Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
COMPOSITIONS AND METHODS INVOLVING AN ESSENTIAL
STAPHYLOCOCCUS AUREUS GENE AND ITS ENCODED PROTEIN
STAAU R4
FIELD OF THE INVENTION
The invention relates to bacterial genes and proteins that are
implicated in the process of fatty acid/phospholipid biosynthesis and also to
bacteriophage genes and their protein products that interact with bacterial
proteins implicated in fatty acid/phospholipid biosynthesis. More
particularly, the
invention relates to compositions and methods involving essential
Staphylococcus
aureus genes and its encoded proteins STAAU_R4. In addition, the invention
relates to screening assays to identify compounds which modulate the level
and/or activity of STAAU_R4 and to such compounds.
BACKGROUND OF THE INVENTION
The Staphylococci make up a medically important genera of
microbes known to cause several types of diseases in humans. S. aureus is a
Gram positive organism which can be found on the skin of healthy human hosts
and it is responsible for a large number of bacteremias.
S. aureus has been successfully treated with the penicillin
derivative Methicillin in the past, but is now becoming increasingly resistant
(MRSA - Methicillin Resistant S. aureus) to this antibiotic [Harbath et al.,
(1998)
Arch. Intern. Med. 158:182-189]. For example, S, aureus endocarditis mortality
can range from 26-45%, and combined f3-lactam/aminoglycoside therapy is
proving increasingly ineffective in disease eradication [Ruder et al., (1999)
Arch.
Intern. Med. 159:462-469].
It is no longer uncommon to isolate S. aureus strains which are
resistant to most of the standard antibiotics, and thus there is an. unmet
medical
need and demand for new anti-microbial agents, vaccines, drug screening
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methods, and diagnostic tests for this organism. Antibiotics can be grouped
into
broad classes of activities against surprisingly few targets within the cell.
Generally, the target molecule is a cellular protein that provides an
essential
function. The inhibition of activity of the essential protein leads either to
death of
_ the bacterial cell or to its inability to proliferate. Critical cellular
functions against
which antibiotics are currently in use include cell wall synthesis, folate and
fatty
acid metabolism, protein synthesis, and nucleic acid synthesis. Of particular
relevance for the current invention is fatty acidlphospholipid biosynthesis.
A proven approach in the discovery of~a new drug, referred to
as target-based drug discovery to distinguish it from cell-based drug
discovery,
is to obtain a target protein and to develop in vitro assays to interfere with
the
biological function of the protein.
There remains a need to identify new bacterial targets and new
target domains, and more particularly S. aureus bacterial targets which could
be
used to screen for and identify antibacterial and more particularly anti-S.
aureus
agents. There also remains a need to identify new antimicrobial agents,
vaccines,
drug screening methods and diagnosis and therapeutic methods.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the
content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention relates to new antimicrobial agents,
vaccines, drug screening methods and diagnosis and therapeutic methods.
More particularly, the invention relates to compounds which
interact with STAAU_R4 and in particular to bacterial growth-inhibitory (or
inhibitor) bacteriophage gene products that interacts with the S. aureus
STAAU R4 polypeptides.
The invention also relates to a pair of interaction proteins and
parts or fragments thereof. More specifically, the invention relates to the
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interacting domains of the S. aureus STAAU_R4 protein and to proteins which
interact with same and block or inhibit a STAAU_R4 biological activity. In a
particular embodiment, the invention relates to a pair of interacting domains
comprised of that of STAAU_R4 and a polypeptide encoded by a bacteriophage
ORF which specifically interacts therewith, such as the S. aureus
bacteriophage
3A ORF 33. In one particularly preferred embodiment of the present invention,
the
interaction of these domains and a modulation thereof forms the basis for
screening assays to identify modulators of STAAU_R4 biological function and
more particularly of antimicrobials.
The present invention also relates to polynucleotides and
polypeptides of a multiprotein complex . believed to be involved in fatty
acid/phospholipid biosynthesis replication containing STAAU_R4 as a subunit,
as
well as variants and portions thereof.
In another aspect, the invention relates to methods for using
such polypeptides, polynucleotides, peptidomimetics and the like, including
treatment and diagnosis of microbial diseases, amongst others.
In a further aspect, the invention relates to methods for
identifying agonists and antagonists using the materials provided by the
invention.
In a related aspect, the invention relates to methods for treating microbial
infections and conditions associated with such infections with the identified
agonist or antagonist.
In a still further aspect, .the invention relates to diagnostic
assays for detecting diseases associated with microbial infections and
conditions
associated with such infections. In one embodiment, the diagnostic assay
detects
STAAU R4 expression and/or activity.
In one particular embodiment of the invention, there is
provided a method of identifying a compound that is active on a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2 or a biologically active
fragment, or variant thereof, wherein SEQ ID NO: 2 or a biologically active
fragment or variant thereof is capable of binding specifically with a
polypeptide
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comprising the sequence selected from SEQ ID NO: 4, a biologically active
fragment thereof, and variant thereof, wherein the fragment or variant retains
its
capability of binding to SEQ ID N0:2, fragment, or variant thereof.
In one preferred embodiment of the invention, the identification
of a compound active on a STAAU_R4 polypeptide is provided by a method
comprising: contacting a first and a second polypeptide in the presence or
absence of a candidate compound, wherein the first polypeptide comprises the
amino acid sequence of SEQ ID NO: 2, a fragment or variant thereof which
specifically binds to a second polypeptide derived from a bacteriophage ORF
which is capable of binding specifically with one of SEQ ID NO: 2, a fragment,
or
variant thereof. In one particular embodiment, the second polypeptide is a
phage
ORF, a fragment thereof or variant thereof, wherein this second polypeptide
maintains its biological activity; and detecting a biological activity of the
first and/or
second polypeptide, wherein a decrease in the biological activity in the
presence
thereof relative to the biological activity in.the absence of the candidate
compound
identifies the candidate compound as a compound that is active on a
polypeptide
comprising the amino acid sequence of SEQ ID NO:2, fragment or variant
thereof.
In one particular embodiment, the biological activity is the
binding of the first and second polypeptides to each other, the method
comprising:
contacting an assay mixture comprising a) a first polypeptide which comprises
the
amino acid sequence of SEQ ID NO:2 or a biologically active fragment, or
variant
thereof, and b) a second polypeptide selected from the group consisting of SEQ
ID NO: 4, a fragment thereof, and a variant thereof; with a test compound;
measuring the binding of the first and the second polypeptides in the presence
of
the candidate compound relative to the binding in the absence thereof and;
determining the ability of the candidate compound to interact with a STAAU_R4
polypeptide, fragment or variant thereof, wherein a decrease in the binding of
the
first and the second polypeptides in the presence of a candidate compound that
interacts with the first polypeptide, relative to the binding in the absence
thereof,
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identifies the candidate compound as a compound that is active on a STAAU_R4
polypeptide, fragment or variant thereof. While in a preferred embodiment, the
method identifies a compound active on STAAU_R4, the method can also identify
compounds active on a bacteriophage ORF which specifically interacts with
STAAU R4.
In one embodiment, the step of detecting comprises the step
of measuring the binding of the first and second proteins, wherein the first
or the
second protein is directly or indirectly detectably labeled.
The invention also encompasses a method of identifying an
antimicrobial agent comprising determining whether a test compound is active
on
a S. aureus polypeptide, namely STAAU R4 as set forth in SEQ ID NO: 2, or
parts thereof.
In a further embodiment, identifying a.compound active on a
STAAU_R4 polypeptide is provided by a method which comprises: contacting a
candidate compound with a polypeptide comprising the amino acid sequence of
SEQ ID NO: 2; a fragment thereof, or a variant thereof, the fragment or
variant
retaining its biological activity, and detecting binding of the candidate
compound
thereto, wherein detection of binding is indicative that the compound is
active on
the polypeptide.
In different embodiments, the step of detecting includes
measuring the binding of a candidate compound to the polypeptide, wherein the
compound is directly or indirectly detectably labeled, by a method comprising,
but
not limited to, time-resolved fluorescence resonance energy transfer (TR-
FRET),
fluorescence polarization changes, measurement by surface plasmon resonance,
scintillation proximity assay, biosensor assay, and phage display.
In one embodiment, a library of compounds is used. Non-
limiting examples of candidate compound include a small molecule, a
peptidomimetic compound, a peptide, and a fragment or derivative of a
bacteriophage inhibitor protein.
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In one embodiment, the candidate compound is a peptide
synthesized by expression systems and purified, or artificially synthesized.
The invention further encompasses a method of identifying a
compound that is active on a STAAU_R4 polypeptide, a fragment or a variant
thereof, comprising the steps of contacting a candidate compound (or library
thereof) with cells expressing a polypeptide comprising the amino acid
sequence
as set forth in SEQ ID NO: 2; and detecting STAAU_R4 activity in the cells,
wherein a decrease in activity relative to STAAU_R4 activity in cells not
contacted
with a candidate compound is indicative of inhibition of STAAU_R4 activity.
The
invention also encompasses such a method but using a fragment or variant of
SEQ ID N0:2.
Of course, the invention further encompasses methods of
identifying a compound that modulates the activity of a STAAU R4 polypeptide,
wherein a compound increasing the activity relative to STAAU_R4 activity in
cells
not contacted with the candidate compound, is selected as a compound which is
a stimulator of STAAU_R4 activity.
The invention further encompasses a method of identifying a
compound that is active on a STAAU_R4 polypeptide, a fragment or a variant
thereof, comprising the steps of contacting a candidate compound (or library
20. thereof) in a cell-free assay, with a STAAU R4 protein or biologically
active
portion thereof, either naturally occurring or recombinant in origin; and
detecting
STAAU_R4 activity, wherein a decrease in activity relative to STAAU_R4
activity
in cell-free 'assay not contacted with a candidate compound is indicative of
inhibition of STAAU_R4 activity.
The invention further encompasses an agonist or an
antagonist of the activity of a STAAU_R4 polypeptide or a nucleic acid or gene
encoding the polypeptide.
The assays described herein may be used as initial or primary
screens to detect promising lead compounds for further development. The same
assays may also be used in a secondary screening assay to measure the activity
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of candidate compounds on a STAAU_R4 polypeptide. Often, lead compounds
will be further assessed in additional, different screens. This invention also
includes secondary STAAU_R4 screens which may involve biological assays
utilizing S. aureus strains or other suitable bacteria.
Tertiary screens may involve the study of the effect of the agent
in an animal. Accordingly, it is within the scope of this invention to further
use an
agent identified as described herein in an appropriate animal model. For
example,
a test compound identified as described herein (e.g., a STAAU_R4 inhibiting
agent, an antisense STAAU R4 nucleic acid molecule, a STAAU_R4-specific
antibody, or a STAAU_R4-binding partner) can be used in an animal model to
determine the efficacy, toxicity, or side effects of treatment with such an
agent.
Alternatively, an agent identified as described herein can be used in an
animal
model to determine the mechanism of action of such an agent. Furthermore, this
invention pertains to uses of novel agents identified by the above-described
screening assays for treatment (e.g. bacterial infections), as described
herein.
The invention further encompasses a method of making an
antibacterial compound, comprising the steps of: a) determining whether a
candidate compound is active on a polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, fragment or variant thereof, or a gene encoding the
polypeptide; and b) synthesizing or purifying the candidate compound in an
amount sufficient to provide a therapeutic effect when administered to an
organism infected by a bacterium naturally producing a polypeptide comprising
the amino acid sequence of SEQ ID NO: 2, fragment or variant thereof.
The invention further encompasses a method for inhibiting a
bacterium, comprising contacting the bacterium with a compound active on a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, fragment or
variant thereof, or a nucleic acid encoding the polypeptide.
In one embodiment, the step of contacting is performed in
vitro.
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_$_
In another embodiment, the step of contacting is performed in
vivo in an animal.
In another embodiment, bacterium is contacted with the active
compound in combination with existing antimicrobial agents. Thus, the
invention
also relates to antimicrobial compositions comprising a compound of the
present
invention in combination with an existing antimicrobial agent. Of course, more
than one active compound of the present invention could be combined with or
without existing antimicrobial agent(s).
The invention further encompasses a method for treating or
preventing a bacterial infection in an animal suffering from an infection or
susceptible of suffering from same, comprising administering thereto a
therapeutically or prophylactically effective amount of a compound active on a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, variant or
fragment thereof, or nucleic acid sequence encoding same. The animal is
preferably, but not necessarily a mammal, and more preferably a human. In one
embodiment, the active compound is administred to the animal in combination
with existing antimicrobial agents. Thus, the invention also relates to
antimicrobial
compositions comprising a compound of the present invention in combination
with
an existing antimicrobial agent.
The invention further encompasses a method of prophylactic
treatment to prevent bacterial infection comprising contacting an indwelling
device
with a compound active on a polypeptide comprising the amino acid sequence of
SEQ ID NO: 2, variant or fragment thereof before its implantation into a
mammal,
such contacting being sufficient to prevent S. aureus infection at the site of
implantation.
The invention further encompasses a method of prophylactic
treatment to prevent infection of an animal by a bacterium comprising
administering to the animal a prophylactically effective amount of a compound
that
is active on a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
variant or fragment thereof or a gene encoding the polypeptide in an amount
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sufficient to prevent infection of the animal. In a particular embodiment, the
prophylactically effective amount reduces adhesion of the bacterium to a
tissue
surface of the mammal. '
The invention further encompasses a method of diagnosing in
an animal an infection with S. aureus, comprising: determining the presence in
the
animal of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
part thereof, variant thereof, fragment thereof, epitope thereof or nucleic
acid
encoding same. Preferably the polypeptide is capable of specifically
interacting
with 3A ORF 33 . Preferably, the animal is a human.
In one embodiment, the determining step comprises
contacting a biological sample of the animal or individual with an antibody
specific
for an epitope present on a polypeptide comprising the amino acid sequence of
SEQ ID NO: 2, variant or fragment thereof.
The invention further encompasses a method of diagnosing in
an animal or individual an infection with S. aureus, comprising determining
the
presence in the animal or individual of a nucleic acid sequence encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, variant or
fragment thereof, wherein the polypeptide is capable of specifically
interacting
with 3A ORF 33 .
In one embodiment, the determining step comprises
contacting a nucleic acid sample of the animal or individual with an isolated,
purified or enriched nucleic acid probe of at least 15 nucleotides in length
that
hybridizes under stringent hybridization conditions with the sequence of SEQ
ID
NO: 1, or the complement thereof.
The invention further encompasses an isolated, purified or
enriched polynucleotide comprising a nucleotide sequence encoding a
polypeptide, which can interact with a bacterial growth-inhibitory (or
inhibitor)
bacteriophage 3A ORF 33 gene product or part thereof.
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In one particular embodiment, the isolated, purified or enriched
polynucleotide comprises a nucleotide sequence encoding a polypeptide
corresponding to SEQ ID NO: 2, or complement thereof.
In another particular embodiment of the present invention, the
isolated, purified or enriched polynucleotide comprises a nucleotide sequence
having at least 60 %, at least 70 %, at least 80 %, and more preferably at
least
90% identity to the sequence of SEQ ID NO: 1, or to the complement thereof.
The invention further encompasses an isolated, purified or
enriched polypeptide comprising the amino acid sequence of SEQ ID NO: 2, a
variant or fragment thereof.
The invention further encompasses an isolated, purified or
enriched polypeptide comprising or consisting in the amino acid sequence of
SEQ
ID NO: 2, or a fragment or. variant thereof.
In one particular embodiment, the isolated, purified or enriched
polypeptide comprises or consists of an amino acid sequence having at least
50%, at least 55%, at least 60%, at least 70%, and more preferably at least
80%,
at least 90%; at least 95% or at least 99% identity to the amino acid sequence
of
SEQ ID NO: 2.
In one particular embodiment, the isolated, purified or enriched
polypeptide of the present invention comprises or consists of an amino acid
sequence having least 60%, at least 70%, at least 75%, at least 80%, more
preferably at least 90%, and more preferably at least 95% or at least 99%
similarity to the amino acid sequence of SEQ ID NO: 2.
In one particular embodiment, the sequence of SEQ ID N0:2 or
fragment or variant thereof is part of a chimeric protein.
The invention further encompasses an isolated, purified or
enriched antibody specific for an epitope encoded by the amino acid sequence
set forth in SEQ ID NO: 2.
The invention further encompasses a composition comprising
two polypeptides, a bacteriophage-encoded polypeptide and a S. aureus
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STAAU_R4 polypeptide corresponding to SEQ ID NO: 2, or variant or fragment
thereof. In another embodiment, the invention encompasses a composition
comprising two interacting polypeptides derived from a bacteriophage encoded
polypeptide and a S. aureus STAAU_R4 polypeptide. As such, the invention
encompasses a composition comprising two nucleic acid sequences encoding
these directly interacting polypeptides.
In another embodiment, the invention encompasses a
composition comprising a pair of specifically interacting domains, the pair
comprising: a STAAU_R4 polypeptide and a polypeptide encoded by a
bacteriophage ORF which specifically interacts with the STAAU_R4 polypeptide.
Further, the invention encompasses a process for producing
a pharmaceutical composition comprising: a) carrying out a screening assay of
the present invention aimed at identifying a compound that is active on a
STAAU_R4 polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
biologically active fragment thereof, or variant thereof; and b) mixing the
compound identified in a) with a suitable pharmaceutical carrier.
In a particular embodiment, the process for producing a
pharmaceutical composition comprises a) identifying a compound that is active
on a STAAU_R4 polypeptide comprising the amino acid sequence of SEQ ID NO:
2, biologically active fragment thereof, or variant thereof that binds
specifically with
a second polypeptide derived from a bacteriophage ORF; and b) mixing the
compound identified in a) with a suitable pharmaceutical carrier.
In a further embodiment of this process of producing a
pharmaceutical composition, the process further includes a scaling-up of the
preparation for isolating of the identified compound active on the STAAU R4
polypeptide. In yet another embodiment of this process of producing a
pharmaceutical composition, the pharmaceutical composition prepared comprises'
a derivative or homolog of the compound identified in a).
Also, the invention encompasses the use of one of: a) a
STAAU_R4 polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
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a biologically active fragment thereof or variant thereof, b) a composition
comprising a pair of specifically interacting domains comprised of a
polypeptide
of STAAU R4, biologically active fragment thereof or variant thereof and a
polypeptide encoded by a bacteriophage ORF which specifically interacts with
STAAIJ_R4; or c) an assay mixture comprising a first polypeptide which
comprises the amino acid sequence of SEQ ID N0:2, biologically active fragment
thereof or variant thereof and a second polypeptide encoded by a bacteriophage
ORF which specifically interact with each other; for the identification of a
compound that is active on a polypeptide comprising the amino acid sequence of
SEQ ID N0:2, biologically active fragment thereof or variant thereof.
In yet another embodiment of the present invention, there is
provided an isolated polypeptide or polynucleotide sequence which comprises a
bacteriophage ORF interacting domain which enables specific binding of the
encoded bacteriophage ORF interacting domain to a bacteriophage ORF, the
sequence being mutagenized in the non-bacteriophage ORF interacting domain.
Further features and advantages of the invention will become
more fully apparent in the following description of the embodiments and
drawings
thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference will
now be made to the accompanying drawings, showing by way of illustration a
preferred embodiment thereof, and in which:
Fig. 1 shows the nucleotide (SEQ ID NO: 1) and amino acid
(SEQ ID NO: 2) sequences of S. aureus STAAU R4.
Fig. 2 shows the nucleotide (SEQ ID NO: 3) and the amino
acid (SEQ ID NO: 4) sequences of S. aureus bacteriophage 3A ORF 33.
