U.S. patent application number 10/201444 was filed with the patent office on 2003-04-24 for blocking expression of virulence factors in s. aureus.
Invention is credited to Beavis, Ronald, Ji, Guangyong, Novick, Richard P..
Application Number | 20030078378 10/201444 |
Document ID | / |
Family ID | 27405996 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030078378 |
Kind Code |
A1 |
Novick, Richard P. ; et
al. |
April 24, 2003 |
Blocking expression of virulence factors in S. aureus
Abstract
This invention provides peptides which inhibit agr transcription
in S. aureus and thereby block the expression of virulence factors
in S. aureus, pharmaceutical compositions comprising these
peptides, as well as methods for treating or preventing an
infection or disease caused by S. aureus using the peptides of the
present invention.
Inventors: |
Novick, Richard P.; (New
York, NY) ; Ji, Guangyong; (Elmhurst, NY) ;
Beavis, Ronald; (Indianapolis, IN) |
Correspondence
Address: |
Elie H. Gendloff
Amster, Rothstein & Ebenstein
90 Park Avenue
New York
NY
10016
US
|
Family ID: |
27405996 |
Appl. No.: |
10/201444 |
Filed: |
July 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10201444 |
Jul 23, 2002 |
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08861476 |
May 22, 1997 |
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6447786 |
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08861476 |
May 22, 1997 |
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08651226 |
May 22, 1996 |
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08651226 |
May 22, 1996 |
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08318499 |
Oct 4, 1994 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 16/1271 20130101;
C07K 14/31 20130101; A61K 38/00 20130101; A61K 39/00 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 001/00; C07K
014/00; C07K 017/00 |
Claims
What is claimed is:
1. A purified peptide which inhibits agr-rnaIII transcription in S.
aureus, said peptide containing six to twelve amino acids in length
and comprising amino acid number 28 from the AgrD region of a
staphylococci bacterium, or an analogue thereof which inhibits
agr-rnaIII transcription in S. aureus.
2. The peptide of claim 1, wherein the staphylococci bacterium is
selected from the group consisting of S. aureus, S. capitis, S.
caprae, S. carnosus, S. caseolyticus, S. epidermidis, S.
haemolyticus, S. hominis, S. hyicus subsp. hyicus, S. hyicus subsp.
chromo, S. kloosii, S. lentus, S. lugdunensis, S. sciuri, S.
simulans and S. xylosus.
3. The peptide of claim 2, wherein the staphylococci bacterium is
S. lugdunensis.
4. The peptide of claim 3, having the amino acid sequence
NH.sub.2-Asp-Ile-Cys-Asn-Ala-Tyr-Phe-COOH and a cyclic thioester
bond between Cys and COOH.
5. The peptide of claim 2, wherein the staphylococci bacterium is
S. aureus.
6. The peptide of claim 5 having the amino acid sequence
NH.sub.2-Tyr-Ser-Thr-Cys-Asp-Phe-Ile-Met-COOH and a cyclic
thioester bond between Cys and COOH.
7. The peptide of claim 5 having the amino acid sequence
NH.sub.2-Gly-Val-Asn-Ala-Cys-Ser-Ser-Leu-Phe-COOH and a cyclic
thioester bond between Cys and COOH.
8. The peptide of claim 5 having the amino acid sequence
NH.sub.2-Tyr-Ile-Asn-Cys-Asp-Phe-Leu-Leu-COOH and a cyclic
thioester bond between Cys and COOH.
9. A peptide composition comprising a peptide which inhibits
agr-rnaIII transcription in S. aureus and a pharmaceutically
acceptable carrier, wherein said peptide contains six to twelve
amino acids in length and comprises amino acid number 28 from the
AgrD region of a staphylococci bacterium, or an analogue thereof
which inhibits agr-rnaIII transcription in S. aureus.
10. The peptide composition of claim 5, wherein the staphylococci
bacterium is selected from the group consisting of S. aureus, S.
capitis, S. caprae, S. carnosus, S. caseolyticus, S. epidermidis,
S. haemolyticus, S. hominis, S. hyicus subsp. hyicus, S. hyicus
subsp. chromo, S. kloosii, S. lentus, S. lugdunensis, S. sciuri, S.
simulans and S. xylosus.
