058471A3 (fr) * 2000-02-10 2002-03-07 Intermune Inc Nouveaux inhibiteurs d'acides amines et de peptides de virulence de staphylococcus
EP1188831A2 (fr) * 1998-09-15 2002-03-20 The Regents of the University of California Cible de la protéine qui active RNAIII (TRAP)
US6447786B1 (en) 1994-10-04 2002-09-10 New York University Blocking expression of virulence factors in S. aureus
US6455323B1 (en) 1997-07-03 2002-09-24 Pharmacia & Upjohn Company Anti-bacterial methods and materials
US6689878B2 (en) 1998-09-15 2004-02-10 The Regents Of The University Of California Target of RNAIII activating protein (TRAP)
US6764823B2 (en) 2000-04-06 2004-07-20 Pharmacia & Upjohn Company Antimicrobial methods and materials
EP1862549A2 (fr) 1998-09-15 2007-12-05 Naomi Balaban Cible de protéine d'activation RNAIII (TRAP)
US7323179B2 (en) 1997-12-19 2008-01-29 Naomi Balaban Methods and compositions for the treatment and prevention of Staphylococcus and other bacterial infections
US7534857B2 (en) 1997-12-19 2009-05-19 Centegen, Inc. Methods and compositions for the treatment and prevention of staphylococcal infections
US7824691B2 (en) 2005-04-04 2010-11-02 Centegen, Inc. Use of RIP in treating staphylococcus aureus infections

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FEMS MICROBIOLOGY LETTERS, Volume 133, issued 12 September 1995, N. BALABAN et al., "Translation of RNAIII, the Staphylococcus Aureus agr Regulatory RNA Molecule, Can be Activated by a 3'-end Deletion", pages 155-161. *
JOURNAL OF BACTERIOLOGY, Volume 173, No. 20, issued October 1991, F. VANDENESCH et al., "A Temporal Signal, Independent of agr, is Required for hla But Not spa Transcription in Staphylococcus Aureus", pages 6313-6320. *
JOURNAL OF BACTERIOLOGY, Volume 176, No. 13, issued July 1994, A. CHEUNG et al., "Cloning and Sequencing of sarA of Staphylococcus Aureus, a Gene Required for the Expression of agr", pages 4168-4172. *
THE EMBO JOURNAL, Volume 12, No. 10, issued 1993, R. NOVICK et al., "Synthesis of Staphylococcal Virulence Factors is Controlled by a Regulatory RNA Molecule", pages 3967-3975. *
THE EMBO JOURNAL, Volume 14, No. 18, issued 1995, E. MORFELDT, "Activation of Alpha-toxin Translation in Staphylococcus Aureus by the Trans-encoded Antisense RNA, RNAIII", pages 4569-4577. *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6447786B1 (en) 1994-10-04 2002-09-10 New York University Blocking expression of virulence factors in S. aureus
WO1996040979A1 (fr) * 1995-06-07 1996-12-19 Microcide Pharmaceuticals, Inc. Procedes d'evaluation de cibles antimicrobiennes
WO1997011690A3 (fr) * 1995-09-29 1997-09-12 Microcide Pharmaceuticals Inc Inhibiteurs des voies de regulation
EP0932613A4 (fr) * 1996-05-22 2002-01-09 Univ New York Blocage de l'expression de l'aggressine dans s. aureus
EP0932613A1 (fr) * 1996-05-22 1999-08-04 New York University BLOCAGE DE L'EXPRESSION DE L'AGGRESSINE DANS $i(S. AUREUS)
WO1997044349A1 (fr) * 1996-05-22 1997-11-27 New York University BLOCAGE DE L'EXPRESSION DE L'AGGRESSINE DANS $i(S. AUREUS)
US6455323B1 (en) 1997-07-03 2002-09-24 Pharmacia & Upjohn Company Anti-bacterial methods and materials
US6485899B1 (en) 1997-07-03 2002-11-26 Pharmacia & Upjohn Company Anti-bacterial methods and materials
US6740485B1 (en) 1997-07-03 2004-05-25 Imperial College Innovations Limited Anti-bacterial methods and materials
WO1999026968A1 (fr) * 1997-11-26 1999-06-03 The University Of Nottingham Oligopeptides et leur utilisation comme agents antibacteriens contre des souches de staphylococcus
US6291431B1 (en) 1997-12-19 2001-09-18 Panorama Research Methods and compositions for the treatment and prevention of Staphylococcal infections
US7067135B2 (en) 1997-12-19 2006-06-27 Naomi Balaban Methods and compositions for the treatment and prevention of Staphylococcus aureus infections
US8067015B2 (en) 1997-12-19 2011-11-29 Naomi Balaban Methods and compositions for the treatment and prevention of Staphylococcus and other bacterial infections
US7534857B2 (en) 1997-12-19 2009-05-19 Centegen, Inc. Methods and compositions for the treatment and prevention of staphylococcal infections
US7323179B2 (en) 1997-12-19 2008-01-29 Naomi Balaban Methods and compositions for the treatment and prevention of Staphylococcus and other bacterial infections
EP1188831A3 (fr) * 1998-09-15 2004-01-21 The Regents of the University of California Cible de la protéine qui active RNAIII (TRAP)
US6747129B1 (en) 1998-09-15 2004-06-08 The Regents Of The University Of California Target of RNAIII activating protein(TRAP)
EP1862549A2 (fr) 1998-09-15 2007-12-05 Naomi Balaban Cible de protéine d'activation RNAIII (TRAP)
US6689878B2 (en) 1998-09-15 2004-02-10 The Regents Of The University Of California Target of RNAIII activating protein (TRAP)
EP1862549A3 (fr) * 1998-09-15 2008-02-13 Naomi Balaban Cible de protéine d'activation RNAIII (TRAP)
EP1188831A2 (fr) * 1998-09-15 2002-03-20 The Regents of the University of California Cible de la protéine qui active RNAIII (TRAP)
WO2001058471A3 (fr) * 2000-02-10 2002-03-07 Intermune Inc Nouveaux inhibiteurs d'acides amines et de peptides de virulence de staphylococcus
US6764823B2 (en) 2000-04-06 2004-07-20 Pharmacia & Upjohn Company Antimicrobial methods and materials
US7824691B2 (en) 2005-04-04 2010-11-02 Centegen, Inc. Use of RIP in treating staphylococcus aureus infections

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Publication number Publication date
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