Fig. 3 shows the bacterial inhibitory potential of bacteriophage
3A ORF 33 and the expression vector used to induce its expression in S.
aureus.
A) Schematic diagram of expression vector pTM/ORF used to induce expression
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of 3A ORF 33 in S. aureus cells; B) Results of a screen to assess the
inhibitory
potential of 3A ORF 33 when expressed in S. aureus grown on semi-solid support
media; and C), D) Results of assays for inhibitory potential of 3A ORF 33 when
expressed in S. aureus grown in liquid medium followed by plating on semi-
solid
medium either containing (C) or not containing (D) the antibiotic necessary to
maintain the selective pressure for the plasmid.
Fig. 4 shows affinity chromatography using GST/3A ORF 33
ligands with a 10.0 mg/ml S. aureus extract. Eluates from affinity columns
containing the ligands at 0, 0.1, 0.5, 1.0, and 2.0 mg/ml resin were resolved
by
14% SDS-PAGE and the gel was stained with silver nitrate. Micro-columns were
sequentially eluted with 100 mM ACB containing 0.1 % Triton X-100 (SDS-PAGE
not shown), 1 M NaCI ACB, and 1 % SDS. Each molecular weight marker is
approximately 200 ng. The lanes labeled ACB indicate eluates from a 2.0 mg/ml
ligand column loaded only with ACB buffer containing 100 mM NaCI. The arrow
designated PT40 indicates the position of the band that was excised for
protein
identification.
Fig. 5 shows affinity chromatography using 3A ORF 33 fused
to a polyhistidine sequence (MSC2/3A ORF 33) as ligand with a 10.0 mg/ml S.
aureus extract. Eluates from affinity columns containing the ligands at 0,
0.1, 0.5,
1.0, and 2.0 mg/ml resin were resolved by 14% SDS-PAGE and the gel was
stained with silver nitrate. Micro-columns were sequentially eluted with 100
mM
ACB containing 0.1 % Triton X-100 (SDS-PAGE not shown), 1 M NaCI ACB, and
1 % SDS. Each molecular weight marker is approximately 200 ng. The lanes
labeled ACB indicate eluates from a 2.0 mg/ml ligand column loaded only with
ACB buffer containing 100 mM NaCI. The arrow designated PT40 indicates the
position of the band that was excised for protein identification.
Fig. 6 shows affinity chromatography using control GST as
ligand with a 10.0 mg/ml S. aureus extract. Eluates from affinity columns
containing the ligands at 0, 0.1, 0.5, 1.0, and 2.0 mg/ml resin were resolved
by
14% SDS-PAGE and the gel was stained with silver nitrate. Micro-columns were
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sequentially eluted with 100 mM ACB containing 0.1 % Triton X-100 (SDS-PAGE
not shown), 1 M NaCI ACB, and 1 % SDS. Each molecular weight marker is
approximately 200 ng. The lanes labeled ACB indicate eluates from a 2.0 mg/ml
ligand column loaded only with ACB buffer containing 100 mM NaCI.
Fig. 7 shows the tryptic peptide mass spectrum analysis of the
PT40 protein that interacted with 3A ORF 33 in affintity chromatography. The
PT40 band was identified as an open reading frame, herein referred as
STAAU_R4, found in Contig782 of the University of Oklahoma genome
sequencing project database (http://www.genome.ou.edu/staph.html).
Fig. 8 shows the results of the BLAST searching analysis of
publically available databases including NCBI (nr). STAAU_R4 is significantly
similar to PIsX protein from a variety of bacteria, including 8. subtilis
(gi~6686325~sp~P71018~PLSX BACSU: FATTY ACID/PHOSPHOLIPID
SYNTHESIS PROTEIN PLSX).
Fig. 9 shows the global optimal alignment of the amino acid
sequences of STAAU_R4 and 8. subtilis PIsX and the identification of the start
codon of the S. aureus gene. A) Result of the global optimal alignment reveals
a
53% identity between STAAU_R4 and PIsX of 8. subtilis; B) Results of the
analysis of the S. aureus stop codon-to-stop codon DNA region containing
STAAU_R4 revealing the presence of an ATG start codon associated with a
predicted ribosomal binding site (RBS) sequence (in bold on the nucleotide
sequence).
Fig. 10 shows A) an overview of the cloning of S. aureus
STAAU_R4 and B) an overview of the cloning of phage 3A ORF 33 in the pGEX-
6PK vector; C) the vector pGEX-6PK containing GST in fusion with PreScission
protease cleavage site and the heart muscle kinase (HMK) phosphorylation site,
followed by BamHI,EcoRl and Sall cloning sites; and D) the results of the Far
western affinity blotting analysis that was designed to test the interaction
between
purified 3A ORF 33 and [32P]-ATP-radiolabelled STAAU_R4 polypeptides. Lanes
1 to 4 represent increasing amount (0.125, 0.250, 0.5 and 1.0 ug) of 3A ORF 33
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resolved on SDS-polyacrylamide gel electrophoresis and blotted onto
nitrocellulose membrane.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non-
restrictive
description of preferred embodiments with reference to the accompanying
drawing which is exemplary and should not be interpreted as limiting the scope
of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The invention relates to the discovery of an essential gene and
its encoded polypeptide in S. aureus and portions thereof useful for example
in
screening, diagnostics, and therapeutics. More specifically, the invention
also
relates to S. aureus STAAU_R4 polypeptides and polynucleotides as described
in greater detail below, and to a pair of polynucleotides encoding a pair of
interacting polypeptides, to the pair of polypeptides themselves, or
interacting
domains thereof. In a particular embodiment, the pair includes a S. aureus
STAAIJ_R4 polypeptide or interacting domain thereof and a 3A ORF 33 or
interacting domain thereof. In one embodiment, the invention relates to
STAAIJ_R4 having the nucleotide or amino acid sequence disclosed as SEQ ID
NO: 1 or SEQ ID NO: 2, respectively. The sequences presented as SEQ ID NOs:
1 and 2 represent an exemplification of the invention, since those of ordinary
skill
will recognize that such sequences can be usefully employed in polynucleotides
in general, including ribopolynucleotides.
The methodology of two previous inventions (U.S. Provisional
Patent Application 60/110,992, filed December 3, 1998, and PCT International
Application W01999/IB99/02040, filed December 3, 1999) has been used to
identify and characterize essential polynucleotide and polypeptide sequences
from S. aureus.
Thus, in a particular embodiment of the present invention,
there is provided polynucleotide and polypeptide sequences isolated from S.
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aureus that can be used in a drug screening assay to identify compounds with
anti-microbial activity. The polynucleotide and polypeptide sequences can be
isolated using a method similar to those described herein, or using another
method. In addition, such polynucleotide and polypeptide sequences can be
chemically synthesized. The identification of the S. aureus STAAU_R4 sequence
as a target for a bacteriophage validates the approach of the present
invention to
identify bacterial targets and also validates STAAU_R4 as a key target for
antibacterial drug development as well as diagnosis and treatment methods
based thereon.
DEFINITIONS
In order to provide a clear and consistent understanding of
terms used in the present description, a number of definitions are provided
hereinbelow.
The terminology "active on", with reference to a particular
cellular target, such as the product of a particular gene, means that the
target is
an important part of a cellular pathway which includes that target and that an
agent or compound acts on that pathway. Thus, in some cases the agent or
compound may~act on a component upstream or downstream of the stated target
(i.e. indirectly on the target), including a regulator of that pathway or a
component
of that pathway. In general, an antibacterial agent is active on an essential
cellular
function, often on a product of an essential gene (i.e. directly on the
target).
The terminology "active on" also refers to a measurable effect
of the compound on the target it is active on (as compared to the activity'of
the
target in the absence of the compound). The activity referred thereto is any
one
of a biological activity of one of the polypeptides of the present invention.
As used herein, the terms "inhibit", "inhibition", 'inhibitory", and
"inhibitor" all refer to a function of reducing a biological activity or
function. Such
reduction in activity or function can, for example, be in connection with a
cellular
component (e.g., an enzyme), or in connection with a cellular process (e.g.,
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synthesis of a particular protein), or in connection with an overall process
of a cell
(e.g., cell growth). In reference to cell growth, the inhibitory effects may
be
bacteriocidal (killing of bacterial cells) or bacteriostatic (i.e. - stopping
or at least
slowing bacterial cell growth). The latter slows or prevents cell growth such
that
fewer cells of the strain are produced relative to uninhibited cells over a
given time
period. From a molecular standpoint, such inhibition may equate with a
reduction
in the level of, or elimination of, the transcription and/or translation
and/or stability
of a specific bacterial target(s), and/or reduction or elimination of activity
of a
particular target biomolecule.
As used herein, the terminologies "STAAU_R4 polypeptide",
"plsX polypeptide" or the like refer to a polypeptide encompassing S. aureus
STAAU_R4 (SEQ ID NO: 2), variant thereof or an active domain of S. aureus
STAAU_R4. As used herein, the term "active domain of S. aureus STAAU_R4",
"biologically active polypeptide of STAAU'R4" or the like refers to a
polypeptide
fragment or portion of S. aureus STAAU_R4 that retains an activity of S.
aureus
STAAU_R4. The term "STAAU_R4 polypeptide" is meant to encompass S.
aureus STAAU_R4 or an active domain of S. aureus STAAU_R4 that is fused to
another, non-STAAU_R4 polypeptide sequence.
"STAAU_R4 activity" "polypeptide comprising the amino acid
sequence SEQ ID NO: 2 activity" "plsX polypeptide activity" or "biological
activity"
of STAAU_R4 or other polypeptides of the present invention is defined as a
detectable biological activity of a gene, nucleic acid sequence, protein or
polypeptide of the present invention. This includes any physiological function
attributable to the specific biological activity of STAAU'R4, or phage ORF of
the
present invention. Non-limiting examples of the biological activities may be
made
directly or indirectly. STAAU_R4 biological activity, for example, is not
limited,
however, to its function in fatty acid/phospholipid biosynthesis. Biological
activities
may also include simple binding to other factors (polypeptides or otherwise),
including compounds, substrates, and of course interacting proteins. Thus, for
STAAU_R4, biological activity includes any standard biochemical measurement
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of STAAU_R4 such as conformational changes, phosphorylation status or any
other feature of the protein that can be measured with techniques known in the
art. STAAU_R4 biological activity also includes activities related to
STAAIJ_R4
gene transcription or translation, or any biological activities of such
transcripts or
translation products. The instant invention is also concerned with STAAlI_R4
interaction with an inhibitory polypeptide of the present invention,
biological
activity of STAAU R4 and fragment thereof also includes assays which monitor
binding and other biochemical measurements of these polypeptides. Furthermore,
for certainty, the terminology "biological activity" also includes
measurements
based on the interaction of domains of interacting proteins of the present
invention (e.g. the phage ORF interacting domain of STAAIJ_R4, or STAAU_R4
interacting domain of a phage ORF domains thereof). Non-limiting examples of
"biological activity" include one or more of the following:
i) Binding to a bacterial growth inhibitory ORF derived from a bacteriophage
including a 3A ORF 33 polypeptide or part thereof.
Determining the binding between polypeptides of the present
invention can be accomplished by one of the methods described below or known
in the art for determining direct binding. While it might be advantageous in
certain
embodiments of the present invention to provide a binding assay which is
amenable to automation and more particularly to high-throughput, the present
invention is not so limited. The binding or physical interaction between a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, provided
herein, or fragment thereof and a bacteriophage protein 3A ORF 33 or portion
thereof may be between. isolated polypeptides consisting essentially of the
sequence necessary for binding, or, alternatively, the respective polypeptide
sequence may be comprised within a larger polypeptide.
A number of non-limiting methods, useful in the invention, to
measure the binding of bacteriophage 3A ORF 33 to a polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or fragment thereof are described below.
Binding can be measured by coupling one molecule to a surface or support such
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as a membrane, a microtiter plate well, or a microarray chip, and monitoring
binding of a second molecule by any number of means including but not limited
to optical spectroscopy, fluorometry, and radioactive label detection.
For example, Time-Resolved Fluorescence Resonance
Energy Transfer (TR-FRET), in which the close proximity of two fluorophores,
whether intrinsic to, as in the case of a naturally-fluorescent amino acid
residue
such as tryptophan, or either covalently or non-covalently bound to a separate
molecule, causes the emission spectrum of one fluorophore to overlap with the
excitation spectrum of the second, and thus dual fluorescence following
excitation
of only one fluorophore is indicative of binding. An additional assay useful
in the
present invention is fluorescence polarization, in which the quantifiable
polarization value for a given fluorescently-tagged molecule is altered upon
binding to a second molecule. Surtace plasmon resonance assays can be used
as a quantitative method to measure binding between two molecules by the
change in mass near an immobilized sensor caused by the binding of one protein
from the aqueous phase to a second immobilized on the sensor. A scintillation
proximity assay can also be used to measure binding of a polypeptide
comprising
the amino acid sequence of SEQ ID NO2, and fragment thereof and a
bacteriophage ORF or fragment thereof in which binding in the proximity to a
scintillant converts radioactive particles into a photon signal that is
detected by a
scintillation counter or other detector. Additionally, binding can be
evaluated by
a Bio Sensor assay, which is based on the ability of the sensor to register
changes in admittance induced by ion-channel modulation following binding.
Phage display is also a powerful quantitative assay to measure protein:protein
interaction using colourimetric ELISA (enzyme-linked immunosorbent assay).
ii) The complementation of glycerol-3-phosphate auxotrophy
A method, useful in the invention, is to measure the ability of
a STAAU R4 polypeptide to relieve glycerol-3-phosphate auxotrophy of an E.
coli
strain that possesses the plsB26 and pIsX50 mutations. A strain of E. coli
bearing
mutations in two distinct loci, plsB and plsX, depends upon the presence of
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glycerol-3-phosphate in the medium for its growth. Introduction, into this
strain, of
a polynucleotide sequence encoding wild-type PIsX circumvents the glycerol-3-
phosphate growth requirement. Strains are assessed for the relief of their
auxotrophic requirement by plating, in parallel, onto minimal medium
containing
or not containing glycerol-3-phosphate. Growth on the plates lacking glycerol-
3-
phosphate indicates complementation of the auxotrophic phenotype.
This assay has proven useful to assess whether homologues
of E. coli plsX from other bacterial species, including the Gram-negative
bacterium Salmonella typhimurium and the Gram-positive bacteria Clostridium
butyricum and Bacillus subtilis can functionally replace PIsX in E. coli.
Similar
assays can be developed to test the activity of PIsX from S. aureus and to
screen
for inhibitors of such activity.
As used herein, the term "polynucleotide encoding a
polypeptide" or equivalent language encompasses polynucleotides that include
a sequence encoding a polypeptide of the invention, particularly a bacterial
polypeptide and more particularly a polypeptide of S. aureus STAAU_R4 protein
having an amino acid sequence set out in Fig. 1, SEQ ID NO: 2. The term also
encompasses polynucleotides that include a single continuous region or
discontinuous regions encoding the polypeptide (for example, polynucleotides
interrupted by integrated phage, an integrated insertion sequence, an
integrated
vector sequence, an integrated transposon sequence, or otherwise altered due
to RNA editing or genomic DNA reorganization) together with additional regions
that also may contain coding andlor non-coding sequences.
As used herein, the term "STAAU_R4 gene" "PIsX gene" is
meant to encompass a polynucleotide encoding a S. aureus STAAU_R4
polypeptide. Any additional nucleotide sequences necessary to direct
transcription
of RNA encoding a S. aureus STAAU_R4 polypeptide, either in a cell or in
vitro,
will be termed "regulatory sequences", which include but are not limited to
transcriptional promoters and enhancers, and transcription terminators.
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As used herein, the term "ORF 33" or "phage 3A ORF 33" or
"3A ORF 33" encompasses a polynucleotide comprising or consisting of the
sequence provided in Fig. 2 (SEQ ID NO: 3), which encodes a gene product
known as the 3A ORF 33 gene product.
As used herein, the term "polynucleotide(s)" generally refers
to any polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified
RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without
limitation, single- and double-stranded DNA, DNA that is a mixture of single-
and
double-stranded regions or single-, double- and triple-stranded regions,
single-
and double-stranded RNA, and RNA that is mixture of single- and double-
stranded regions, hybrid molecules comprising DNA and RNA that may be single-
stranded or, more typically, double-stranded, or triple-stranded regions, or a
mixture of single- and double-stranded regions. In addition, "polynucleotide"
as
used herein refers to triple-stranded regions comprising RNA or DNA or both
RNA
and DNA. The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of the
molecules,
but more typically involve only a region of some of the molecules. One of the
molecules of a triple-helical region often is an oligonucleotide. As used
herein, the
term "polynucleotide(s)"also includes DNAs or RNAs as described above that
contain one or more modified bases. Thus, DNAs or RNAs with backbones
modified for stability or for other reasons are "polynucleotide(s)" as that
term is
intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just two
examples,
are polynucleotides as the. term is used herein. It will be appreciated that a
great
variety of modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term "polynucleotide(s)" as
it is
employed herein embraces such chemically, enzymatically or metabolically
modified forms of polynucleotides, as well as the chemical forms of DNA and
RNA
characteristic of viruses and cells, including, for example, simple and
complex
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cells. "Polynucleotide(s)" also embraces short polynucleotides often referred
to
as oligonucleotide(s). Polynucleotides can also be DNA and RNA chimeras.
As used herein, the term "polypeptide(s)" refers to any peptide
or protein comprising two or more amino acids joined to each other by peptide
bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains,
commonly referred to as peptides, oligopeptides and oligomers and to longer
chains generally referred to as proteins. Polypeptides may contain amino acids
other than the 20 gene-encoded amino acids. "Polypeptide(s)" include those
modified either by natural processes, such as processing and other post-
translational modifications, but also by chemical modification techniques.
Such
modifications are well described in basic texts and in more detailed
monographs,
as well as in a voluminous research literature, and they are well known to
those
of skill in the art. It will be appreciated that the same type of modification
may be
present in the same or varying degree at several sites in a given polypeptide.
Also, a given polypeptide may contain many types of modifications.
Modifications
can occur anywhere in a polypeptide, including the peptide backbone, the amino
acid side-chains, and the amino or carboxyl termini. Modifications include,
for
example, acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment
of flavin, covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid
derivative, covalent attachment of phosphotidylinositol, cross-linking,
cyclization,
disulfide bond formation, demethylation, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic
processing, phosphorylation, prenylation, racemization, glycosylation, lipid
attachment, sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation, selenoylation, sulfation and transfer-RNA mediated addition of
amino acids to proteins, such as arginylation, and ubiquitination. See, for
instance: Proteins - Structure and Molecular Properties, 2nd Ed., T. E.
Creighton,
W. H. Freeman and Company, New York (1993); Wold, F., Posttranslational
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Protein Modifications: Perspectives and Prospects, pgs. 1-12 in
Posttranslational
Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York
(1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); and Rattan et al.,
Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad.
Sci.
663: 48-62(1992). Polypeptides may be branched or cyclic, with or without
branching. Cyclic, branched and branched circular polypeptides may result from
post-translational natural processes and may be made by entirely synthetic
methods, as well.
As used herein, the term "variant(s)" refers to a polynucleotide
or polypeptide that differs from a reference polynucleotide or polypeptide,
respectively, but retains one or more of the biological activities of the
initial (e.g.
non-variant) polynucleotide or polypeptide of the present invention (e.g.