11. The peptide composition of claim 10, wherein the staphylococci
bacterium is S. lugdunensis.
12. The peptide composition of claim 11, wherein the peptide has
the amino acid sequence NH.sub.2-Asp-Ile-Cys-Asn-Ala-Tyr-Phe-COOH
and a cyclic thioester bond between Cys and COOH.
13. The peptide composition of claim 10, wherein the staphylococci
bacterium is S. aureus.
14. The peptide composition of claim 13, wherein the peptide has
the amino acid sequence
NH.sub.2-Tyr-Ser-Thr-Cys-Asp-Phe-Ile-Met-COOH and a cyclic
thioester bond between Cys and COOH.
15. The peptide composition of claim 13, wherein the peptide has
the amino acid sequence
NH.sub.2-Gly-Val-Asn-Ala-Cys-Ser-Ser-Leu-Phe-COOH and a cyclic
thioester bond between Cys and COOH.
16. The peptide composition of claim 13, wherein the peptide has
the amino acid sequence
NH.sub.2-Tyr-Ile-Asn-Cys-Asp-Phe-Leu-Leu-COOH and a cyclic
thioester bond between Cys and COOH.
17. A method for treating or preventing an infection or disease
caused by S. aureus in a subject comprising administering to the
subject a peptide which inhibits agr-rnaIII transcription in S.
aureus in an amount effective to treat or prevent the infection or
disease caused by S. aureus, wherein said peptide contains six to
twelve amino acids in length and comprises amino acid number 28
from the AgrD region of a staphylococci bacterium, or an analogue
thereof which inhibits agr-rnaIII transcription in S. aureus.
18. The method of claim 17, wherein the staphylococci bacterium is
selected from the group consisting of S. aureus, S. capitis, S.
caprae, S. carnosus, S. caseolyticus, S. epidermidis, S.
haemolyticus, S. hominis, S. hyicus subsp. hyicus, S. hyicus subsp.
chromo, S. kloosii, S. lentus, S. lugdunensis, S. sciuri, S.
simulans and S. xylosus.
19. The method of claim 18, wherein the staphylococci bacterium is
S. lugdunensis.
20. The method of claim 19, wherein the peptide has the amino acid
sequence NH.sub.2-Asp-Ile-Cys-Asn-Ala-Tyr-Phe-COOH and a cyclic
thioester bond between Cys and COOH.
21. The method of claim 18, wherein the staphylococci bacterium is
S. aureus.
22. The method of claim 21, wherein the peptide has the amino acid
sequence NH.sub.2-Tyr-Ser-Thr-Cys-Asp-Phe-Ile-Met-COOH and a cyclic
thioester bond between Cys and COOH.
23. The method of claim 21, wherein the peptide has the amino acid
sequence NH.sub.2-Gly-Val-Asn-Ala-Cys-Ser-Ser-Leu-Phe-COOH and a
cyclic thioester bond between Cys and COOH.
24. The method of claim 21, wherein the peptide has the amino acid
sequence NH.sub.2-Tyr-Ile-Asn-Cys-Asp-Phe-Leu-Leu-COOH and a cyclic
thioester bond between Cys and COOH.
Description
[0001] This application is a continuation-in-part of copending
application Ser. No. 08/651,226, filed May 22, 1996, which is a
continuation-in-part of application Ser. No. 08/318,499, filed Oct.
4, 1994, the contents of which are incorporated by reference
herein.
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made under NIH Grant No. A1-R01-30138. As
such, the Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0003] 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. Sa also causes diseases such as toxic shock
syndrome.
[0004] Like other Gram-positive pathogens, Sa 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.
[0005] 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.
[0006] 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 Sa 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.
[0007] 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.
[0008] In S. aureus, expression of virulence factors is controlled
by a global regulator known as agr (Peng, H., et al. J. Bacteriol.