STAAU_R4). A typical variant of a polynucleotide differs in nucleotide
sequence
from another reference polynucleotide. Changes in the nucleotide sequence of
the variant may or may not alter the amino acid sequence of a polypeptide
encoded by the reference polynucleotide. Nucleotide changes may result in
amino
acid substitutions, additions, deletions, and truncations in the polypeptide
encoded by the reference sequence, or in the formation of fusion proteins, as
discussed below. A typical variant of a polypeptide differs in amino acid
sequence
from another reference polypeptide. Generally, differences are limited so that
the
sequences of the reference polypeptide and the variant are closely similar
overall
and, in many regions, identical. A variant and reference polypeptide may
differ in
amino acid sequence by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or may not be
one
encoded by the genetic code. The present invention also includes variants of
each of the polypeptides of the invention, that is polypeptides that vary from
the
referents by conservative amino acid substitutions whereby a residue is
substituted by another with like characteristics. Typically, such
substitutions are
among Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and
Glu; and among the basic residues Lys and Arg; or aromatic residues Phe and
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Tyr. Particularly preferred are variants in which 1-10, 1-5, 1-3, 2-3, or 1
amino acid
or amino acids are substituted, deleted, or added in any combination. A
variant
of a polynucleotide or polypeptide may be a naturally occurring such as an
allelic
variant, or it may be a variant that is not known to occur naturally. Non-
naturally
occurring variants of polynucleotides and polypeptides may be made by
mutagenesis techniques, by direct synthesis, and by other recombinant methods
known to skilled artisans. As used herein, the term "fragment", when used in
reference to a polypeptide, is a polypeptide having an amino acid sequence
that
is entirely the same as part but not all of the amino acid sequence of the
polypeptide according to the invention from which it "derives". As with S.
aureus
STAAIJ_R4 polypeptides, fragments may be "free-standing" ("consisting of"), or
comprised within a larger polypeptide of which they form a part or region,
most
preferably as a single continuous region in a single larger polypeptide.
The term "isolated", when used in reference to a nucleic acid
means that a naturally occurring sequence has been removed from its normal
cellular (e.g., chromosomal) environment or is synthesized in a non-natural
environment (e.g., artificially synthesized). Thus, the sequence may be in a
cell-
free solution or placed in a different cellular environment. The term does not
imply
that the sequence is the only nucleotide chain present, but that it is
essentially
free (about 90-95% pure at least) of non-nucleotide material naturally
associated
with it, and thus is distinguished from isolated chromosomes.
The term "enriched", when used in reference to a
polynucleotide means that the specific DNA or RNA sequence constitutes a
significantly higher fraction (2-5 fold) of the total DNA or RNA present in
the cells
or solution of interest than in normal or diseased cells or in cells from
which the
sequence was originally taken. This could be.caused by a person, by
preferential
reduction in the amount of other DNA or RNA present, or by a preferential
increase in the amount of the specific DNA or RNA sequence, or by a
combination of the two. However, it should be noted that enriched does not
imply
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that there are no other DNA or RNA sequences present, just that the relative
amount of the sequence of interest has been significantly increased.
As used herein, the term "significantly higher fraction"
indicates that the level of enrichment is useful to the person making such an
enrichment and indicates an increase in enrichment relative to other nucleic
acids
of at least about 2-fold, or 5- to 10-fold or even more. The term also does
not
imply that there is no DNA or RNA from other sources. The other source of DNA
may, for example, comprise DNA from a yeast or bacterial genome, or a cloning
vector such as pUC19. This term distinguishes from naturally occurring events,
such as viral infection, or tumor type growths, in which the level of one mRNA
may
be naturally increased relative to other species of mRNA. That is, the term is
meant to cover only those situations in which a person has intervened to
elevate
the proportion of the desired nucleic acid.
As used herein, the term "purified" in reference to nucleic acid
does not require absolute purity (such as a homogeneous preparation). Instead,
it represents an indication that the sequence is relatively more pure than in
the
natural environment (compared to the natural level, this level should be at
least
2-5 fold greater, e.g., in terms of mg/mL). Individual clones isolated from a
genomic or cDNA library may be purified to electrophoretic homogeneity. The
claimed DNA molecules obtained from these clones could be obtained directly
from total DNA or from total RNA. cDNA clones are not naturally occurring, but
rather are preferably obtained via manipulation of a partially purified
naturally
occurring substance (messenger RNA). The construction of a cDNA library from
mRNA involves the creation of a synthetic substance (cDNA) and pure individual
cDNA clones can be isolated from the synthetic library by clonal selection of
the
cells carrying the cDNA library. Thus, the process which includes the
construction
of a cDNA library from mRNA and isolation of distinct cDNA clones yields an
approximately 106-fold purification of the native message over its proportion
in
naturally occurring cells. Thus, purification of at least one order of
magnitude,
preferably two or three orders, and more preferably four or five orders of
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magnitude is expressly contemplated. A genomic library can be used in the same
way and yields the same approximate levels of purification.
The terms "isolated", "enriched", and ~ "purified" used with
respect to nucleic acids, above, may similarly be used to denote the relative
purity
and abundance of polypeptides. These, too, may be stored in, grown in,
screened
in, and selected from libraries using biochemical techniques familiar in the
art.
Such polypeptides may be natural, synthetic or chimeric and may be extracted
using any of a variety of methods, such as antibody immunoprecipitation, other
"tagging" techniques, conventional chromatography and/or electrophoretic
methods. Some of the above utilize the corresponding nucleic acid sequence.
As used herein, the term "complement" when used in
reference to a given polynucleotide sequence refers to a sequence of
nucleotides
which can form a double-stranded heteroduplex in which every nucleotide in the
sequence of nucleotides is base-paired by hydrogen bonding to a nucleotide
opposite it in the heteroduplex with the given polynucleotide sequence. The
term
may refer to a DNA or an RNA sequence that is the complement of another RNA
or DNA sequence. As used herein, the term "hybridizes" refers to the formation
of a hydrogen-bonded heteroduplex between two nucleic acid molecules.
Generally, a given nucleic acid molecule will hybridize with its complement,
or with
a molecule that is sufficiently complementary to the given molecule to permit
formation of a hydrogen-bonded heteroduplex between the two molecules.
As used herein, the term "probe" refers to a polynucleotide of
at least 15 nucleotides (nt), 20 nt, 30 nt, 40 nt, 50 nt, 75 nt, 100 nt, 200
nt, 500 nt,
1000 nt, and even up to 5000 to 10,000 nt in length.
"Identity" and "similarity," as used herein and as known in the
art, are relationships between two or more polypeptide sequences or two or
more
polynucleotide sequences, as the case may be, as determined by comparing the
sequences.
Amino acid or nucleotide sequence "identity" and "similarity"
are determined from an optimal global alignment between the two sequences
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being compared. A non-limiting example of optimal global alignment can be
carried-out using the Needleman - Wunsch algorithm [Needleman and Wunsch,
1970, J. Mol. Biol. 48:443-453]. "Identity" means that an amino acid or
nucleotide
at a particular position in a first polypeptide or polynucleotide is identical
to a
corresponding amino acid or nucleotide in a second polypeptide or
polynucleotide
that is in an optimal global alignment with the first polypeptide or
polynucleotide.
In contrast to identity, "similarity" encompasses amino acids that are
conservative
substitutions.
The term "conservative" substitution is well-known in the art
and broadly refers to a substitution which does not significantly change the
chemico-physical properties of the substituted amino acid. For example, a
"conservative" substitution is any substitution that has a positive score in
the
blosum62 substitution matrix [Hentikoff and HentikofF, 1992, Proc. Natl. Acad.
Sci.
USA 89: 10915-10919]. By the statement "sequence A is n°l°
similar to sequence
B" is meant that n% of the positions of an optimal global alignment between
sequences A and B consists of conservative substitutions. By the statement
"sequence A is n% identical to sequence B" is meant that n% of the positions
of
an optimal global alignment between sequences A and B consists of identical
residues or nucleotides. Optimal global alignments in this disclosure used the
following parameters in the Needleman-Wunsch alignment algorithm:
For polypeptides:
Substitution matrix: blosum62.
Gap scoring function: -A -B*LG, where A=11 (the gap penalty),
B=1 (the gap length penalty) and LG is the length of the gap.
For nucleotide sequences:
Substitution matrix: 10 for matches, 0 for mismatches.
Gap scoring function: -A -B*LG where A=50 (the gap penalty),
B=3 (the gap length penalty) and LG is the length of the gap.
The term ' identity' and 'similarity' between sequences can be
extended to their fragments. An optimal local alignment between sequences A
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and B is the highest scoring alignment of fragments of A and B. By the
statement
"sequence A is n% similar locally to B" is meant that n% of the positions of
an
optimal local alignment between sequences A and B consists of conservative
substitutions. By the statement "sequence A is n% identical locally to B" is
meant
that n% of the position of an optimal local alignment between sequences A and
B consists of identical residues or nucleotides. A non-limiting example of
optimal
local alignment can be carried-out using the Smith-Waterman algorithm [Smith,
T.F and Waterman, M.S. 1981. Identification of common molecular
subsequences. J. Mol. Biol. 147:195-197].
Of course, the above-listed parameters are but one specific
example of alignment algorithm parameters. Numerous algorithms and
parameters are available and known to the person of ordinary skill.
Typical conservative substitutions are among Met, Val, Leu
and 11e; among Ser and Thr; among the residues Asp, Glu and Asn; among the
residues Gln, Lys and Arg; or aromatic residues Phe and Tyr. In calculating
the
degree (most often as a percentage) of similarity between two polypeptide
sequences, one considers the number of positions at which identity or
similarity
is observed between corresponding amino acid residues in the two polypeptide
sequences in relation to the entire lengths of the two molecules being
compared.
As used herein, the term "antibody" is meant to encompass
constructions using the binding (variable) region of such an antibody, and
other
antibody modifications. Thus, an antibody useful in the invention may comprise
a whole antibody, an antibody fragment, a polyfunctional antibody aggregate,
or
in general a substance comprising one or more specific binding sites from an
antibody. The antibody fragment may be a fragment such as an Fv, Fab or
F(ab')z
fragment or a derivative thereof, such as a single chain Fv fragment. The
antibody
or antibody fragment may be non-recombinant, recombinant or humanized. The
antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth. In
addition, an aggregate, polymer, derivative and conjugate of an immunoglobulin
or a fragment thereof can be used where appropriate. Neutralizing antibodies
are
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especially useful according to the invention for diagnostics, therapeutics and
methods of drug screening and drug design.
As used herein, the term "specific for an epitope present on a
S. aureus STAAU_R4 polypeptide", when used in reference to an antibody,
means that the antibody recognizes and binds an antigenic determinant present
on a S. aureus STAAU_R4 polypeptide or fragment thereof according to the
invention.
As used herein, the term "antigenically equivalent
derivative(s)" encompasses a polypeptide, polynucleotide, or the equivalent of
either which will be specifically recognized by certain antibodies which, when
raised to the protein, polypeptide or polynucleotide according to the
invention,
interferes with the immediate physical interaction between pathogen and
mammalian host.
As used herein, the term "essential", when used in connection
with a gene or gene product, means that the host cannot survive without, or is
significantly growth compromised, in the absence or depletion of functional
product. An "essential gene" is thus one that encodes a product that is
beneficial,
or preferably necessary, for cellular growth in vitro in a medium appropriate
for
growth of a strain having a wild-type allele corresponding to the particular
gene
in question. Therefore, if an essential gene is inactivated or inhibited, that
cell will
grow significantly more slowly than a wild-type strain or even not at all.
Preferably,
growth of a strain in which such a gene has been inactivated will be less than
20%, more preferably less than 10%, most preferably less than 5% of the growth
rate of the wild-type, or the rate will be zero, in the growth medium.
Preferably, in
the absence of activity provided by a product of the gene, the cell will not
grow at
all or will be non-viable, at least under culture conditions similar to normal
in vivo
growth conditions. For example, absence of the biological activity of certain
enzymes involved in bacterial cell wall synthesis can result in the lysis of
cells
under normal osmotic conditions, even though protoplasts can be maintained
under controlled osmotic conditions. Preferably, but not necessarily, if such
a
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gene is inhibited, e.g., with an antibacterial agent or a phage product, the
growth
rate of the inhibited bacteria will be less than 50%, more preferably less
than 30%,
still more preferably less than 20%, and most preferably less than 10% of the
growth rate of the uninhibited bacteria. As recognized by those skilled in the
art,
the degree of growth inhibition will generally depend upon the concentration
of the
inhibitory agent. In the context of the invention, essential genes are
generally the
preferred targets of antimicrobial agents. Essential genes can encode "target"
molecules directly or can encode a product involved in the production,
modification, or maintenance of a target molecule.
As used herein, "target" refers to a biomolecule or complex of
biomolecules that can be acted on by an exogenous agent or compound, thereby
modulating, preferably inhibiting, growth or viability of a bacterial cell. A
target may
be a nucleic acid sequence or molecule, or a polypeptide or a region of a
polypeptide.
As used herein, the term "signal that is generated by
interaction of a S. aureus polypeptide comprising the amino acid sequence of
SEQ ID NO: 2, or fragments thereof to a 3A ORF 33 or fragment thereof' or the
like refers to the measurable indicator of polypeptide interaction in a
binding
assay, wherein the interacting polypeptide comprises the amino acid sequence
of SEQ ID NO: 2, fragment thereof or variant thereof and 3A ORF 33, fragment
thereof or variant thereof. As used herein, the term "signal that is generated
by
activation or inhibition of a S. aureus polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, or fragments thereof" refers to the measurable
indicator of polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
fragment or variant thereof; activity in an assay of STAAIJ_R4 activity. For
example, the signal may include, but is not limited to (i) a signal resulting
from
binding of 3A ORF 33 to a STAAU_R4 polypeptide, including a fluorescence
signal (time-resolved fluorescence resonance energy transfer assay;
fluorescence
polarization assay), spectrophotometer absorbance measurement of a
colourimetric signal (phage display ELISA), mass change measurement (surface
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plasmon resonance analysis), or a viability measurement on selective medium
(yeast two-hybrid analysis)
As used herein, the term "standard", used in reference to
polypeptide activity, means the amount of activity observed or detected
(directly
or indirectly) in a given assay performed in the absence of a candidate
compound.
A "standard" serves as a reference to determine the effect, positive or
negative,
of a candidate compound on polypeptide activity.
As used herein, the term "increase in activity" refers to an
enhanced level of measurable activity of a polypeptide in a given assay in the
presence of a candidate compound relative to the measurable level of activity
in
the absence of a candidate compound. Activity is considered increased
according
to the invention if it is at least 10% greater, 20% greater, 50% greater, 75%
greater, 100% greater or more, up to 2-fold, 5-fold, 10-fold, 20-fold, 50-
fold, 100-
fold or more than in the absence of a candidate compound.
As used herein, the term "decrease in activity" refers to a
reduced level of measurable activity of a polypeptide in a given assay in the
presence of a candidate compound relative to the measurable level of activity
in
the absence of a candidate compound. Activity is considered decreased
according to the invention if it is at least 10% less, preferably 15% less,
20% less,
50% less, 75% less, or even 100% less (i.e., no activity) than that observed
in the
absence of a candidate compound.
As used herein, the term "conditions that permit their
interaction", when used in reference to a S. aureus polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or fragments thereof, and a candidate
compound means that the two entities are placed together, whether both in
solution or with one immobilized or restricted in some way and the other in
solution, wherein the parameters (e.g., salt, detergent, protein or candidate
compound concentration, temperature, and redox potential, among others) of the
solution are such that the S. aureus polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, or fragments thereof, and the candidate compound
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may physically associate. Conditions that permit protein:candidate interaction
include, for example, the conditions described herein for TR-FRET, fluorescent
polarization, Surface Plasmon Resonance and Phage display assays.
As used herein, the term "detectable change in a measurable
parameter of STAAU_R4" refers to an alteration in a quantifiable
characteristic of
a S. aureus STAAU_R4 polypeptide.
As used herein, the term "agonist" refers to an agent or
compound that enhances or increases the activity of a S. aureus STAAU R4
polypeptide or polynucleotide. An agonist may be directly active on a S.
aureus
STAAU R4 polypeptide or polynucleotide, or it may be active on one or more
constituents in a pathway that leads to enhanced or increased activity of a S.
aureus STAAU_R4 polypeptide or polynucleotide.
As used herein, the term "antagonist" refers to an agent or
compound that reduces or decreases the activity of a S. aureus STAAU_R4
polypeptide or polynucleotide. An antagonist may be directly active on a S.
aureus
STAAU_R4 polypeptide or polynucleotide, or it may be active on one or more
constituents in a pathway that leads to reduced or decreased activity of a S.
aureus STAAU R4 polypeptide or polynucleotide.
As used herein, the term "antibacterial agent',' or "antibacterial
compound" refers to an agent or compound that has a bacteriocidal or
bacteriostatic effect on one or more bacterial strains, preferably such an
agent or
compound is bacteriocidal or bacteriostatic on at least S. aureus.
As used herein, the term "synthesizing" refers to a process of
chemically synthesizing a compound.
As used in the context of treating a bacterial infection a
"therapeutically effective amount", "pharmaceutically effective amount" or
"amount
sufficient to provide a therapeutic effect" indicates an amount of an
antibacterial
agent which has a therapeutic effect. This generally refers to the inhibition,
to
some extent, of the normal cellular functioning of bacterial cells required
for
continued bacterial infection. Further, as used herein, a therapeutically
effective
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amount means an amount of an antibacterial agent that produces the desired
therapeutic effect as judged for example by clinical trial results and/or
animal
models. This amount can be routinely determined by one skilled in the art and
will
vary depending on several factors, such as the particular bacterial strain
involved
and the particular antibacterial agent used. In the same context, an "amount
sufficient to reduce adhesion" of a bacterium to a tissue or tissue surface
indicates an amount of an antibacterial agent that is effective for
prophylactically
preventing or reducing the extent of bacterial infection of the given tissue
or tissue
surface. The same principle applies to the terminology "prophylactically
effective
amount".
As used in the context of treating a bacterial infection,
contacting or administering the antimicrobial agent 'in combination with
existing
antimicrobial agents' refer to a concurrent contacting or administration of
the
active compound with antibiotics to provide a bactericidal or growth
inhibitory
effects beyond the individual bactericidal or growth inhibitory effects of the
active
compound or the antibiotic. Existing antibiotic refers for example to the
group
consisting of penicillins, cephalosporins, imipenem, monobactams,
aminoglycosides, tetracyclines, sulfonamides, trimethoprim/sulfonamide,
fluoroquinolones, macrolides, vancomycin, polymyxins, chloramphenicol and
lincosamides.
As used herein, a "tissue" refers to an aggregation of cells of
one or more cell types which together perform one or more specific functions
in
an organism. As used herein, a "tissue surface" refers to that portion of a
tissue
that forms a boundary between a given tissue and other tissues or the
surroundings of the tissue. A tissue surface may refer to an external surface
of an
animal, for example the skin or cornea, or, alternatively, the term may refer
to a
surface that is either internal, for example, the lining of the gut, or to a
surface that
is exposed to the outside surroundings of the animal only as the result of an
injury
or a surgical procedure.
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As used herein, the term "measuring the binding of a
candidate compound" refers to the use of an assay preferably permitting the
quantitation of the amount of a candidate compound physically associated with
a S. aureus STAAU_R4 polypeptide, fragment or variant thereof.
A "candidate compound" as used herein, is any compound
with a potential to modulate the expression or activity of a S. aureus
STAAU,R4
polypeptide.
As used herein, the term "directly or indirectly delectably
labeled" refers to the attachment of a moiety to a candidate compound that
renders the candidate compound either directly detectable (e.g., an isotope or
a
fluorophore) or indirectly detectable (e.g., an enzyme activity, allowing
detection
in the presence of an appropriate substrate, or a specific antigen or other
marker
allowing detection by addition of an antibody or other specific indicator).