179: 4365-4372 (1988); Regassa, L. B., et al. Infect. Immun. 60:
3381-3383 (1992)). Agr is a genetic locus that contains several
genes. Two of these, agrA and agrC, 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, agr-rnaIII (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 agr-rnaIII, 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)).
[0009] 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 agr 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 aeruginosa. It
has not been shown, however, whether these chemicals would have any
effect on Sa, or offer any potential clinical utility.
SUMMARY OF THE INVENTION
[0010] The present invention is based upon the discovery of
peptides which interfere with the activation of rnaIII
transcription and thus prevent expression of VF. Prevention of the
expression of VF by S. aureus using peptides which inhibit
activation of rnaIII transcription are expected to prevent or treat
diseases caused by Staphylococcal infections. Finally, the peptides
of the present invention, in addition to treating or preventing
diseases or infection caused by S. aureus, also can be used in
vitro for preventing colonization of S. aureus.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 represents the agr locus of S. aureus. Schematic map
of the agr locus showing the major transcripts RNAII and RNAIII
(arrows) and the genes indicated by boxes.
[0012] FIG. 2 represents a comparison of the predicted AgrD
sequences of S. aureus strains RN6390B (laboratory strain), RN7690
(clinical isolate), RN6607 (clinical isolate), RN8463 (clinical
isolate) and S. lugunensis. The underlined region corresponds to
the identification of the activator peptide from RN6309B and the
inhibitor peptide from S. lugunensis.
[0013] FIG. 3 represents the effects of purified peptide from S.
aureus strains RN6390B, RN6607 and RN8463 on the RNAIII
transcription of early exponential phase (EEP) and mid-exponential
phase (MEP) cultured S. aureus strains RN6390B, RN6607 and RN8463.
The RNAIII transcription was measured as described in Ji, G., et
al. PNAS USA 92:12055-12059 (1995) with 10 mM Tris-HCl, pH 7.5 as
the control.
[0014] FIGS. 4A and 4B represent the effect of S. lugdunensis
pheromone on the RNAIII transcription of various S. aureus strains:
RN6390B (laboratory strain), RN6596 (laboratory strain), RN6607
(clinical isolate), RN7690 (clinical isolate), RN7843 (clinical
isolate), RN8462 (TSST) and RN8463 (TSST). FIG. 4A, activation
assays; FIG. 4B, inhibition assays.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention provides purified and isolated
peptides which inhibit agr-rnaIII transcription in S. aureus. The
peptides of the present invention are cyclic, comprise about six to
about twelve amino acids in length, and include amino acid number
28 from the AgrD region of a staphylococci bacterium. As used
herein, amino acid number 28 of the agrD region of a staphylococci
bacterium corresponds to the "cysteine" shown in FIG. 2, which is
believed to be conserved in the corresponding AgrD regions of
various staphylococci bacterium. The staphylococci bacterium
includes but is not limited to S. aureus, S. capitis, S. caprae, S.
carnosus, S. caseolyticus, S. epidermidis, S. haemolyticus, S.
hominis, S. hyicus subsp. hyicus, S. hyicus subsp. chromo, S.
kloosii, S. lentus, S. lugdunensis, S. sciuri, S. simulans and S.
xylosus.
[0016] The inhibitor peptides of the present invention include
sequences corresponding to the native peptide from staphylococci
bacterium, as well as analogues thereof which contain amino acid
substitutions which also result in the peptides being able to
inhibit agr-rnaIII transcription in S. aureus. The purified
inhibitor peptides of the present invention may be isolated
directly from staphylococci bacterium, recombinantly produced, or
synthesized chemically using procedures known in the art.
Preferably, the peptides are synthesized chemically. Specific
examples of the inhibitor peptides include but are not limited to
the following amino acid sequences:
NH.sub.2-Asp-Ile-Cys-Asn-Ala-Tyr-Phe-COOH,
NH.sub.2-Tyr-Ser-Thr-Cys-Asp-Phe-Ile-Met-COOH,
NH.sub.2-Gly-Val-Asn-Ala-C- ys-Ser-Ser-Leu-Phe-COOH and
NH.sub.2-Tyr-Ile-Asn-Cys-Asp-Phe-Leu-Leu-COOH, wherein each peptide
contains a cyclic thioester bond between the cysteine and the COOH
end. The synthesis of peptides containing cyclic thioester bonds
between the cysteine and the COOH end is within the purview of one
skilled in the art. It is also within the confines of the present
invention that the cyclic bond can be a bond other than a thioester
bond, such as a disulfide bond, for example, which can be obtained
by adding a cysteine residue at the carboxyl end, so long that such
a modification results in a peptide having inhibitor activity.
[0017] The present invention also provides a peptide composition
comprising one or more of the inhibitor peptides and a
pharmaceutically or physiologically acceptable carrier. The carrier
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof. Examples of suitable pharmaceutical carriers
include lactose, sucrose, starch, talc, magnesium stearate,
crystalline cellulose, methyl cellulose, carboxymethyl cellulose,
glycerin, sodium alginate, gum arabic, powders, saline, water,
among others. The choice of carrier will depend upon the route of
administration. The formulations may conveniently be presented in
unit dosage and may be prepared by methods well-known in the
pharmaceutical art, by bringing the peptide(s) into association
with a carrier or diluent, as a suspension or solution, and
optionally one or more accessory ingredients, e.g. buffers, surface
active agents, and the like.
[0018] The present invention also provides a method for treating or
preventing an infection or disease caused by S. aureus in a subject
comprising administering to the subject one or more of the
inhibitor peptides in an amount effective to treat or prevent the
infection or disease caused by S. aureus. The subject may be human
or animal and is preferably is human. The peptide(s) alone, or
conjunction with a suitable pharmaceutically acceptable carrier,
may be administered by procedures known to those skilled in the
art, including but not limited to parenteral (i.e., intravenous,
intramuscular, subcutaneous, or intraperitoneal administration),
oral, sublingual and topical administration. The actual dose of the
peptide(s) administered will depend upon the route of
administration, the pharmacokinetics properties of the individual
treated, as well as the results desired, and is readily
determinable by one skilled in the art. It is also within the
confines of the present invention that the peptide(s) may be
administered in combination with traditional antibiotics which are
used to treat diseases or infections caused by S. aureus.
[0019] The present invention is described in the following Examples
which are set forth 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.
EXAMPLE 1
Effect of Conditioned-Media of Staphylococci on the Agr Expression
of S. aureus RN6390B
[0020] Various culture supernatants from 172 S. aureus and 15 other
staphylococci were grown in CYGP broth at 37.degree. C. for 6 hours
starting at a cell density of 2.times.10.sup.9 cells/ml. Cells were
removed by centrifugation at 4.degree. C. The supernatant was
boiled for 10 minutes, filtered (0.22 .mu.m filter), centrifuged,
filtered with a Centricon 3 filter (3 kDa cutoff). The supernatant
from these S. aureus strains and other staphylococci were then
analyzed using the procedures described in Ji, G., et al. PNAS USA
92:12055-12059 (1995) for their ability to activate or inhibit agr
transcription of S. aureus RN6309B. The results are presented in
Tables 1 and 2. These strains can be divided into four groups as
follows. In groups I, II and III (all S. aureus), the members of
any one group produce a substance that activates the virulence
response (agr transcription) in any other member of the same group
but inhibits the response in any member of either of the other two
groups. It is believed that this substance may have activation or
inhibition properties depending on the strain being tested. In
group IV (several staphylococcal species other than S. aureus),
each of the members produces a substance that inhibits the response
in RN6390B, the standard strain from group I. The substances
produced by group IV strains have little or no agr-activating
activity with any member of the group.