A "method of screening" refers to a method for evaluating a
relevant activity or property of a large plurality of compounds, rather than
just one
or a few compounds. For example, a method of screening can be used to
conveniently test at least 100, more preferably at least 1000, still more
preferably
at least 10,000, and most preferably at least 100,000 different compounds, or
even more. In a particular embodiment, the method is amenable to automated,
cost-effective high throughput screening on libraries of compounds for lead
development.
In a related aspect or in preferred embodiments, the invention
provides a method of screening for potential antibacterial agents by
determining
whether any of a plurality of compounds, preferably a plurality of small
molecules,
is active on STAAU R4. Preferred embodiments include those described for the
above aspect, including embodiments which involve determining whether one or
more test compounds bind to or reduce the level of activity of a bacterial
target,
and embodiments which utilize a plurality of different targets as described
above.
As used herein, the term "simultaneously" when used in
connection with the assays of the present invention, refers to the fact that
the
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specified components or actions at least overlap in time, and is thus not
restricted
to the fact that the initiation and termination points are identical. For
certainty, a
simultaneous contact of a STAAU R4 polypeptide with a candidate compound
and a bacteriophage polypeptide, for example, is an overlap in contact
periods,
which can, but does not necessarily reflect the fact that the latter two are
introduced into an assay mixture at the exact same time.
The term "compounds" preferably includes, but is not limited
to, small organic molecules, peptides, polypeptides and antibodies that bind
to a
polynucleotide and/or polypeptide of the invention, such as for example
inhibitory
ORF gene product or target thereof, and thereby inhibit, extinguish or enhance
its
activity or expression. Potential compounds may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or antibody that
binds the
same sites) on a binding molecule, such as a bacteriophage gene product,
thereby preventing bacteriophage gene product from binding to STAAU R4
polypeptides.
The term "compounds" is also meant to include small
molecules that bind to and occupy the binding site of a polypeptide, thereby
preventing binding to cellular binding molecules, such that normal biological
activity is prevented. Examples of small molecules include but are not limited
to
small organic molecules, peptides or peptide-like molecules. Preferred
potential
compounds include compounds related to and variants of inhibitory ORF encoded
by a bacteriophage and of STAAU R4 and any homologues and/or peptido-
mimetics and/or fragments thereof. Other examples of potential polypeptide
antagonists include antibodies or, in some cases, oligonucleotides or proteins
which are closely related to the ligands, substrates, receptors, enzymes,
etc., as
the case may be, of the polypeptide, e.g., a fragment of the ligands,
substrates,
receptors, enzymes, etc.; or small molecules which bind to the polypeptide of
the
present invention but do not elicit a response, so that the activity of the
polypeptide is prevented. Other potential compounds include antisense
molecules
(see Okano, 1991 J. Neurochem. 56, 560; see also "Oligodeoxynucleotides as
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Antisense Inhibitors of Gene Expression", CRC Press, Boca Raton, FL (1988),
for
a description of these molecules).
As used herein, the term "library" refers to a collection of 100
compounds, preferably of 1000, still more preferably 5000, still more
preferably
10,000 or more, and most preferably of 50,000 or more compounds.
As used herein, the term "small molecule" refers to
compounds having molecular mass of less than 3000 Daltons, preferably less
than 2000 or 1500, still more preferably less than 1000, and most preferably
less
than 600 Daltons. Preferably but not necessarily, a small molecule is not an
oligopeptide.
As used herein, the term "mimetic" refers to a compound that
can be natural, synthetic, or chimeric and is structurally and functionally
related
to a reference compound. In terms of the present invention, a
"peptidomimetic,"
for example, is a non-peptide compound that mimics the activity-related
aspects
of the 3-dimensional structure of a peptide or polypeptide, for example a
compound that mimics the structure of a peptide or active portion of a phage-
or
bacterial ORF-encoded polypeptide.
As used herein, the term "bacteriophage inhibitor protein"
refers to a protein encoded by a bacteriophage nucleic acid sequence, which
inhibits bacterial function in a host bacterium. Thus, it is a bacteria-
inhibiting
phage product. The term "bacteriophage inhibitor protein" encompasses a
fragment, derivative, or active portion of a bacteriophage inhibitor protein.
In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either STAAU R4 or
its
target molecule or ligand to facilitate separation of complexed from
uncomplexed
forms of one or both of the proteins or polypeptides, as well as to
accommodate
automation of the assay. Binding of a test compound to a STAAIJ_R4 protein (or
fragment, or variant thereof) or interaction of a STAAU R4 protein with a
target
molecule or ligand in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the reactants.
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Examples of such vessels include microtitre plates, test tubes
and micro-centrifuge tubes.
In one embodiment a fusion protein can be provided which
adds a domain that allows one or both of the proteins to be bound to a matrix.
For
example, glutathione-S-transferase (GST)/STAAU_R4 fusion proteins or
GST/target fusion proteins (e.g. GST/3A ORF 33 ) can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione
derivatized microtitre plates, which are then combined with the test compound
or
the test compound and either the non-adsorbed target protein or STAAU_R4
protein and the mixture incubated under conditions conducive to complex
formation (e.g. at physiological conditions for salt and pH). Following
incubation
the beads or microtitre plate wells are washed to remove any unbound
components, the matrix immobilized in the case of beads, complex determined
either directly or indirectly, for example, as described above. Alternatively,
the
complexes can be dissociated from the matrix, and the level of STAAU_R4
binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices (and
well-known in the art) can also be used in the screening assays of the
invention.
For example, either a STAAU R4 protein or a STAAU R4 target molecule or
ligand can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated STAAU_R4 protein or target molecules or ligand can be prepared
from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art
(e.g.,
biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the
wells of
streptavidin-coated 96 well plates (Pierce Chemical). Alternatively,
antibodies
reactive with target molecules or ligand but which do not interfere with
binding of
the STAAU_R4 protein (or part thereof) to its target molecule or ligand can be
derivatized to the wells of the plate, and unbound target or STAAU_R4 protein
trapped in the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies reactive with
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the STAAU_R4 protein or target molecule, as well as enzyme-linked assays which
rely on detecting an enzymatic activity associated with the STAAU_R4 protein.
As used herein, the term "active portion", when refering to a
bacteriophage-derived sequence, relates to an epitope, a catalytic or
regulatory
domain, or a fragment of a bacteriophage inhibitor protein that is responsible
for,
or a significant factor in, bacterial target inhibition. The active portion
preferably
may be removed from its contiguous sequences and, in isolation, still effect
inhibition.
As used herein, the term "treating a bacterial infection" refers
to a process whereby the growth and/or metabolic activity of a bacterium or
bacterial population in a host, preferably a mammal, more preferably a human,
is
inhibited or ablated. Similarly, the term "preventing a bacterial infection"
refers to
a process whereby the growth and/or metabolic activity of a bacterium or
bacterial
population in a host, preferably a mammal, more preferably a human, at risk of
being infected is inhibited or ablated.
As used herein, the term "bacterium" refers to a single
bacterial strain and includes a single cell and a plurality or population of
cells of
that strain unless clearly indicated to the contrary. In reference to bacteria
or
bacteriophage, the term "strain" refers to bacteria or phage having a
particular
genetic content. The genetic content includes genomic content as well as
recombinant vectors. Thus, for example, two otherwise identical bacterial
cells
would represent different strains if each contained a vector, e.g., a plasmid,
with
different inserts.
As used herein, the term "diagnosing" refers to the
identification of an organism or strain of an organism responsible for a
bacterial
infection.
As used herein, the term "infection with Staphylococcus
aureus" refers to the presence, growth or proliferation of cells of a S.
aureus strain
within, or on a surface of, an animal, such as a mammal, preferably a human.
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As used herein, the term "bacteriophage 3A ORF 33 -encoded
polypeptide" refers to a polypeptide encoded by SEQ ID NO: 3 or to a fragment
or derivative thereof encompassing an active portion of a bacteriophage 3A ORF
33 -encoded polypeptide of sequence disclosed in SEQ ID NO: 4.
As used herein, the term "polypeptide complex" refers to a
combination of two or more polypeptides in a physical association with each
other.
It is preferred that such a physical association be required for some aspect
of the
activity of one or more of the polypeptides in such a polypeptide complex.
As used herein, the term "physical association" refers to an
interaction between two moieties involving contact between the two moieties.
As used herein, the term "bodily material(s)" means any
material derived from an individual or from an organism infecting, infesting
or
inhabiting an individual, including but not limited to, cells, tissues and
waste, such
as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,
organ tissue, skin, urine, stool or autopsy materials. .
As used herein, the term "disease(s)" means any disease
caused by or related to infection by a bacterium, including, for example,
otitis
media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural
empyema and endocarditis, and most particularly meningitis, such as for
example
infection of cerebrospinal fluid.
As used herein, the term "fusion protein(s)" refers to a protein
- encoded by a gene comprising amino acid coding sequences from two or more
separate proteins fused in frame such that the protein comprises fused amino
acid sequences from the separate proteins.
As used herein, the term "host cell(s)" is a cell which has been
transformed or transfected, or is capable of transformation or transfection by
an
exogenous polynucleotide sequence.
As used herein, the term "immunoiogically equivalent
derivative(s)" encompasses a polypeptide, polynucleotide, or the equivalent of
either which when used in a suitable formulation to raise antibodies in a
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vertebrate, results in antibodies that act to interfere with the immediate
physical
interaction between pathogen and mammalian host.
As used herein, the term "immunospecific" means that
characteristic of an antibody whereby it possesses substantially greater
affinity for
the polypeptides of the invention or the polynucleotides of the invention than
its
affinity for other related polypeptides or polynucleotides respectively,
particularly
those polypeptides and polynucleotides in the prior art.
~As used herein, the term "individual(s)" means a multicellular
eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a
bovid,
a simian, a primate, and a human.
As used herein, the term "Organism(s)" means a (i)
prokaryote, including but not limited to, a member of the genus Streptococcus,
Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria,
Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia,
Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,
Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,
Bacillus,
Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus,
Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella!
Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and
further including, but not limited to, a member of the species or group, Group
A
Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D
Streptococcus, Group G Streptococcus, Streptococcus pneumoniae,
Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis,
Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria
meningitidis, Staphylococcus aureus, Staphylococcus epidermidis,
Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,
Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,
Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella
parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella
dysenteriae,
Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae,
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Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii,
Proteus
mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia
marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenteric,
Shigella
flexneri, Pseudomortas aeruginosa, Franscisella tularensis, Brucella abortis,
Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium
tetani,
Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia
trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and
(iii) a
unicellular or filamenous eukaryote, including but not limited to, a
protozoan, a
fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and
a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or
Candida albicans.
As used herein, the term "recombinant expression system(s)"
refers to a system in which vectors comprising sequences encoding polypeptides
of the invention or portions thereof, or polynucleotides of the invention are
introduced or transformed into a host cell or host cell lysate for the
production of
the polynucleotides and polypeptides of the invention.
As used herein, the term "artificially synthesized" when used
in reference to a peptide, polypeptide or polynucleotide means that the amino
acid
or nucleotide subunits were chemically joined in vitro without the use of
cells or
polymerizing enzymes. The chemistry of polynucleotide and peptide synthesis is
well known in the art.
In addition to the standard single and triple letter
representations for amino acids, the term "X" or "Xaa" may also be used in
describing certain polypeptides of the invention. "X" and "Xaa" mean that any
of
the twenty naturally occurring amino acids may appear at such a designated
position in the polypeptide sequence.
As used herein, the term "specifically binding" in the context of
the interaction of two polypeptides means that the.two polypeptides physically
interact via discrete regions or domains on the polypeptides, wherein the
interaction is dependent upon the amino acid sequences of the interacting
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domains. Generally, the equilibrium binding concentration of a polypeptide
that
specifically binds another is in the range of about 1 mM or lower, more
preferably
1 uM or lower, preferably 100 nM or lower, 10 nM or lower, 1 nM or lower, 100
pM
or lower, and even 10 pM or lower.
As used herein, the term "decrease in the binding" refers to a
drop in the signal that is generated by the physical association between two
polypeptides under one set of conditions relative to the signal under another
set
of reference conditions. The signal is decreased if it is at least 10% lower
than the
level under reference conditions, and preferably 20%, 40%, 50%, 75%, 90%, 95%
or even as much as 100% lower (i.e., no detectable interaction).
As used herein, the term "detectable marker", when used in
the context of a yeast two-hybrid assay, refers to a polypeptide that confers
a trait
upon a cell expressing that polypeptide that signals the presence or amount of
that polypeptide expressed. Detectable markers are encoded on plasmids that
may exist episomally or may be integrated into the genome of a host cell.
Detectable markers include, but are not limited to, polypeptides encoding
enzymes allowing colorimetric or fluorescent detection (e.g., E. coli LacZ,
which
catalyzes the conversion of the substrate analog X-gal to generate a blue
color),
polypeptides encoding enzymes conferring antibiotic resistance, and
polypeptides
encoding enzymes conferring the ability of a yeast strain to grow on medium
lacking a given component (i.e., critical for the relief of auxotrophy).
As used herein, the term "results in the expression of a
detectable marker" means that the interaction of factors necessary to permit
the
expression of a detectable marker (e.g., two-hybrid transactivation domain and
DNA binding domain fusion proteins) causes the transactivation and translation
of detectable levels of a detectable marker. A "detectable level" is that
level of
expression that can be differentiated from background expression occurring in
the
substantial absence of one or more factors or conditions necessary for marker
expression. Detectable levels will vary depending upon the nature of the
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detectable marker, but will generally consist of levels at least about 10% or
more
greater than the background level of a given marker.
As used herein, the term "decrease in the expression" refers
to a drop in the expression of a detectable marker under one set of conditions
, relative to the expression under another set of reference conditions. The
expression of a detectable marker is decreased if it is at least 10% lower
than the
level under reference conditions, and preferably 20%, 40%, 50%, 75%, 90%, 95%
or even as much as 100% lower (i.e., not expressed).
Identification of the S. aureus STAAU R4 seauence
The methodology used to identify the STAAU R4 polypeptide
is described in detail in U.S. Provisional Patent Application 60/110,992,
filed
December 3, 1998, and PCT International Application W01999/IB99/02040, filed
December 3, 1999. Briefly, this PCT application concerns bacteriophages that
can
infect a selected bacterium. The sequencing and characterization of the phage
genetic information allow the identification of all open reading frames (ORFs)
encoded by the phage, including those that are essential or instrumental in
inhibiting their host. Each ORF is identified using computer softwares and
individually expressed in the host. The effect of this expression on host
viability
is then measured. Identification of ORFs from the phage genome which inhibit
the
host bacterium both provides a compound that could be used as a bacterial
inhibitor compound per se (or derivatized or modified to obtain further
inhibitors)
and as a tool for the identification of the bacterial target affected by the
phage-
encoded inhibitor.
Using methodology described in detail in Example 1 and 2, a
S. aureus polypeptide that specifically bound the bacterial growth inhibitory
3A
phage ORF 33 protein was isolated. Briefly, the 3A ORF 33 protein was used as
a ligand in an affinity chromatography binding step with S. aureus protein
extract.
The selected S. aureus interacting polypeptide was purified and further
analyzed
by tryptic digestion and mass spectrometry using MALDI-ToF technology [Qin,
J.,
et al. (1997) Anal. Chem. 69:3995-4001]. Computational analysis
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(http://prowl.rockfeller.edu/cgi-bin/ProFound) of the mass spectrum obtained
identifies the corresponding ORF in the S. aureus nucleotide sequence in the
University of Oklahoma S. aureus genomic database at
http://www.genome.ou.edu/staph.html. The interaction between 3A ORF 33 and
the candidate target protein, herein referred as STAAU R4, was also confirmed
in affinity blotting assays. Comparison of the ORF of the S. aureus contig
that
encodes a Cryptic peptide similar to that identified in the S. aureus phage 3A
ORF
33 binding studies with all other sequences in the public domain databases
revealed that STAAU_R4 is related to PIsX, a protein implicated in fatty
acid/phospholipid synthesis (Fig. 8). As shown in Fig. 9A, the degree of
relatedness of the identified 8. subtilis ORF to the STAAU R4 protein is 53%
identity and 71 % similarity at the amino acid level across the entire
sequence.
Function of PIsX (STAAU R4)
Fatty acid biosynthesis is essential to the viability of all cells
because it yields a wide variety of lipid molecules some of which are key
structural
components of membranes and others which have important roles in signalling
and in energy storage. The importance of phospholipids, for example, is
underscored by the lack of genetic defects in the metabolism of these lipids
in
humans. In bacteria, lipid metabolism is similarly of critical importance to
the cell.
In E. coli, eight enzyme-catalyzed reactions convert acetyl-coenzyme A into
fatty
acids. Three subsequent reactions append acyl chains onto glycerol-3-phosphate
to yield CDP diacylglycerol, a key branchpoint molecule for the biosynthesis
of
important cellular phospholipids including phosphatidylserine,
phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin.
The initial reaction of phospholipid biosynthesis in E. coli is
catalyzed by the product of the pls8 gene, glycerol-3-phosphate
acyltransferase.
This enzyme preferentially utilizes saturated fatty acyl derivatives
(palmitoyl-
coenzymeA or palmitoyl-acyl carrier protein) for the initial acylation of
glycerol-3-
phosphate. Interestingly, the glycerol-3-phosphate auxotrophic mutant of E.
coli
that was originally described by Bell [Bell, R.M. (1974) J. Bacteriol. 117:
1065-
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1076] was found to contain mutations at two loci; both mutations were
necessary
to observe the dependence of the strain on glycerol-3-phosphate for growth
[Larson, T.J., Ludtke, D.N., and Bell, R.M. (1984) J. Bacteriol. 160: 711-
717].
Independent transposon insertions from wild-type cells, mediated by phage P1
cotransduction, were sought which could correct the auxotrophy of the mutant
strain. As expected, a portion of the transposon insertions were
cotransducible
with markers in the plsB region. However, the remainder mapped to a locus
termed plsX in a distinct region of the chromosome. Indeed, both p1sB26 and
pIsX50 mutations are necessary and sufficient for conferral of glycerol-3
phosphate auxotrophy.
Wild-type PIsX may function either to elevate the intracellular
levels of glycerol-3-phosphate or to increase the affinity of the defective
glycerol-
3-phosphate acyltransferase, encoded by plsB26, for its substrate, allowing
the
mutant PIsB to function [Larson, T.J., Ludtke, D.N., and Bell, R.M. (1984) J.
Bacteriol. 160: 711-717]. PIsX apparently does not influence glycerol-3-
phosphate
acyltransferase activity in vitro since the VmaX and the apparent Km defects
resulting from the pIsB26 mutation were independent of the presence of wild
type
PIsX. However, restoration of a wild-type copy of PIsX to a strain of E. coli
bearing
the pIsB26 pIsX50 mutations and a temperature-sensitive mutation in PIsC,
encoding the 1-acyl glycerol-3-phosphate acyltransferase, was not sufficient
to
restore lysophosphatidic acid synthesis in vivo at the nonpermissive
temperature
[Heath, R.J., Goldfine, H., and Rock, C.O. (1997) J. Bacteriol. 179: 7257-
7263].
Thus, PIsX cannot restore glycerol-3-phosphate activity in vivo.
Functional homologues to E. coli plsXexist in several bacterial
species, including Salmonella typhimurium [Zhang, Y., and Cronan~Jr., J.E.