EXAMPLE 2
Purification of RN6390B S. aureus Peptide Using C18 Reverse Phase
HPLC
[0021] S. aureus strain RN 7668 (pRN6911) was grown in tryptophan
assay medium plus 50 .mu.g/ml of L-tryptophan and 5 .mu.g/ml CBAP
starting at 2.times.10.sup.9 cells/ml. Before use, the medium was
dialyzed with a 2 kDA cutoff membrane, discarding the contents of
the membrane sac. After 6 hours of growth at 37.degree. C., cells
were removed by centrifugation and the culture supernatant was
filtered (0.22 .mu.m filter), boiled for 10 minutes, lyophilized
and resuspended in 2.5% acetonitrile/0.1% trifluoroacetic acid
(1/40 volume of the culture supernatant). This material (3 ml) was
loaded onto an HPLC C18 column in 2.5% acetonitrile/0.1%
trifluoroacetic acid, and eluted with an acetonitrile gradient
(16-48%) at 0.27% acetonitrile per minute. The collected fractions
(1.5 ml per fraction) were lyophilized and suspended in 0.1 ml of
20 mM Tris-HCL buffer (pH 7.5). Fractions with activator pheromone
activity were pooled and filtered through a Centricon 3 filter with
3 kDa cutoff. The filtrate (1 ml) was rerun on the HPLC C-18 column
and eluted with an acetonitrile gradient at 0.2% acetonitrile per
minute over the interest range (20-30%).
[0022] The activator peptide, eluting at an acetonitrile
concentration of about 28.5%, was analyzed by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) (Hillenkamp, F.,
et al. Anal. Chem. 63:1193A-1203A (1991)) and its amino acid
sequence was determined to be Tyr-Ser-Thr-Cys-Asp-Phe-Ile-Met by a
Procise Edman Sequencer (Perkin-Elmer), which is located within the
AgrD gene as shown in FIG. 2. MALDI-MS was performed using a linear
time-of-flight mass spectrometer with a nitrogen laser ion source
custom-built at New York University. The matrix used was
.alpha.-cyano-4-hydroxycinnamic acid and the sample was prepared
using the droplet method.
[0023] The purified activator peptide was then analyzed for its
ability activate and inhibit RNAIII transcription of S. aureus
strains RN6390B, RN6607 and RN8463, as described in Ji, G., et al.
PNAS USA 92:12055-12059 (1995). The results are presented in FIG.
3, which show that the activator peptide had an activator effect on
strain RN6390B, and an inhibitor effect on strains RN6607 and
RN8463.
EXAMPLE 3
Purification of Other S. aureus Peptides Using C18 Reverse Phase
HPLC
[0024] S. aureus strain RN 7667 containing cloned agrBD genes of
either RN6607 or RN8463 was grown in tryptophan assay medium plus
50 .mu.g/ml of L-tryptophan and 5 .mu.g/ml CBAP starting at
2.times.10.sup.9 cells/ml. After 6 hours of growth, cells were
removed by centrifugation and the culture supernatant was filtered
(0.22 .mu.m filter), boiled for 10 minutes, lyophilized and
suspended in solution A (2.5% acetonitrile plus 0.1%
trifluoroacetic acid). This material was loaded onto a Sephasil C18
(Pharmacia) column (3 cm.times.5 cm), washed once with solution A,
once with solution B (15% acetonitrile plus 0.1% trifluoroacetic
acid) and eluted with solution C (40% acetonitrile plus 0.1%
trifluoroacetic acid). The eluted material was lyophilized,
suspended in 20 mM Tris-HCL buffer (pH 7.5) and filtered through a
Centricon 3 filter (Amicon). The filtrate was then loaded onto an
HPLC C-18 column and eluted with an acetonitrile gradient (16-32%)
at 0.2% acetonitrile per minute. Fractions with activity were
pooled, lyophilized, suspended in 20 mM Tris-HCL buffer (pH 7.5)
and analyzed by MALDI-MS, and its amino acid sequence was
determined by a Perkin-Elmer Procise Edman Sequencer. The amino
acid sequences for RN6607 and RN8463 were determined to be
Gly-Val-Asn-Ala-Cys-Ser-Ser-Leu-P- he and
Tyr-Ile-Asn-Cys-Asp-Phe-Leu-Leu, respectively, which are located in
the same region of agrD as the activator peptide from S. aureus
strain RN6390B as shown in FIG. 2.