(1998)
J. Bacteriol. 180: 3295-3303] and the Gram-positive bacteria Clostridium
butyricum [Heath, R.J., Goldfine, H., and Rock, C.O. (1997) J. Bacteriol. 179:
7257-7263] and Bacillus subtilis [Morbidoni, H., de Mendoza, D., and Cronan,
Jr.,
J.E. (1996) J. Bacteriol. 178: 4794-4800]. To date, no homologue of the S,
aureus
PIsX has been described or isolated. In Gram-positive bacteria, while the
function
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of PIsX remains to be determined, plsX is likely to be an essential gene based
on
indications that repeated' attempts to disrupt the gene were unsuccessful
[Morbidoni, H., de Mendoza, D., and Cronan, Jr., J.E. (1996) J. Bacteriol.
178:
4794-4800].
The demonstration that bacteriophage have adapted to
inhibiting a host bacterium by acting on a particular cellular component or
target
provides a strong indication that that component is an appropriate target for
developing and using antibacterial agents, e.g. in therapeutic treatments. The
present invention provides additional guidance over mere identification of
bacterial essential genes, as the present invention also provides an
indication of
accessibility of the target to an inhibitor, and an indication that the target
is
sufficiently stable over time (e.g., not subject to high rates of mutation) as
phage
acting on that target were able to develop and persist. Thus the present
invention
identifies STAAU_R4 as particularly likely to bean appropriate target for
development of antibacterial agents.
Identification of the surface of interaction on STAAU R4
This invention relates, in part, to a specific interaction between
a growth-inhibitory protein encoded by the S. aureus bacteriophage genome and
an essential S. aureus protein. In one embodiment, this interaction forms the
basis for drug screening assays. More specifically, the invention relates to
the
interacting domains of the protein encoded by the S. aureus STAAU_R4 and the
S. aureus bacteriophage 3A ORF 33 proteins, forming the basis for screening
assays. The. invention provides a method for the identification of 3A ORF 33
and,
more preferably, STAAU_R4 polypeptide fragments which are involved in the
interaction between STAAU R4 and 3A ORF 33.
Several approaches and techniques known to those skilled in
the art can be used to identify and to characterize interacting fragments of
STAAU_R4 and 3A ORF 33. These fragments may include, for example,
truncation polypeptides having a portion of an amino acid sequence of any of
the
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two proteins, or variants thereof, such as a continuous series of residues
that
includes an amino- and/or carboxyl-terminal amino acid sequence.
Fragments of STAAU_R4 and 3A ORF 33 can be cloned by
genetic recombinant technology and tested for interaction using a yeast two
s hybrid assay as exemplified below.
Partial proteolysis of proteins in solution is one method to
delineate the domain boundaries in multi-domain proteins. By subjecting
proteins
to limited digestion, the most accessible cleavage sites are preferentially
hydrolyzed. These cleavage sites preferentially reside in less structured
regions
which include loops and highly mobile areas typical of the joining amino acids
between highly structures domains. Purified STAAU_R4 and 3A ORF 33 proteins
can be subjected to partial proteolysis. The proteolysis can be performed with
low
concentrations of proteases (trypsin, chymotrypsin, endoproteinase Glu-C, and
Asp-N) with STAAU_R4 or 3A ORF 33 in solution, resulting in the generation of
defined proteolytic products as observed by SDS-PAGE. An acceptable
concentration and reaction time is defined by the near complete conversion of
the
full-length protein to stable proteolytic products. The proteolytic products
are then
subjected to affinity chromatography containing the appropriated partner of
interaction (3A ORF 33 or STAAU_R4 purified proteins) to determine a protein
sub-region able to interact. Interacting domains are identified by mass
spectrometry to determine both the intact fragment mass and the completely
digested with trypsin (by in-gel digestion) to better determine the amino acid
residues contained within the partial proteolytic fragment. Using both sets of
data,
the amino acid sequence of the partial proteolytic fragment can be precisely
determined.
Another approach is based on peptide screening using
different portions of 3A ORF 33 or STAAU_R4 to identify minimal peptides from
each polypeptide that are able to disrupt the interaction between the two
proteins.
It is assumed that fragments able to prevent interaction between STAAU_R4 and
3A ORF 33 correspond to domains of interaction located on either of the two
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interacting proteins. The different peptide fragments can be screened as
competitors of interaction in protein: protein binding assays such as the ones
described below. Fine mapping of interaction sites) within a protein can be
performed by an extensive screen of small overlapping fragments or peptides
spanning the entire amino acid sequence of the protein.
Suitable STAAU R4 and 3A ORF 33 -derived amino acid
fragments representative of the complete sequence of both proteins can be
chemical synthesis. For instance, in the multipin approach, peptides are
simultaneously synthesis by the assembly of small quantities of peptides (ca.
50
nmol) on plastic pins derivatized with an ester linker based on glycolate and
4-
(hydroxymethyl) benzoate [Maeji 1991 Pept Res, 4:142-6].
S. aureus STAAU R4 polypeptides
In one aspect of the invention there are provided polypeptides
of S. aureus referred to herein as "STAAU R4" and "STAAU_R4 polypeptides"
as well as biologically, diagnostically, prophylactically, clinically or
therapeutically
useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of the invention
are variants of S. aureus STAAU_R4 polypeptides encoded by naturally occurring
alleles of the STAAU_R4 gene. The present invention provides for an isolated
polypeptide which comprises or consists of: (a) an amino acid sequence which
has at least 50% identity, preferably at least 55% identity, preferably at
least 60%
identity, preferably at least 70% identity, preferably at least 80% identity,
more
preferably at least 90%, yet more preferably at least 95%, most preferably at
least
97-99%, or exact identity, over the entire length of SEQ ID N0: 2; or b) an
amino
acid sequence that has at least 70% similarity, at least 75% similarity, at
least
80% similarity, at least 90% similarity, at least 95% similarity, at least 97-
99%
similarity or even 100% similarity over the entire length of SEQ ID NO: 2.
The polypeptides of the invention include a polypeptide of Fig.
1 (SEQ ID NO: 2) (in particular the mature polypeptide) as well as
polypeptides
and fragments, particularly those which have a biological activity of
STAAU_R4,
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and also those which have at least 50% identity over 50 or more amino acids to
a polypeptide of SEQ ID NO: 2 or the relevant portion, preferably at least
55%,
60%, 70%, or 80% identity over 50 or more amino acids to a polypeptide of SEQ
ID NO: 2, more preferably at least 90% identity over 50 or more amino acids to
a
polypeptide of SEQ ID NO: 2 and still more preferably at least 95% identity
over
50 or more amino acids to a polypeptide of SEQ ID NO: 2 and yet still more
preferably at least 99% identity or exact identity over 50 or more amino acids
to
a polypeptide of SEQ ID NO: 2.
The polypeptides of the invention also include a polypeptide or
protein fragment that has at least 70%, 75%, 80% or 90% similarity, 95%
similarity
or even 97-99% similarity over 50 or more amino acids to a polypeptide of SEQ
ID NO: 2.
It is most preferred that a polypeptide of the invention is
derived from S. aureus, however, it may be obtained from other organisms of
the
same taxonomic genus. A polypeptide of the invention may also be obtained, for
example, from organisms of the same taxonomic family or order.
Fragments of STAAU_R4 also are included in the invention.
These fragments may include, for example, truncation polypeptides having a
portion of an amino acid sequence of Fig. 1 (SEQ ID NO: 2), a fragment or a
variant thereof, such as a continuous series of residues that includes an
amino-
and/or carboxyl-terminal amino acid sequence. Degradation forms of the
polypeptides of the invention produced by or in a host cell, particularly S.
aureus,
are also preferred. Further preferred are fragments characterized by
structural or
functional attributes such as fragments that comprise alpha-helix and alpha-
helix-
forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-
forming
regions, coil and coil-forming regions, hydrophilic regions, hydrophobic
regions,
alpha amphipathic regions, beta amphipathic regions, flexible regions, surface
forming regions, substrate binding region, and high antigenic index regions.
Fragments of STAAU R4 may be expressed as fusion proteins with other
proteins or protein fragments.
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Preferred fragments also include an isolated polypeptide
comprising an amino acid sequence having at least 10, 15, 20, 30, 39, 50, 100,
200, 250, 300 or or more contiguous amino acids from the amino acid sequence
of SEQ ID NO: 2.
Also preferred are biologically "active" fragments which are
those fragments that mediate activities of S. aureus STAAU_R4, including those
with a similar activity or an improved activity, or with a decreased
undesirable
activity. Also included are those fragments that are antigenic or immunogenic
in
an animal, especially in a human. Particularly preferred are fragments
comprising
domains that confer a function essential for viability of S. aureus.
Fragments of the polypeptides of the invention may be
employed for producing the corresponding full-length polypeptide by peptide
synthesis; therefore, these variants may be employed as intermediates for
producing the full-length polypeptides of the invention.
S. aureus Polynucleotides
It is an object of the invention to provide polynucleotides that
encode STAAU_R4 polypeptides, particularly polynucleotides that encode the
poljrpeptide herein designated S. aureus STAAU_R4.
In one aspect of the invention, a polynucleotide is provided
that comprises a region encoding a S. aureus STAAU_R4 polypeptide, the
polynucleotide comprising a sequence set out in SEQ ID NO: 1. Such a
polynucleotide encodes a full length STAAU R4 gene, a fragment or a variant
thereof. It is contemplated that this full-length gene is essential to the
growth
and/or survival of an organism which possesses it, such as S. aureus.
As a further aspect of the invention there are provided isolated
nucleic acid molecules encoding and/or expressing a fragment of a full-length
STAAU R4 polypeptide, particularly a S. aureus STAAU_R4 polypeptide, a
fragment or a variant thereof. Further embodiments of the invention include
biologically, diagnostically, prophylactically, clinically or therapeutically
useful
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polynucleotides, polypeptides, variants thereof, and compositions comprising
same.
A polynucleotide of the invention is obtained using S. aureus
cells as starting material, the nucleotide sequence information disclosed in
SEQ
ID N0:1, and standard cloning and screening methods, such as those for cloning
and sequencing chromosomal DNA fragments from bacteria. For example, to
obtain a polynucleotide sequence of the invention, such as the polynucleotide
sequence disclosed as in SEQ ID NO: 1, a library of clones of chromosomal DNA
of S. aureus in E. .coli or another suitable host is probed with a
radiolabeled
oligonucleotide, preferably a 17-mer or longer, derived from a partial
sequence.
Clones carrying DNA identical to that of the probe can be distinguished using
stringent hybridization conditions. As herein used, the terms "stringent
conditions"
and "stringent hybridization conditions" mean hybridization occurring only if
there
is at least 95% and preferably at least 97% identity between the sequences. A
specific example of stringent hybridization conditions is of an overnight
incubation
of a hybridization support (e.g., a nylon or nitrocellulose membrane) at
42°C in a
solution comprising: 1 X 10~ cpm/ml labeled probe, 50% formamide, 5x SSC
(150mM NaCI, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured,
sheared salmon sperm DNA, followed by washing the hybridization support in
0.1 x SSC at 65°C. Hybridization and wash conditions are well known to
those
skilled in the art and are exemplified in Sambrook, et al., Molecular Cloning:
A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),
particularly
Chapter 11 therein. Solution hybridization may also be used with the
polynucleotide sequences provided by the invention. By sequencing the
individual
clones thus identified by hybridization, it is possible to confirm the
identity of the
clone.
Alternatively, an amplification process can be utilized to isolate
the polynucleotide. In this approach, the sequence disclosed as SEQ ID N0:1 is
targeted by two o(igonucleotides, one identical to a sequence on the. coding
DNA
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strand at or upstream of the ATG initiation colon and the other which anneals
to
the opposite strand at or downstream of the stop colon. Priming from these
oligonucleotides in a polymerase chain reaction yields a full-length gene
coding
sequence. Such suitable techniques are described by Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2"d Edition, Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y. (1989).
In a further aspect, the present invention provides for an
isolated polynucleotide comprising or consisting of: (a) a polynucleotide
sequence
which has at least 60% identity, preferably at least 70% identity, more
preferably
at least 80% identity, more preferably at least 90% identity, yet more
preferably
at least 95%, most preferably at least 97-99% or exact identity, to that of
SEQ ID
NO: 1; (b) a polynucleotide sequence encoding a polypeptide which has at least
50% identity, preferably at least 55% identity, preferably at least 60%
identity,
more preferably at feast 70% identity, more preferably at least 80% identity,
more
preferably at least 90%, yet more preferably at least 95%, most preferably at
least
97-99% or exact identity to SEQ ID NO: 2 over the entire length of SEQ ID NO:
2; or the complement of a sequence of (a) or (b) above.
The invention provides a polynucleotide sequence identical
over its entire length to the coding sequence of SEQ ID N0:1. Also provided by
the invention is a coding sequence for a mature polypeptide or a fragment
thereof
by itself as well as a coding sequence for a mature polypeptide or a fragment
in
reading frame with another coding sequence, such as a sequence encoding a
leader or secretory sequence, a pre-, or pro-, or prepro-protein sequence. The
polynucleotide of the invention may also contain at least one non-coding
sequence, including for example, but not limited to at least one non-coding 5'
and
3' sequence, such as the transcribed but non-translated sequences, termination
signals (such as rho-dependent and rho-independent termination signals),
ribosome binding sites, Kozak sequences, sequences that stabilize or
destabilize
mRNAs, introns, and polyadenylation signals. The polynucleotide sequence may
also comprise additional coding sequence encoding additional amino acids. For
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example, a marker sequence that facilitates purification of the fused
polypeptide
can be encoded. In certain embodiments of the invention, the marker sequence
is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and
described in Gentz et al., Proc. Natl. Acad. Sci. 86: 821-824 (1989), or an HA
peptide tag [Wilson et al., Cell 37: 767 (1984)], both of which may be useful
in
purifying polypeptide sequences fused to them. Polynucleotides of the
invention
also include, but are not limited to, polynucleotides comprising a structural
gene
and its naturally associated sequences that control gene expression.
While it is most preferred that a polynucleotide of the invention
be derived from S. aureus, it may also be obtained from other organisms of the
same taxonomic genus. A polynucleotide of the invention may also be obtained,
for example, from organisms of the same taxonomic family or order.
Further preferred embodiments are polynucleotides encoding
S. aureus STAAU_R4 variants that have the amino acid sequence of S. aureus
STAAU_R4 polypeptide of SEQ ID NO: 2 in which several, a few, 10 to 50, 5 to
10, ~ to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified,
deleted
and/or added, in any combination. Especially preferred among these
polynucleotides are those encoding silent nucleotide alterations that do not
alter
the coding sequence or activities of S. aureus STAAU_R4 polypeptides they.
encode.
Preferred embodiments are polynucleotides encoding
polypeptides that retain substantially the same biological function or
activity as the
mature polypeptide encoded by a DNA of SEQ ID NO: 1.
In accordance with certain preferred embodiments of this
invention there are provided polynucleotides that hybridize, particularly
under
stringent conditions, to S. aureus STAAU_R4 polynucleotide sequences, such as
those polynucleotides in Fig. 1.
The polynucleotides of the invention are useful as
hybridization probes for RNA, cDNA and genomic DNA to isolate full-length
cDNAs and genomic clones encoding genes that have a high degree of sequence
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identity to the STAAU R4 gene. Such probes generally will comprise at least 15
to about 100 residues or base pairs, although such probes will preferably have
about 20 to 50 nucleotide residues or base pairs. Particularly preferred
probes are
about 20 to about 30 nucleotide residues or base pairs in length.
A coding region of a related STAAU_R4 gene from a bacterial
species other than S. aureus may be isolated by screening a library using a
DNA
sequence provided in SEQ ID NO: 1 to synthesize an oligonucleotide probe. A
labeled oligonucleotide having a sequence complementary to that of a gene of
the
invention is then used to screen a library of cDNA, genomic DNA or mRNA to
determine to which members) of the library the probe hybridizes.
There are several methods available and well known to those
skilled in the art to obtain full-length DNAs, or extend short DNAs, for
example
those based on the method of Rapid Amplification of cDNA Ends (RACE) [see,
for example, Frohman, et al., Proc. Natl. Acad. Sci. USA 85:8998-9002, 1988].
Recent modifications of the technique, exemplified by the MARATHONTM
technology (Clontech Laboratories Inc.) for example, have significantly
simplified
the search for longer cDNAs. In the MARATHON technology, cDNAs are
prepared from mRNA extracted from a chosen cell and an 'adaptor' sequence is
ligated onto each end. Nucleic acid amplification by PCR is then carried out
to
amplify the "missing" 5' end of the DNA using a combination of gene specific
and
adaptor specific oligonucleotide primers. The PCR reaction is then repeated
using
"nested" primers, that is, primers designed to anneal within the amplified
product
(typically an adaptor-specific primer that anneals further 3' in the adaptor
sequence and a gene-specific primer that anneals further 5' in the selected
gene
sequence). The products of this reaction can then be analyzed by DNA
sequencing and a full-length DNA constructed either by joining the product
directly
to the existing DNA to give a complete sequence, or by carrying out a separate
full-length PCR using the new sequence information for the design of the 5'
primer.
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The polynucleotides and polypeptides of the invention may be
employed, for example, as research reagents and materials for discovery of
treatments of and diagnostics for diseases, particularly human diseases, as
further discussed herein relating to polynucleotide assays.
The pofynucleotides of the invention that are oligonucleotides
derived from a sequence of SEQ ID N0:1 are useful for the design of PCR
primers in reactions to determine whether or not the polynucleotides
identified
herein in whole or in part are transcribed in bacteria in infected tissue.
That is, the
polynucleotides of the invention are useful for diagnosis of infection with a
bacterial strain carrying those sequences. It is recognized that such
sequences
also have utility in diagnosis of the stage of infection and type of infection
the
pathogen has attained.
The invention also provides polynucleotides that encode a
polypeptide that is the mature protein plus additional amino or carboxyl-
terminal
amino acids, or amino acids interior to the mature polypeptide. Such sequences
may play a role in processing of a protein from precursor to a mature form,
may
allow protein transport, may lengthen or shorten protein half-life or may
facilitate
manipulation of a protein for assay or production, among other things. As
generally is the case in vivo, the additional amino acids may be processed
away
from the mature protein by cellular enzymes.
A precursor protein, having a mature form of the polypeptide
fused to one or more prosequences may be an inactive form of the polypeptide.
When prosequences are removed such inactive precursors generally are
activated. Some or all of the prosequences may be removed before activation.
Generally, such precursors are called proproteins.
A polynucleotide of the invention thus may encode a mature
protein, a mature protein plus a leader sequence (which may be referred to as
a
preprotein), a precursor of a mature protein having one or more prosequences
that are not the leader sequences of a preprotein, or a preproprotein, which
is a
precursor to a proprotein, having a leader sequence and one or more
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prosequences, which generally are removed during processing steps that produce
active and mature forms of the polypeptide.
In addition to the standard A, G, C, T/U representations for
nucleotides, the term "N" may also be used in describing certain
polynucleotides
of the invention. "N" means that any of the four DNA or RNA nucleotides may
appear at such a designated position in the DNA or RNA sequence, except it is
preferred that N is not a nucleotide that when taken in combination with
adjacent
nucleotide positions, read in the correct reading frame, would have the effect
of
generating a premature termination codon in such reading frame.
For each and every polynucleotide of the invention there is
also provided a polynucleotide complementary to it.
Vectors. Host Cells, and Expression Systems
The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that are
genetically
engineered with vectors of the invention and the production of polypeptides of
the
invention by recombinant techniques. Cell-free translation systems can also be
employed to produce such proteins using RNAs derived from the DNA constructs
of the invention
Recombinant STAAU_R4 and bacteriophage polypeptides of
the present invention may be prepared by processes well known to those skilled
in the art from genetically engineered host cells comprising expression
systems.
Accordingly, in a further aspect, the present invention relates to expression
systems that comprise a STAAU R4 or bacteriophage polynucleotide or
polynucleotides of the present invention, to host cells which are genetically
engineered with such expression systems, and to the production of polypeptides
of the invention by recombinant techniques.
For recombinant production of a STAAU_R4 polypeptide of
the invention, host cells can be genetically engineered to incorporate
expression
systems or portions thereof or polynucleotides of the invention.
Representative
examples of appropriate hosts include bacterial cells (Gram positive and Gram
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negative), fungal cells, insect cells, animal cells and plant cells.
Polynucleotides
are introduced to bacteria by standard chemical treatment protocols, such as
the
induction of competence to take up DNA by treatment with calcium chloride
(Sambrook et al., supra). Introduction of polynucleotides into fungal (e.g.,
yeast)
host cells is effected, if desired, by standard chemical methods, such as
lithium
acetate-mediated transformation.
A great variety of expression systems are useful to produce
polypeptides of the invention. Such vectors include among others, chromosomal-
,
episomal- and virus-derived vectors. For example, vectors derived from
bacterial
plasmids, from bacteriophages, from transposons, from yeast episomes, from
insertion elements, from yeast chromosomal elements, from viruses, and from
vectors derived from combinations thereof, are useful in the invention.
Polypeptides of the invention are recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid or urea extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography, and
lectin chromatography. Well known techniques for refolding may be employed to
regenerate an active conformation when the polypeptide is denatured during
isolation and/or purification.
Diagnostic, Prognostic, Serotyping, and Mutation Assays
This invention is also related to the use of STAAU R4
polynucleotides and polypeptides of the invention for use as diagnostic
reagents.
Detection of S. aureus STAAU_R4 polynucleotides and/or polypeptides in a
eukaryote, particularly a mammal, and especially a human, will provide a
diagnostic method for diagnosis of disease, staging of disease or response of
an
infectious organism to drugs. Eukaryotes, particularly mammals, and especially
humans, particularly those infected or suspected to be infected with an
organism
comprising the S. aureus STAAU_R4 gene or protein, may be detected at the
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nucleic acid or amino acid level by a variety of well known techniques as well
as
by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or infected
individual's bodily materials. Polynucleotides from any of these sources,
particularly DNA or RNA, may be used directly for detection or may be
amplified
enzymatically by using PCR or any other amplification technique prior to
analysis.
RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same
ways. Using amplification, characterization of the species and strain of
infectious
or resident organism present in an individual, may be made by an analysis of
the
genotype of a selected polynucleotide of the organism. Deletions and
insertions
can be detected by a change in size of the amplified product in comparison to
a
genotype of a reference sequence selected from a related organism, preferably
a different species of the same genus or a different strain of the same
species.
Point mutations can be identified by hybridizing amplified DNA
to labeled STAAU R4 polynucleotide sequences. Perfectly or significantly
matched sequences can be distinguished from imperfectly or more significantly
mismatched duplexes by DNase or RNase digestion, for DNA or RNA
respectively, or by detecting differences in melting temperatures or
renaturation
kinetics. Polynucleotide sequence differences may also be detected by
alterations
in the electrophoretic mobility of polynucleotide fragments in gels as
compared to
a reference sequence. This may be carried out with or without denaturing
agents.
Polynucleotide differences may also be detected by direct DNA or RNA
sequencing. See, for example, Myers et al, (1985) Science 230, 1242. Sequence
changes at specific locations also may be revealed by nuclease protection
assays, such as RNase, V1 and S1 protection assay or a chemical cleavage
method. See, far example, Cotton et al., (1985) Proc. Natl. Acad. Sci., USA
85,
4397-4401.
In another embodiment, an array of oligonucleotide probes
comprising STAAU R4 nucleotide sequence or fragments thereof can be
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constructed to conduct efficient screening of, for example, genetic mutations,
serotype, taxonomic classification or identification. Array technology methods
are
well known and have general applicability and can be used to address a variety
of questions in molecular genetics including gene expression, genetic linkage,
and genetic variability [see, for example, Chee et al., (1996) Science 274,
610].
Thus in another aspect, the present invention relates to a
diagnostic kit which comprises: (a) a polynucleotide of the present invention,
preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof; (b)
a nucleotide sequence complementary to that of (a); (c) a polypeptide of the
present invention, preferably the polypeptide of SEQ ID NO: 2 or a fragment
thereof; or (d) an antibody to a polypeptide of the present invention,
preferably to
the polypeptide of SEQ ID NO: 2 or fragment thereof.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may
comprise a substantial component. Such a kit will be of use in diagnosing a
disease or susceptibility to a disease, among others.
This invention also relates to the use of STAAU R4
polynucleotides of the present invention as diagnostic reagents. Defection of
a
mutated form of a polynucleotide of the invention, preferably, SEQ ID NO: 1,
which is associated with a disease or pathogenicity will provide a diagnostic
tool
that can add to, or define, a diagnosis of a disease, a prognosis of a course
of
disease, a determination of a stage of disease, or a susceptibility to a
disease,
which results from under-expression, over-expression or altered expression of
the
polynucleotide. Organisms, parfiicularly infectious organisms, carrying
mutations
in such polynucleotide may be detected at the polynucleotide level by a
variety of
techniques, such as those described elsewhere herein.
The STAAU_R4 nucleotide sequences of the present
invention are also valuable for organism chromosome identification. The
sequence is specifically targeted to, and can hybridize with, a particular
location
on an organism's chromosome, particularly to a S. aureus chromosome. The
mapping of relevant sequences to chromosomes according to the present
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invention may be an important step in correlating those sequences with
pathogenic potential andlor an ecological niche of an organism and/or drug
resistance of an organism, as well as the essentiality of the gene to the
organism.
Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic map data. Such data may be found on-line in a sequence database. The
relationship between genes and diseases that have been mapped to the same
chromosomal region are then identified through known genetic methods, for
example, through linkage analysis (coinheritance of physically adjacent genes)
or
mating studies, such as by conjugation.
The differences in a polynucleotide and/or polypeptide
sequence between organisms possessing a first phenotype and organisms
possessing a different, second different phenotype can also be determined. (f
a
mutation is observed in some or all organisms possessing the first phenotype
but
not in any organisms possessing the second phenotype, then the mutation is
likely to be the causative agent of the first phenotype.
Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or infected
individual's bodily materials. Particularly DNA or polynucleotides, from any
of
these sources may be used directly for detection or may be amplified
enzymatically using PCR or other amplification technique with oligonucleotide
amplification primers derived from the polynucleotide sequence of S. aureus
STAAU R4. RNA, particularly mRNA, or RNA reverse transcribed to cDNA, is
also useful for diagnostics. Following amplification of a S. aureus STAAU_R4-
related polynucleotide from a sample, characterization of the species and
strain
of infecting or resident organism is made by an analysis of the amplified
polynucleotide relative to one or more reference pofynucleotides or sequences
relative to a standard from a related organism (i.e. a known strain of S.
aureus).
The invention further provides a process for diagnosing
bacterial infections such as those caused by S. aureus, the process comprising
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determining from a sample derived from an individual, such as a bodily
material,
an increased level of expression of a polynucleotide having a sequence
disclosed
in SEQ ID NO: 1 relative to a sample taken from a non-diseased individual.
Increased or decreased expression of a STAAU_R4 polynucleotide can be
measured using any one of the methods well known in the art for the
quantitation
of polynucleotides, such as, 'for example, PCR, RT-PCR, RNase protection,
Northern blotting and other hybridization methods, and spectrometry.
In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of STAAU_R4 polypeptide compared to
normal control tissue samples may be used to detect the presence of an
infection,
for example. Assay techniques that can be used to determine levels of a S.
aureus STAAU R4 polypeptide, in a sample derived from a host, such as a bodily
material, are well-known to those of skill in the art. Such assay methods
include
radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody
sandwich assays, antibody detection and ELISA assays.
Gridding and Polynucleotide Subtraction of S. aureus Genomic Seauences
The STAAU_R4 polynucleotides of the invention may be used
as components of polynucleotide arrays, preferably high density arrays or
grids.
These high density arrays are particularly useful for diagnostic and
prognostic
purposes. For example, a set of spots each comprising a different gene, and
further comprising a polynucleotide or polynucleotides of the invention, may
be
used for probing, such as hybridization or nucleic acid amplification, using a
probe
obtained or derived from a bodily sample, to determine the presence a
parfiicular
polynucleotide sequence or related sequence in an individual.
Antibodies Specific for S. aureus Peptides or Polypeptides
The STAAU_R4 polypeptides and polynucleotides of the
invention or variants thereof, or cells expressing them are useful as
immunogens
to produce antibodies immunospecific for such polypeptides or polynucleotides,
respectively.
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In certain preferred embodiments of the invention there are
provided antibodies against S. aureus STAAU_R4 polypeptides or
polynucleotides encoding them. Antibodies against STAAU R4-polypeptide or
STAAU_R4-polynucleotide are useful qfor treatment of infections, particularly
bacterial infections.
Antibodies generated against the polypeptides or
polynucleotides of the invention are obtained by administering the
poiypeptides
and/or polynucleotides of the invention or epitope-bearing fragments of either
or
both, analogues of either or both, or cells expressing either or both, to an
animal,
, preferably a nonhuman, using routine protocols. For preparation of
monoclonal
antibodies, any technique known in the art that provides antibodies produced
by
continuous cell line cultures is useful. Examples include various techniques,
such
as those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et
al.,
Immunology Today 4: 72 (1983); and Cole et al., pg. 96-96 in Monoclonal
Anbitodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (US
Patent No: 4,946,968) can be adapted to produce single chain antibodies to
poiypeptides or polynucleotides of this invention. Also, transgenic mice, or
other
mammals, are useful to express humanized antibodies immunospecific to the
polypeptides or polynucleotides of fhe invention.
When antibodies are administered therapeutically, the
antibody or variant thereof is preferably modified to make it less immunogenic
in
the individual. For example, if the individual is human the antibody is most
preferably "humanized," where the complementarity determining region or
regions
of the hybridoma-derived antibody has been transplanted into a human
monoclonal antibody, for example as described in Jones et al. (1986), Nature
321,
522-525 or Tempest et al., (1991 ) Biotechnology 9, 266-273.
Alternatively, phage display technology is useful to select
antibody genes with binding activities towards a STAAU_R4 polypeptide of the
invention. In one possible scheme, antibody fragments specific for S. aureus
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STAAU R4 are selected from an immune library of antibody genes expressed as
fusions with coat protein of filamentous phage. Alternatively, naive libraries
are
screened by phage display techniques to identify genes encoding antibodies
specified for STAAU_R4 or from naive libraries [McCafferty, et al., (1990),
Nature
348, 552-554; Marks, et al., (1992) Biotechnology 10, 969-783; a recent
reference
is de Haard et al. (1999) J. Biol. Chem. 274: 18218-18230]. The ability to
recover,
for various targets, antibodies with subnanomolar affinities obviates the need
for
immunization. The affinity of these antibodies can also be improved by, for
example, chain shuffling [Clackson et al., (1991) Nature 352: 628].
The above-described antibodies may be employed to isolate
or to identify clones expressing the polypeptides or polynucleotides of the
invention, for example to purify the polypeptides or polynucleotides by
immunoaffinity chromatography.
A variant polypeptide or polynucleotide of the invention, such
as an antigenically or immunologically equivalent derivative or a fusion
protein of
the polypeptide is also useful as an antigen to immunize a mouse or other
animal
such as a rat or chicken. A fused protein provides stability to the
polypeptide
acting as a carrier, or acts as an adjuvant or both. Alternatively, the
antigen is
associated, for example by conjugation, with an immunogenic carrier protein,
such
as bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid.
Alternatively, when antibodies are to be administered therapeutically,
alternatively
a' multiple antigenic polypeptide comprising multiple copies of the
polypeptide, or
an antigenically or immunologically equivalent polypeptide thereof may be
sufficiently antigenic to improve immunogenicity so as to obviate the use of a
' carrier.
In accordance with an aspect of the invention, there is
provided the use of a STAAU_R4 polynucleotide of the invention for therapeutic
or prophylactic purposes, in particular genetic immunization. The use of a
STAAU_R4 polynucleotide of the invention in genetic immunization preferably
employs a suitable delivery method such as direct injection of plasmid DNA
into
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muscles [Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum.
Gene
Ther. (1983) 4: 419], delivery of DNA complexed with specific protein carriers
[Wu
et al., JBiol Chem. (1989) 264: 16985], coprecipitation of DNA with calcium
phosphate [Benvenisty and Reshef, Proc. Natl. Acad. Sci. USA, (1986) 83:
9551],
encapsulation of DNA in various forms of liposomes [Kaneda et al., Science
(1989) 243: 375], particle bombardment [Tang et al., Nature (1992) 356:152,
Eisenbraun et al., DNA Cell Biol (1993) 12: 791] or in vivo infection using
cloned
retroviral vectors [Seeger et al., Proc. Natl, Acad. Sci. USA (1984) 81:
5849].
Antagonists and Agonists: Assays and Molecules
The invention is based in part on the discovery that
STAAU_R4 is a target for the bacteriophage 3A ORF 33 inhibitory factor.
Applicants have recognized the utility of the interaction in the development
of
antibacterial agents. Specifically, the inventors have recognized that 1)
STAAU R4 is a critical target for bacterial inhibition; 2) 3A ORF 33 or
derivatives
1'5 or functional mimetics thereof are useful for inhibiting bacterial growth;
and 3) the
interaction between STAAU R4 or fragment thereof of S. aureus and 3A ORF 33
may be used as a target for the screening and rational design of drugs or
antibacterial agents. In addition to methods of directly inhibiting STAAU_R4
activity, methods of inhibiting STAAU R4 expression are also attractive for
antibacterial activity.
In several embodiments of the invention, there are provided
methods for identifying compounds which bind to or otherwise interact with and
inhibit or activate an activity or expression of a polypeptide and/or
poiynucleotide
of the invention comprising: contacting a polypeptide and/or polynucleotide of
the
invention with a compound to be screened under conditions to permit binding to
or other interaction between the compound and the polypeptide and/or
polynucleotide to assess the binding to or other interaction with the
compound,
such binding or interaction preferably being associated with a second
component
capable of providing a detectable signal in response to the binding or
interaction
of the polypeptide and/or polynucleotide with the compound; and determining
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whether the compound binds to or otherwise interacts with and activates or
inhibits an activity or expression of the polypeptide and/or polynucleotide by
detecting the presence or absence of a signal generated from the binding or
interaction of the compound with the polypeptide and/or polynucleotide.
Potential antagonists include, among others, small organic
molecules, peptides, polypeptides and antibodies that bind to a polynucleotide
and/or polypeptide of the invention and thereby inhibit or extinguish its
activity or
expression. Potential antagonists also may be small organic molecules, a
peptide,
a polypeptide such as a closely related protein or antibody that binds the
same
sites on a binding molecule, such as a binding molecule, without inducing
STAAU_R4-induced activities, thereby preventing the action or expression of S.
aureus STAAU_R4 polypeptides and/or polynucleotides by excluding S. aureus
STAAU R4 polypeptides and/or polynucleotides from binding.
Potential antagonists also include a small molecule that binds
to and occupies the binding site of the polypeptide thereby preventing binding
to
cellular, binding molecules, such that normal biological activity is
prevented.
Examples of small molecules include but are not limited to small organic
molecules, peptides or peptide-like molecules. Other potential antagonists
include
antisense molecules [see Okano, (1991 ) J. Neurochem. 56, 560; s.ee also
Oligodeoxynucleotides As Antisense Inhibitors of Gene Expression, CRC Press,
Boca Raton, FL (1988), for a description of these molecules]. Preferred
potential
antagonists include compounds related to and variants of 3A ORF 33 and of
STAAU R4. Other examples of potential polypeptide antagonists include
antibodies or, in some cases, oligonucleotides or proteins which are closely
related to the ligands, substrates, receptors, enzymes, etc., as the case may
be,
of the polypeptide, e.g., a fragment of the ligands, substrates, receptors,
enzymes, etc.; or small molecules which bind to the polypeptide of the present
invention but do not elicit a response, so that the activity of the
polypeptide is
prevented.
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Compounds may be identified from a variety of sources, for
example, cells, cell-free preparations, chemical libraries, and natural
product
mixtures. These substrates and ligands may be natural substrates and ligands
or
may be structural or functional mimetics. See, e.g., Coligan et al., Current
Protocols in Immunology 1 (2): Chapter 5 (1991 ). Peptide modulators can also
be
selected by screening large random libraries of all possible peptides of a
certain
length.
Compounds derived from the polypeptide sequence of 3A
ORF 33 could represent fragments representing small overlapping peptides
spanning the entire amino acid sequence of these ORFs. Fragments of 3A ORF
33 can be produced as described above.
Certain of the polypeptides of the invention are biomimetics,
functional mimetics of the natural S. aureus STAAU,R4 polypeptide. These .
functional mimetics are useful for, among other things, antagonizing the
activity
of S. aureus STAAU_R4 polypeptide or as an antigen or immunogen in a manner
described above. Functional mimetics of the polypeptides of the invention
include
but are not limited to truncated polypeptides. Polynucleotides encoding each
of
these functional mimetics may be used as expression cassettes to express each
mimetic polypeptide. It is preferred that these cassettes comprise 5' and 3'
restriction sites to allow for a convenient means to ligate the cassettes
together
when desired. It is further preferred that these cassettes comprise gene
expression signals known in the art or described elsewhere herein.
Screening Assays According to the Invention
It is desirable to devise screening methods to identify
compounds which stimulate or which inhibit the function of the STAAU_R4
polypeptide or polynucleotide of the invention. Accordingly, the present
invention
provides for a method of screening compounds to identify those that modulate
the
function of a polypeptide or polynucleotide of the invention. In general,
antagonists may be employed for therapeutic and prophylactic purposes. It is
contemplated that an agonist of STAAU_R4 may be useful, for example, to
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enhance the growth rate of bacteria in a sample being cultured for diagnostic
or
other purposes.
It has been determined that STAAU_R4 is a target for
bacteriophage 3A ORF 33 product, which acts as an inhibitory factor.
Applicants
have recognized the utility of the interaction in the development of
antibacterial
agents. Polypeptide and/or polynucleotide targets such as STAAU R4 are
critical
targets for bacterial inhibition. S. aureus bacteriophage 3A ORF 33 or
derivatives
or functional mimetics thereof are useful for inhibiting bacterial growth and
the
interaction, binding, inhibition and/or activation which occurs between
polypeptides, such as for example STAAU~R4 of S. aureus and 3A ORF 33 may
be used for the screening and rational design of drugs or antibacterial
agents. In
addition to methods for directly inhibiting a target such as STAAU R4
activity,
methods of inhibiting a target such as STAAU_R4 expression are also attractive
for antibacterial activity.
In preferred embodiments, the method involves the interaction
of an inhibitory ORF product or fragment thereof with the corresponding
bacterial
target or fragment thereof that maintains the interaction with the ORF product
or
fragment. Interference with the interaction between the components can be
monitored, and such interference is indicative of compounds that may inhibit,
activate, or enhance the activity of the target molecule.
a. Binding Assays
There are a number of methods of examining binding of a
candidate compound to a protein target such as STAAU_R4 and a polypeptide
comprising amino acid sequence of SEQ ID NO: 2, or fragment thereof.
Screening methods that measure the binding of a candidate compound to a
STAAU_R4 polypeptide or polynucleotide, or to cells or supports bearing the
polypeptide or a fusion protein comprising the polypeptide, by means of a
label
directly or indirectly associated with the candidate compound, are useful in
the
invention.