[0025] The purified peptide were then analyzed for their ability
activate and inhibit RNAIII transcription of S. aureus strains
RN6390B, RN6607 and RN8463, as described in Ji, G., et al. PNAS USA
92:12055-12059 (1995). The peptide from RN6607 had an activator
effect on strain RN6607, and an inhibitor effect on strains RN6390B
and RN8463, while the peptide from RN8463 had an activator effect
on strain RN8463, and an inhibitor effect on strains RN6390B and
RN6607, as shown in FIG. 3.
EXAMPLE 4
Purification and Analysis of Inhibitor Peptide From S.
lugdunensis
[0026] S. aureus strain RN 7668 (pSLBD) was grown in methionine
assay medium plus 50 .mu.g/ml of L-methionine and 5 .mu.g/ml CBAP
starting at 2.times.10.sup.9 cells/ml. Before use, the medium was
dialyzed with a 2 kDA cutoff membrane, discarding the contents of
the membrane sac. After 6 hours of growth at 37.degree. C., cells
were removed by centrifugation and the culture supernatant was
boiled for 10 minutes and filtered (0.22 .mu.m filter). This
material was loaded onto a Sephasil C18 column (3.5.times.4 cm),
washed with (i) 2.5% acetonitrile, 0.1% trifluoroacetic acid, (ii)
10.5% acetonitrile, 0.1% trifluoroacetic acid. The inhibitor was
eluted with 42.5% acetonitrile, 0.1% trifluoroacetic acid,
lyophilized, suspended in 20 mM Tris-HCl buffer (pH 7.5) and
filtered through a Centricon 3 filter with a 3 kDa cutoff. This
material was then loaded onto an HPLC C-18 column and eluted with
an acetonitrile gradient. Fractions with inhibitor pheromone
activity were pooled, lyophilized, dissolved in 20 mM Tris-HCL
buffer (pH 7.5) and stored at -80.degree. C. The fractions were
analyzed by MALDI-MS, and using Edman degradation procedure, this
peptide was determined to have the amino sequence
Asp-Ile-Cys-Asn-Ala-Tyr-Phe, which is located in the same region of
agrD as the activator peptide from S. aureus strain RN6390B as
shown in FIG. 2.
[0027] The purified S. lugdunensis pheromone was then analyzed for
its ability activate and inhibit RNAIII transcription of various S.
aureus strains. S. aureus strains were grown to 30 Klett units (for
activation assays) and to 60 Klett units (for inhibition assays).
The purified S. lugdunensis pheromone was added and the mixtures
were incubated at 37.degree. C. for 30 minutes. Whole cell lysates
were prepared from these cultures and analyzed by Northern blot
hybridization using a RN6390B RNAIII-specific probe. The S.
lugdunensis pheromone inhibits the agr response in 5 of 6 S. aureus
strains tested as shown in FIGS. 4A and 4B.
EXAMPLE 5
Commercial Synthesis of Synthetic Peptide Corresponding to RN6390B
and S. lugdunensis Sequences
[0028] Peptides having the same amino acid sequences as the native
peptides from RN6390B and S. lugdunensis were synthesized
commercially (Yale University, New Haven, Conn.) and analyzed by
MALDI-MS. Unlike the purified peptides, the synthesized peptides
did not have activity. Mass spectroscopy showed that the synthetic
peptides were dimeric, whereas the native peptide molecules were
monomeric and had molecular masses that were 18.+-.1 atomic mass
units less than those predicted by their respective amino acid
sequences. Taken together, these results suggest that the cysteines
in the synthetic peptides had spontaneously formed intermolecular
disulfides, whereas those in the native peptides were involved in
an intramolecular bond, most likely a cyclic thioester introduced
post-translationally and involving the C-terminal carboxyl, since
there is no other conserved carboxyl group in the molecule.
Consistent with this possibility were the results of treatment of
the native peptides with iodoacetic acid and hydroxylamine.
Iodoacetic acid, expected to react with free --SH groups, had no
effect, whereas hydroxylamine, expected to react with thioesters,
abolished activity.
EXAMPLE 6
Synthesis of a Synthetic Peptide Corresponding to the RN8463
Sequence
[0029] We have succeeded in synthesizing a small quantity of a
cyclic thioester derivative of the RN8463 octapeptide and have
shown that the synthetic material inhibits agr expression by
RN6390B. It is expected that the introduction of the cyclic
thioester bond to the other peptide will have a similar effect on
activity.