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The screening method may involve competition for binding of
a labeled competitor such as 3A ORF 33 or a fragment that is competent to bind
STAAU_R4 or fragment thereof.
Non-limiting examples of screening assays in accordance with
the present invention include the following [Also reviewed in Sittampalam et
al.
1997 Curr. Opin. Chem. Biol. 3:384-91]:
i.) Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
A method of measuring inhibition of binding of two proteins
using fluorescence resonance energy transfer [FRET; de Angelis, 1999,
Physiological Genomics]. FRET is a quantum mechanical phenomenon that
occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in
close proximity (usually < 100 A of separation.) if the emission spectrum of D
overlaps with the excitation spectrum of A. Variants of the green fluorescent
protein (GFP) from the jellyfish Aequorea~victoria are fused to a polypeptide
or
protein and serve as D-A pairs in a FRET scheme to measure protein-protein
interaction. Cyan (CFP: D) and yellow (YFP: A) fluorescence proteins are
linked
with STAAU_R4 polypeptide, or a fragment thereof and a 3A ORF 33 polypeptide
respectively. Under optimal proximity, interaction between the STAAU_R4
polypeptide and a 3A ORF 33 polypeptide causes a decrease in intensity of CFP
fluorescence concomitant with an increase in YFP fluorescence.
The addition of a candidate modulator to the mixture of
appropriately labeled STAAU_R4 and 3A ORF 33 polypeptide, will result in an
inhibition of energy transfer evidenced by, for example, a decease in YFP
fluorescence at a given concentration of 3A ORF 33 relative to a sample
without
the candidate inhibitor.
An extension of the FRET technology, termed time-resolved
FRET (TR-FRET or HTRF [homogeneous time-resolved energy transfer]) fends
itself particularly well to identification of protein-protein interactions in
the context
of high-throughput screening. In brief, TR-FRET constitutes a homogeneous
assay method based on the long=lived fluorescence of rare earth cryptates such
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as Europium (Eu) and amplification by nonradiative energy transfer to a
suitable
acceptor such as allophycocyanin (APC). The TR-FRET principle allows double
discrimination of the emitted signal through temporal and spectral
selectivity.
Since the lifetime of fluorescence emission from APC (acceptor) contains a
contribution equal to the Eu (donor) lifetime in the presence of nonradiative
energy transfer, a long-lived APC acceptor signal can be resolved from its
natural
prompt fluorescence in the absence of energy transfer. Eu and APC are brought
into proximity via a pair of interacting molecules such as polypeptides. To
demonstrate interaction between the STAAU R4 polypeptide, or a fragment
thereof, and a 3A ORF 33 polypeptide, the respective polypeptide is labeled by
recombinant DNA methodology to contain an N- or C-terminal tag that is
recognized by a binding molecule which itself is conjugated to either Eu or
APC.
A variety of binding molecules may be employed, including an antibody
(directed
against an epitope) or streptavidin (directed against biotin). Alternatively,
one or
both of the interacting proteins is conjugated directly to either Eu or APC.
In one of several possible assay formats, STAAU_R4, or a
fragment thereof is expressed as a fusion with a polyhistidine tag and is
recognized by an anti-polyhistidine Eu antibody conjugate; 3A ORF 33 is
expressed as a fusion with GST and is detected by an anti-GST APC antibody
conjugate. Under optimal proximity and in the presence of the anti-
polyhistidine
and anti-GST antibody conjugates, interaction between STAAU_R4, or a fragment
thereof, and 3A ORF 33 induces nonradiative, time-resolved energy transfer
from Eu to APC, detected optimally at 665 nm.
The addition of a candidate modulator to the mixture of
appropriately labeled STAAU_R4 and 3A ORF 33 polypeptide, will result in an
inhibition of energy transfer evidenced by, for example, a decease in APC
fluorescence at a given concentration of 3A ORF 33 relative to a sample
without
the candidate inhibitor.
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ii.) Fluorescence polarization
Fluorescence polarization measurement is another useful
method to quantitate protein-protein binding. The fluorescence polarization
value
for a fluorescently-tagged molecule depends on the rotational correlation time
or
tumbling rate. Protein complexes, such as those formed by a S. aureus
STAAU_R4 polypeptide, or a fragment thereof associating with a fluorescently
labeled polypeptide (e.g., 3A ORF 33 or a binding fragment thereof), have
higher
polarization values than does the fluorescently labeled polypeptide. Inclusion
of
a candidate inhibitor of the STAAU R4 interaction results in a decrease in
fluorescence polarization relative to a mixture without the candidate
inhibitor if the
candidate inhibitor disrupts or inhibits the interaction of STAAU_R4 with its
polypeptide binding partner. It is preferred that this method be used to
characterize small molecules that disrupt the formation of polypeptide or
protein
complexes.
iii.) Surface plasmon resonance
Another powerful assay to screen for inhibitors of a protein:
protein interaction is surface plasmon resonance. Surface plasmon resonance is
a quantitative method that measures binding between two (or more) molecules
by the change in mass near a sensor surface caused by the binding of one
protein or other biomolecule from the aqueous phase (analyte) to a second
protein or biomolecule immobilized on the sensor(ligand). This change in mass
is measured as resonance units versus time after injection or removal of the
second protein or biomolecule (analyte) and is measured using a Biacore
Biosensor (Biacore AB) or similar device. STAAU_R4, or a polypeptide
comprising a fragment of STAAU_R4 , could be immobilized as a ligand on a
sensor chip (for example, research grade CM5 chip; Biacore AB) using a
covalent
linkage method (e.g. amine coupling in 10 mM sodium acetate [pH 4.5]). A blank
surface is prepared by activating and inactivating a sensor chip without
protein
immobilization. Alternatively, a ligand surface can be prepared by noncovalent
capture of ligand on the surface of the sensor chip by means of a peptide
affinity
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tag, an antibody, or biotinylation. The binding of 3A ORF 33 to STAAU_R4, or a
fragment thereof, is measured by injecting purified 3A ORF 33 over the ligand
chip surface. Measurements are performed at any desired temperature between
4°C and 37°C. Conditions used for the assay (i.e., those
permitting binding) are
as follows: 25 mM HEPES-KOH (pH 7.6), 150 mM sodium chloride, 15% glycerol,
1 mM dithiothreitol, and 0.001 % Tween 20 with a flow rate of 10 ul/min.
Preincubation of the sensor chip with candidate inhibitors will predictably
decrease the interaction between 3A ORF 33 and STAAU R4. A decrease in 3A
ORF 33 binding, detected as a reduced response on sensorgrams and
measured in resonance units, is indicative of competitive binding by the
candidate
compound.
iv.) Scintillation Proximity Assay
A scintillation proximity assay (SPA) may be used to
characterize the interaction between a S. aureus STAAU_R4 polypeptide, or a
fragment thereof, for example comprising the amino acid sequence of SEQ ID
NO: 2 and another polypeptide. The SPA relies in a solid-phase substrate, such
as beads or the plastic of a microtitre plate, into which a scintillant has
been
incorporated. For the assay, the target protein, for example a S. aureus
STAAU R4 polypeptide, is coupled to the beads or to the surface of the plate,
either covalently through activated surface chemistries or non-covalently
through
a peptide affinity tag, an antibody, or biotinylation. Addition of a
radiolabeled
binding polypeptide, for example [3zP]-radiolabeled 3A ORF 33 , results in
close
proximity of the radioactive source molecule to the scintillant. As a
consequence,
the radioactive decay excites the scintillant contained within the bead or
within the
plastic of the plate and detectable light is emitted. Compounds that prevent
the
association between immobilized S. aureus STAAU R4 polypeptide and
radiolabeled 3A ORF 33 will diminish the scintillation signal. The SPA thus
represents an example of an ideal technology with which to screen for
inhibitors
of the STAAU_R4-3A ORF 33 interactions because it is readily adapted to high-
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throughput, automated format and because of its sensitivity for detection of
protein-protein interactions with Kovalues in the micromolar to nanomolar
ranges.
v.) Bio Sensor Assay
ICS biosensors have been described by AMBRI (Australian
Membrane Biotechnology Research Institute; http//www.ambri.com.au/). In this
technology, the self-association of macromolecules such as STAAU R4, or
fragment thereof, and bacteriophage 3A ORF 33 or fragment thereof, is coupled
to the closing of gramacidin-facilitated ion channels in suspended membrane
bilayers and hence to a measurable change in the admittance (similar to
impedence) of the biosensor. This approach is linear over six order of
magnitude
of admittance change and is ideally suited for large scale, high through-put
screening of small molecule combinatorial libraries.
vi.) Phage display
Phage display is a powerful assay to measure protein:protein
interaction. In this scheme, proteins or peptides are expressed as fusions
with
coat proteins or tail proteins of filamentous bacteriophage. A comprehensive
monograph on this subject is Phage Display of Peptides and Proteins. A
Laboratory Manual edited by Kay et al. (1,996) Academic Press. For phages in
the
Ff family that include M13 and fd, gene III protein and gene VIII protein are
the
most commonly-used partners for fusion with foreign protein or peptides.
Phagemids are vectors containing origins of replication both for plasmids and
for
bacteriophage. Phagemids encoding fusions to the gene III or gene VIII can be
rescued from their bacterial hosts with helper phage, resulting in the display
of the
foreign sequences on the coat or at the tip of the recombinant phage.
In one example of a simple assay, purified recombinant
STAAU R4 protein, or fragment thereof, could be immobilized in the wells of a
microtitre plate and incubated with phages displaying a 3A ORF 33 sequence in
fusion with the gene III protein. Washing steps are performed to remove
unbound
phages and bound phages are detected with monoclonal antibodies directed
against phage coat protein (gene VIII protein). An enzyme-linked secondary
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antibody allows quantitative detection of bound fusion protein by
fluorescence,
chemiluminescence, or colourimetric conversion. Screening for inhibitors is
performed by the incubation of the compound with the immobilized target before
the addition of phages. The presence of an inhibitor will specifically reduce
the
signal in a dose-dependent manner relative to controls without inhibitor.
It is important to note that in assays of protein-protein
interaction, it is possible that a modulator of the interaction need not
necessarily
interact directly with the domains) of the proteins that physically interact.
It is also
possible that a modulator will interact at a location removed from the site of
protein-protein interaction and cause, for example, a conformational change in
the
STAAU_R4 polypeptide. Modulators (inhibitors or agonists) that act in this
manner
can be termed allosteric effectors and are of interest since the change they
induce
may modify the activity of the STAAU R4 polypeptide.
Testing for inhibitors is performed by the incubation of the
7 5 compound with the reaction mixtures. The presence of an inhibitor will
specifically
reduce the signal in a dose-dependent manner relative to controls without
inhibitor. Compounds selected for their ability to inhibit interactions
between
STAAU R4-3A ORF 33 are further tested in secondary screening assays.
b. Bacterial growth inhibition .
Compounds selected for their ability to inhibit interactions
between a STAAU R4 polypeptide and 3A ORF 33 polypeptide or to inhibit the
STAAU_R4 activity can be further tested in functional assays of bacterial
growth.
Cultures of S. aureus are grown in the presence of varying concentrations of a
candidate compound added directly to the medium or using a vehicle which is
appropriate for the delivery of the compound into the cell. For compounds that
correspond to polypeptides, the nucleotide sequence encoding said polypeptides
can be cloned into a S. aureus expression vector containing an inducible
promotor. The expression of the polypeptide could be induced following
transfection of cells. For example, the polypeptide may include,. but is not
limited
to different 3A ORF 33 -derived fragments.
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Following the induction of expression or the addition of
compound, the cultures are then incubated for an additional 4 h at
37°C. During
that period of time, the effect of inhibitors on bacterial cell growth may be
monitored at 40 min intervals, by measuring, for example, the ODSSS and the
number of colony forming units (CFU) in the cultures. The number of CFU is
evaluated as follows: cultures are serially diluted and aliquots from the
different
cultures are plated.out on agar plates.Following incubation overnight at
37°C, the
number of colonies is counted. Non-treated cultures of S. aureus are included
as
negative control.
In another aspect, the present invention relates to a screening
kit for identifying agonists, antagonists, ligands, receptors, substrates,
enzymes,
etc. for a polypeptide and/or polynucleotide of the present invention; or
compounds which decrease or enhance the production of such polypeptides
and/or polynucleotides, which , comprises: (a) a polypeptide and/or a
polynucleotide of the present invention; (b) a recombinant cell expressing a
polypeptide and/or polynucleotide of the present invention; (c) a cell
membrane
associated with a polypeptide and/or polynucleotide of the present invention;
or
(d) an antibody to a polypeptide and/or polynucleotide of the present
invention;
which polypeptide is preferably that of SEQ ID NO: 2, and for which the
polynucleotide is preferably that of SEQ ID NO: 1.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may
comprise a substantial component.
It will be readily appreciated by the skilled artisan that a
polypeptide and/or polynucleotide of the present invention may also be used in
a
method for the structure-based design of an agonist, antagonist or inhibitor
of the
polypeptide and/or polynucleotide, by: (a) determining .in the first instance
the
three-dimensional structure of the polypeptide and/or polynucleotide, or
complexes thereof; (b) deducing the three-dimensional structure for the likely
reactive site(s), binding sites) or motifs) of an agonist, antagonist or
inhibitor; (c)
synthesizing candidate compounds that are predicted to bind to or react with
the
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deduced binding site(s), reactive site(s), and/or motif(s); and (d) testing
whether
the candidate compounds are indeed agonists, antagonists or inhibitors. It
will be
further appreciated that this will normally be an iterative process, and this
iterative
'process may be performed using automated and computer-controlled steps.
Each of the polynucleotide sequences provided herein may be
used in the discovery and development of antibacterial compounds. The encoded
protein, upon expression, can be used as a target for the screening of
antibacterial drugs. Additionally, the polynucleotide sequences encoding the
amino terminal regions of the encoded protein or Shine-Dalgarno or other
sequence that facilitate translation of the respective mRNA can be used to
construct antisense sequences to control the expression of the coding sequence
of interest.
The invention also provides the use of the polypeptide,
polynucleotide, agonist or antagonist of the invention to interfere with the
initial
physical interaction between a pathogen or pathogens and a eukaryotic,
preferably mammalian, host that is responsible ,for sequelae of infection. In
particular, the molecules of the invention may be used: in the prevention of
adhesion of bacteria, in particular Gram positive and/or Gram negative
bacteria,
to eukaryotic, preferably mammalian, extracellular matrix proteins on in-
dwelling
devices or to extracellular matrix proteins in wounds; to block bacterial
adhesion
between eukaryotic, preferably mammalian, extracellular matrix proteins and
bacterial STAAU R4 proteins that mediate tissue damage and/or; to block the
normal progression of pathogenesis in infections initiated other than by the
implantation of in-dwelling devices or by other surgical techniques. In
accordance
with yet another aspect of the invention, there are provided STAAtJ_R4
antagonists, preferably bacteriostatic or bacteriocidal antagonists.
The antagonists of the invention may be employed, for
instance, to prevent, inhibit and/or treat diseases.
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Compositions, kits and administration
In a further aspect of the invention there are provided
compositions comprising a STAAU R4 polynucleotide and/or a S. aureus
STAAIJ_R4 polypeptide for administration to a cell or to a multicellular
organism.
The present invenfiion provides for pharmaceutical
compositions comprising a therapeutically effective amount of a polypeptide
and/or polynucleotide, such as the soluble form of a polypeptide andlor
polynucleotide of the present invention, antagonist peptide or small molecule
compound, in combination with a pharmaceutically acceptable carrier or
excipient.
Such carriers include, but are not limited to, saline, buffered saline,
dextrose,
water, glycerol, ethanol, and combinations thereof. The pharmaceutical
compositions may be administered in any efFective, convenient manner
including,
for instance, administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes
among others.
In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for example as a
sterile apueous dispersion, preferably isotonic.
Alternatively the composition may be formulated for topical
application for example in the form of ointments, creams, lotions, eye
ointments,
eye drops, ear drops, mouthwash, impregnated dressings and sutures and
aerosols, and may contain appropriate conventional additives, including, for
example, preservatives, solvents to assist drug penetration, and emollients in
ointments and creams. Such topical formulations may also contain compatible
conventional carriers, for example cream or ointment bases, and ethanol or
oleyl
alcohol for lotions. Such carriers may constitute from about 1 % to about 98%
by
weight of the formulation; more usually they will constitute up to about 80%
by
weight of the formulation. Alternative means for systemic administration
include
transmucosal and transdermal administration using penetrants such as bile
salts
or fusidic acids or other detergents. In addition, if a polypeptide or other
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compounds of the present invention can be formulated in an enteric or an
encapsulated formulation, oral administration may also be possible.
Administration of these compounds may also be topical and/or localized, .in
the
form of salves, pastes, gels, and the like.
For administration to mammals, and particularly humans, it is
expected that the daily dosage level of the active agent will be from 0.01
mg/kg
to 10 mg/kg, typically around 1 mg/kg. The physician in any event will
determine
the actual dosage that will be most suitable for an individual and will vary
with the
age, weight and response of the particular individual. The above dosages are
exemplary of the average case. There can, of course, be individual instances
where higher or lower dosage ranges are merited, and such are within the scope
of this invention.
As used herein, the term "in-dwelling device".refers to surgical
implants, prosthetic devices and catheters, i.e., devices that are introduced
to the
body of an individual and remain in position for an extended time. Such
devices
include, but are not limited to, artificial joints, heart valves, pacemakers,
vascular
grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters,
continuous ambulatory peritoneal dialysis (CAPD) catheters.
The composition of the invention may be administered by
injection to achieve a systemic effect against relevant bacteria shortly
before
insertion of an in-dwelling device. Treatment may be continued after surgery
during the in-body time of the device. In addition, the composition could also
be
used to broaden perioperative cover for any surgical technique to prevent
bacterial wound infections, especially S. aureus wound infections.
Many orthopedic surgeons consider that humans with
prosthetic joints should be considered for antibiotic prophylaxis before
dental
treatment that could produce a bacteremia. Deep infection is a serious
complication sometimes leading to loss of the prosthetic joint and is
accompanied
by significant morbidity and mortality. It may therefore be possible to extend
the
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use of the active agent as a replacement for prophylactic antibiotics in this
situation.
In addition to the therapy described above, the compositions
of this invention may be used generally as a wound treatment agent to prevent
adhesion of bacteria to matrix proteins exposed in wound tissue and for
prophylactic use in dental treatment as an alternative to, or in conjunction
with,
antibiotic prophylaxis.
Alternatively, the composition of the invention may be used to
bathe an indwelling device immediately before insertion. The active agent will
preferably be present at a concentration of 1 mg/ml to 10mg/ml for bathing of
wounds or indwelling devices.
A vaccine composition is conveniently in injectable form.
Conventional adjuvants may be employed to enhance the immune response. A
suitable unit dose for vaccination is 0.5-5 microgram of antigen /kg, and such
dose is preferably administered 1-3 times and with an interval of 1-3 weeks.
With
the indicated dose range, no adverse toxicological effects will be observed
with
the compounds of the invention that would preclude their administration to
suitable individuals.
Sequence Databases, Seguences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable
information resource with which to determine their 2- and 3-dimensional
structures
as well as to identify further sequences of homology. These approaches are
most
easily facilitated by storing the sequence in a computer readable medium and
then using the stored data in a known macromolecular structure program or to
search a sequence database using well-known searching tools, such as GCC.