1TABLE 1 Effect of Conditioned-Media of Staphylococci on the Agr
Expression of S. aureus RN6390B Strains Activation Inhibition S.
aureus Animal Mastitis Isolates (6) 4 2 Capsule Strains (3) 0 3
Clinical Isolates (39) 17 22 Coag.sup.- (9) 0 9 Laboratory Strains
(4) 1 3 MRSA (65) 13 52 TSST-1.sup.+ (46) 3 43 S. capitis, S.
caprae, S. camosus, 0 All S. caseolyticus, S epidermidis, S.
haemolyticus, S hominis, S. hyicus subsp. hyicus, S. hyicus subsp.
chromo, S. kloosii, S. lentus, S. lugdunensis, S. sciuri, S.
simulans, S. xylosus
[0030]
2TABLE 2 Effect of conditioned-media on S. aureus Agr expression
Strains Used Supernatant Added for Assays RN6390B RN6596 RN7690
RN6607 RN7843 RN8462 RN8463 RN6390B + + + - - - - RN6596 + + + - -
- - RN7690 + + + - - - - RN6607 - - - + - - - RN7843 - - - - + + +
RN8462 - - - - + + + RN8463 - - - - + + + Note: + Activation -
Inhibition Group: (1) RN6390, RN6596, RN7690 (2) RN6607 (3) RN7843,
RN8462, RN8463
[0031] All publications mentioned hereinabove are hereby
incorporated by reference in their entirety.
[0032] 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.
Sequence CWU 1
1
8 1 7 PRT Staphylococcus lugdunensis 1 Asp Ile Cys Asn Ala Tyr Phe
1 5 2 8 PRT Staphylococcus aureus 2 Tyr Ser Thr Cys Asp Phe Ile Met
1 5 3 9 PRT Staphylococcus aureus 3 Gly Val Asn Ala Cys Ser Ser Leu
Phe 1 5 4 8 PRT Staphylococcus aureus 4 Tyr Ile Asn Cys Asp Phe Leu
Leu 1 5 5 46 PRT Staphylococcus aureus 5 Met Asn Thr Leu Phe Asn
Leu Phe Phe Asp Phe Ile Thr Gly Ile Leu 1 5 10 15 Lys Asn Ile Gly
Asn Ile Ala Ala Tyr Ser Thr Cys Asp Phe Ile Met 20 25 30 Asp Glu
Val Glu Val Pro Lys Glu Leu Thr Gln Leu His Glu 35 40 45 6 47 PRT
Staphylococcus aureus 6 Met Asn Thr Leu Val Asn Met Phe Phe Asp Phe
Ile Ile Lys Leu Ala 1 5 10 15 Lys Ala Ile Gly Ile Val Gly Gly Val
Asn Ala Cys Ser Ser Leu Phe 20 25 30 Asp Glu Pro Lys Val Pro Ala
Glu Leu Thr Asn Leu Tyr Asp Lys 35 40 45 7 46 PRT Staphylococcus
aureus 7 Met Lys Lys Leu Leu Asn Lys Val Ile Glu Leu Leu Val Asp
Phe Phe 1 5 10 15 Asn Ser Ile Gly Tyr Arg Ala Ala Tyr Ile Asn Cys
Asp Phe Leu Leu 20 25 30 Asp Glu Ala Glu Val Pro Lys Glu Leu Thr
Gln Leu His Glu 35 40 45 8 53 PRT Staphylococcus lugdunensis 8 Met
Asn Leu Leu Ser Gly Leu Phe Thr Lys Gly Ile Ser Ala Ile Phe 1 5 10
15 Glu Phe Ile Gly Asn Phe Ser Ala Gln Asp Ile Cys Asn Ala Tyr Phe
20 25 30 Asp Glu Pro Glu Val Pro Gln Glu Leu Ile Asp Leu Gln Arg
Lys Gln 35 40 45 Leu Ile Glu Ser Val 50
* * * * *