The polynucleotide and polypeptide sequences of the
invention are particularly useful as components in databases useful for search
analyses as well as in sequence analysis algorithms. As used in this section
entitled "Sequence Databases, Sequences in a Tangible Medium, and
Algorithms," the terms "polynucleotide of the invention" and "polynucleotide
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sequence of the invention" mean any detectable chemical or physical
characteristic of a polynucleotide of the invention that is or may be reduced
to or
stored in a tangible medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan data
therefrom, called bases, and mass spectrographic data. As used in this section
-entitled Databases and Algorithms, the terms "polypeptide of the invention"
and
"polypeptide sequence of the invention" mean any detectable chemical or
physical
characteristic of a polypeptide of the invention that is or may be reduced to
or
stored in a tangible medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan data
therefrom, and mass spectrographic data.
The invention provides a computer readable medium having
stored thereon polypeptide sequences of the invention and/or polynucleotide
sequences of the invention. The computer readable medium can be any
composition of matter used to store information or data, including, for
example,
commercially available floppy disks, tapes, chips, hard drives, compact disks,
and
video disks.
In a preferred embodiment of the invention there is provided
a computer readable medium having stored thereon a member selected from the
group consisting of: a polynucleotide comprising the sequence of SEQ 1D N0:1;
a polypeptide comprising the sequence of SEQ ID N0:2; a set of polynucleotide
sequences wherein at least one of said sequences comprises the sequence of
SEQ ID N0:1: a set of polypeptide sequences wherein at feast one of said
sequences comprises the sequence of SEQ ID N0:2; a data set representing a
polynucleotide sequence comprising the sequence of SEQ ID N0:1; a data set
representing a polynucleotide sequence encoding a polypeptide sequence
comprising the sequence of SEQ ID N0:2; a polynucleotide comprising the
sequence of,SEQ ID N0:1; a polypeptide comprising the sequence of SEQ ID
N0:2; a set of polynucleotide sequences wherein at least one of said sequences
comprises the sequence of SEQ ID NO: 1; a set of polypeptide sequences
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wherein at least one of said sequences comprises the sequence of SEQ ID NO:
2; a data set representing a polynucleotide sequence comprising the sequence
of SEQ ID NO: 1; a data set representing a polynucleotide sequence encoding a
polypeptide sequence comprising the sequence of SEQ ID NO: 2.
All publications and references, including but not limited to
patents and patent applications, cited in this specification are herein
incorporated
by reference in their entirety as if each individual publication or reference
were
specifically and individually indicated to be incorporated by reference herein
as
being fully set forth. Any patent application to which this application claims
priority
is also incorporated by reference herein in its entirety in the manner
described
above for publications and references.
The present invention is illustrated in further detail by the
following non-limiting examples.
EXAMPLE 1
Identification of the inhibitory ORF from
Staphylococcus aureus bacteriophage 3A
The S. aureus propagating strain 3A (PS 3A obtained from the
Laboratory Center for Disease Control (CDC) Health Canada, Ottawa, Ontario)
was used as a host to propagate phages 3A (also obtained from CDC). The
phage was propagated using the agar layer method described by Swanstorm and
Adams [Swanstrom et al. (1951 ) Proc. Soc. Exptl. Biol. & Med. 78: 372-375].
Phage DNA was prepared from the purified phages as described in Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual, 2"a Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
3A ORF 33 (Fig. 2; SEQ ID NO: 4) was amplified by
polymerase chain reaction (PCR) from their respective phage genomic DNA. The
PCR products were gel purified and digested with BamHl-Hindlll. Digested PCR
products was then ligated into BamHl-Hindlll- digested pTM vector, a S. aureus
vector containing a kanamycin resistance selective marker (Fig. 3A), and used
to
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transform S. aureus strain RN4220 [Kreiswirth et al. (1983) Nature 305: 709-
712].
In the resulting vectors, pTM 3AORF33 (Fig.3A), expression of the phage ORF
is under the control of an arsenite-inducible promoter. Selection of
recombinant
clones was performed on Luria-Bertani (LB) agar plates containing 30 ~,g/ml of
kanamycin.
Sodium arsenite (NaAsO~) was used to induce gene
expression from the ars promoter/operator. The effect of expression of phage
ORF on bacterial cell growth was then evaluated in functional assays on solid
medium and in liquid medium. As shown in Fig. 3B, the induction of expression
of phage 3A ORF 33, by plating three independent transformants (clones 1 to 3)
on semi-solid medium containing 5 uM sodium arsenite, results in the
inhibition
of bacterial growth on solid medium compared to plating in the absence of
inducer
or plating of a control non-inhibitory ORF (phage 44AHJD ORF 114)
transformant.
As shown in Fig. 3C and D, the density of the liquid culture, as assessed by
colony forming units (CFU), for S. aureus clones harboring the 3A ORF 33
increased over time under non-induced conditions. Similar growth rates were
also
observed with transformants harboring a non-inhibitory ORF (labeled as 'non
killer' on the graphs) under both induced and non-induced conditions. Each
timepoint in both graphs represents the average obtained from three
independent
transformants of S. aureus.
The expression of 3A ORF 33 inhibits the bacterial growth as'
observed by the reduction in CFU with time for induced cultures. At 4 h
following
induction, the expression of 3A ORF 33 is cytocidal resulting in a 2.5 log
inhibition
reduction in the number of CFU compared to the number of CFU initially present
in the same culture (Fig. 3C). When colony plating was done in the absence of
kanamycin, the antibiotic necessary to maintain the selective pressure for the
plasmid encoding 3A ORF 33 (Fig. 3D), the extent of growth inhibition was
reduced, resulting in a 1 log cytocidal effect.
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EXAMPLE 2
Identification of a S. aureus protein targeted
by bacteriophage 3A ORF 33
To identify the S. aureus proteins) that interacts with inhibitory
ORF 33 of S. aureus bacteriophage 3A, GST-fusion or polyhistidine-fusion of 3A
ORF 33 were generated. The recombinant protein was purified and utilized to
make a GST/3A ORF 33 affinity column. Cellular extracts prepared from S.
aureus cells were incubated with the affinity matrix and the matrix was washed
with buffers containing increasing concentrations of salt and different
detergents.
The protein elution profile was assessed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). A protein of molecular mass ~ 40 kDa, identified
as PT40, was specifically eluted from the affinity matrix (Fig. 4 and 5) and
was not
detected in eluates from the GST negative control column (Fig. 6). Eluted
proteins
were further characterized to determine the identity of the interacting
protein and
to validate the interaction of the protein with 3A ORF 33 as described in
detail
below.
A. Generation of fusion 3A ORF 33 recombinant protein.
ORF 33 from bacteriophage 3A was sub-cloned into pGEX 4T-
1 (Pharmacia), an expression vector for in-frame translational fusions with
GST
and into pET15b MCS2, an expression vector in frame with a polyhistidine
(histag). Plasmid pTM 3AORF33 (Fig 3A) was purified on a Qiagen column and
digested with Hindlll, treated with Klenow fragment of E. coli DNA polymerase,
and digested with BamHl. The DNA restriction product containing 3A ORF 33 was
gel purified by QiAquick spin column (Qiagen) and ligated into pGEX 4T-1 and
pET15b MCS2 expression vectors (prepared by digestion with Sall, treatment
with
Klenow fragment of E. coli DNA polymerase, and digestion with BamHl, followed
by gel purification by QiAquick spin columns). Recombinant expression vectors
were identified by restriction enzyme analysis of plasmid minipreps. Large-
scale
DNA preparations were performed with Qiagen columns, and the resulting
plasmid was sequenced. Test expressions in E. coli cells (BL21 (DE3) Gold)
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containing the expression plasmids were performed to identify optimal protein
expression conditions. E. coli cells containing'the expression constructs were
grown in 2 L Luria-Bertani broth at 37°C to an OD6oo of 0.4 to 0.6 (1
cm
pathlength) and induced with 1 mM IPTG for the optimized time and at the
optimized temperature (30°C for 3hrs).
B. Fusion of GST/3A ORF 33 and MCS2/3A ORF 33 arotein purifications.
Cells containing GST/3A ORF 33 fusion protein were
suspended in 10 ml GST lysis buffer/liter of cell culture (GST lysis buffer:
20 mM
Hepes pH 7.2, 500 mM NaCI, 10 % glycerol, 1 mM DTT, 1 mM EDTA, 1 mM
benzamidine,. and 1 PMSF) and lysed by French Pressure cell followed by three
bursts of twenty seconds with an ultra-sonicator at 4°C. The lysate was
centrifuged at 4°C for 30 minutes at 10 000 rpm in a Sorval SS34 rotor.
The
supernatant was applied to a 4 ml glutathione sepharose column pre-
equilibrated
with lysis buffer and allowed to flow by gravity. The column was washed with
10
column volumes of lysis buffer and eluted in 4 ml fractions with GST elution
buffer
(20 mM Hepes pH 8.0, 500 mM NaCI, 10 % glycerol, 1 mM DTT, 0.1 mM EDTA,
and 25 mM reduced glutathione). The fractions were analyzed by 15% SDS-
PAGE (Laemmli) and visualized by staining with Coomassie Brilliant Blue 8250
stain to assess the amount of eluted GST/3A ORF 33 protein.
Cells containing MCS2/3A ORF 33 (histag) fusion proteins
were suspended in 10 ml lysis buffer/liter of cell culture with histag lysis
buffer (20
mM Hepes pH 7.5, 500 mM NaCI, 10% glycerol, 1 mM benzamidine, and 1 mM
PMSF) and lysed by passage through a French Pressure cell followed by three
bursts of twenty seconds with an ultra-sonicator at 4°C. The ~ lysate
was
centrifuged at 4°C for 30 minutes at 10 000 rpm in a Sorval SS34 rotor.
The
supernatant was applied to a 4 ml metal chelate sepharose column pre-
equilibrated with Ni2+, and washed with histag lysis buffer and allowed to
flow by
gravity. The column was washed with 10 column volumes of histag lysis buffer
containing 5 mM imidazole, 6 column volumes of histag lysis buffer containing
50
mM imidazole and eluted in 4 ml fractions with histag elution buffer (20 mM
Hepes
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pH 7.5, 500 mM NaCI, 500 mM imidazole, and 10% glycerol). The fractions were
analyzed by 15% SDS-PAGE (Laemmli) and visualized by staining with
Coomassie Brilliant Blue 8250 stain.
C. S. aureus extract preparation.
A Staphylococcus aureus extract was prepared from the cell
pellets using lysostaphin digestion followed by sonication and nuclease
digestion.
The cell pellet (2.9g) was suspended in 8 ml of 20~mM Hepes pH 7.5, 150 mM
NaCI, 10% glycerol, 10 mM MgS04, 10 mM CaCl2, 1 mM DTT, 1 mM PMSF, 1
mM benzamidine, 1000 units of lysostaphin, 0.5 mg RNase A, 750 units
micrococcal nuclease, and 375 units DNase 1. The cell suspension was
incubated at 37°C for 30 minutes, cooled to 4°C, and made up to
a final
concentration of 1 mM EDTA and 500 mM NaCI. The lysate was sonicated on ice
using three bursts of 20 seconds each. The lysate was centrifuged at 20 000
rpm
for 1 hr in a~Ti70 fixed angle Beckman rotor. The supernatant was removed and
dialyzed overnight in a 10 000 Mr dialysis ,membrane against Affinity
Chromatography Buffer (ACB; 20 mM Hepes pH 7.5, 10 % glycerol, 1 mM DTT,
and 1 mM EDTA) containing 100 mM NaCI, 1 mM benzamidine, and 1 mM PMSF.
The dialyzed protein extract was removed from the dialysis tubing and frozen
in
one ml aliquots at -70 °C.
D. Affinity column preparation.
GST, GST/3A ORF 33, and MCS2/3A ORF 33 were dialyzed
overnight against ACB containing 1 M NaCI. Protein concentrations were
determined by Bio-Rad Protein Assay and proteins were crosslinked to Affigel
10
resin (Bio-Rad) at protein/resin concentrations of 0, 0.1, 0.5, 1.0, and 2.0
mg/ml.
The crosslinked resin was sequentially incubated in the presence of
ethanolamine
and bovine serum albumin (BSA) prior to column packing and equilibration with
ACB containing 100 mM NaCI. S. aureus extracts were centrifuged at
4°C in a
micro-centrifuge for 15 minutes and diluted to 5 mg/ml with ACB containing 100
mM NaCI. 400 u1 of extract was applied to 40 u1 columns containing 0, 0.1,
0.5,
1.0, and 2.0 mg/ml ligand and ACB containing 100 mM NaCI (400 u1) was applied
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to an additional column containing 2.0 mg/ml ligand. The columns were washed
with ACB containing 100 mM NaCI (400 u1) and sequentially eluted with ACB
containing 0.1 % Triton X-100 and 100 mM NaCI (100 u1), ACB containing 1 M
NaCI (160 u1), and 1 % SDS (160 u1). 80 u1 of each eluate was resolved by 16
cm
14 % SDS-PAGE [Laemmli, U.K. (1970) Nature 227: 680-685] and the protein
was visualized by silver stain.
E. Affinity chromatography
One, candidate interacting protein of 40 kDa (PT40) was
observed in the 1 M NaCI eluates of the GST/3A ORF 33 chromatography
experiment (Fig. 4). The PT40 protein was also observed in the 1 M NaCI
eluates
of the MCS2/3A ORF 33 experiment (Fig. 5). This candidate protein was not
observed in the GST control affinity chromatography experiment. An estimation
of the relative abundance of PT40 protein in the Staphylococcus aureus extract
relies upon the assumption that nearly quantitative recovery of the candidate
interacting protein has occurred during the affinity chromatography. Affinity
chromatography experiments with the 5 mg/ml extract using ligands MCS2/3A
ORF 33 and GST/3A ORF 33 yielded similar quantities of PT40 with
approximately 25 ng of PT40 in the 1 M NaCI eluate of the 2.0 mg/ml column.
Affinity chromatography experiments with the 10 mg/ml extract using ligands
MCS2/3A ORF33 and GST/3A ORF 33 yielded similar quantities of PT40 with
approximately 70 ng of PT40 in the 1 M NaCI eluate of the 2.0 mg/ml column.
Although protein quantitation from silver stained SDS-PAGE gels is only
approximate, the estimated abundance of PT40 in the extracts is approximately
0.005%.
F. Identification of S, aureus STAAU R4 as an 3A ORF 33 interacting protein
The candidate protein PT40 was excised from the SDS-PAGE
gels and prepared for tryptic peptide mass determination by MALDI-ToF mass
spectrometry. [Qin, J., et al. (1997) Anal. Chem. 69, 3995-4001 ]. As
exemplified
in Fig. 7, high quality mass spectra were obtained. The PT40 proteins observed
in the two affinity chromatography experiments (eluates presented in Figs. 4
and
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5) were identical as determined by the masses of the tryptic peptides. The
PT40
band was identified as an open reading frame found in Contig782 of the
University of Oklahoma genome sequencing project database
(http://www.genome.ou.edu/staph.html) (herein referred as 'STAAU_R4'). G.
Bioinformatic analysis of STAAU R4
Sequence homology (BLAST) and Hidden Markov Model
(HMM) searches were then carried out using an implementation of both programs.
Downloaded public databases used for sequence analysis include:
i) non-redundant GenBank (nr) (www.ncbi.nlm.nih.gov)
ii) pdbaa database (www.ncbi.nlm.nih.gov)
iii) PRODOM (http://protein.toulouse.inra.fr/protein.html)
iv) Swissprot and TREMBL (www.expasy.ch)
v) Block plus and Block prints (http://blocks.fhcrc.org)
vi) Pfam (http://pfam.wustl.edu)
vii) Prosite (www.expasy.ch)
As shown in Fig.B, the results of the BLAST searches
performed on STAAU_R4 revealed similarity to fatty acid/phospholipid synthesis
PIsX protein from a variety of bacteria, including B. subtilis. In particular,
the result
of the global optimal alignment, presented in Fig. 9A, reveals a 53% identity
and
a 71 % similarity between the amino acid sequences of STAAU_R4 and B. subtilis
PIsX (gi~6686325~sp~P71018~PLSX BACSU: FATTY ACID/PHOSPHOLIPID
SYNTHESIS PROTEIN PLSX).
A software program was developed and used on the S, aureus
sequence to identify the start codon of the candidate ORF. The software scans
the primary nucleotide sequence for an appropriate start codon. Two possible
selections can be made for defining the nature of the start codon; a)
selection of
ATG or b) selection of ATG or GTG. The analysis also involved the
identification
of a ribosomal binding site (RBS) sequence that consists on finding the best
ungapped alignment of the Shine-dalgarno sequence 'aaaggaggf within the 25
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nucleotides long sequence located upstream of the start codon. Position
dependent scores are as followed:
aaaggaggt
233664652
As shown in Fig. 9B, the analysis of the S. aureus stop codon-
to-stop codon DNA region containing the STAAU R4 revealed the presence of
two possible ATG start codons associated with a predicted RBS sequence with
a score of 70 % of the perfect match. Based on the sequence alignment of
STAAU_R4 with other PIsX polypeptides, exemplified for 8. subtilis PIsX in
Fig.
9A, the start codon of STAAU_R4 is predicted to correspond to the first
methionine downstream of the RBS sequence identified in bold on Fig 9B.
EXAMPLE 3
Affinity blotting interaction between
STAAU R4 and 3A ORF 33.
The bacterial STAAU R4 and 3A ORF 33 interaction was also
confirmed by an affinity blotting assay. The probe was made of the STAAU_R4
protein by incubation of the heart muscle kinase (HMK)-tagged STAAU_R4 GST-
fusion with [32P]-ATP and HMK. The labeled probe was incubated with the
blocked membrane bearing the immobilized inhibitory phage ORF, and the
remaining signal after extensive washing is detected by exposure to X-ray
film.
A. Generation of GST/3A ORF 33 and GST/STAAU R4recombinant polypeptides
for affinity blotting analysis.
The pGEX-6P1 harboring GST in fusion with PreScission
protease cleavage site was purchased from Pharmacia Amersham Biotech. The
pGEX-6PK was obtained by cloning synthetic annealed oligonucleotides
corresponding to the heart muscle kinase (HMK) phosphorylation site (SEQ ID
NO: 5: 5'- GATCTCGTCGTGCATCTGTTGGATCCCCGGAATTCCCGGG-3' And
SEQ ID NO: 6: 5'-TCGACCCGGGAATTCCGGGGATCCAACAGAT-
GCACGACGA-3') into the unique BamHl-Sall [Kaelin et al. (1992) Cell 70: 351-
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364]. As a consequence, the HMK site is cloned in fusion with GST and is
followed by unique BamHl, EcoRl and Sall cloning sites. Insertion of the
duplex
was confirmed by DNA sequence analysis and the plasmid is referred to as
pGEX-6PK (Fig.10C).
As shown in Fig 10B, bacteriophage 3A ORF 33 was sub-
cloned into pGEX-6,PK by EcoRl and Xhol digestion from a pGADT7 vector
(Clontech Laboratories) harboring the 3A ORF 33 sequence. The DNA fragment
containing 3A ORF 33 was gel purified by Qiagen column and ligated in frame at
the 3'-end of the HMK labeling site into pGEX-6PK (which had been previously
digested with EcoRl and Saln to generate pGEX-6PK3A ORF 33. Recombinant
expression vectors were identified by restriction enzyme analysis of plasmid
minipreps, large-scale DNA preparations were performed with Qiagen columns.
As shown in Fig 10A, full-length, STAAU_R4 was amplified
from S. aureus genomic DNA by PCR using the sense strand primer targeting the
initiation codon preceded by a BamHl restriction site (5'-
cg atccATGGTTAAATTAGCAATTGAT-3') (SEQ ID NO: 7) and the antisense
oligonucleotide targeting the stop codon preceded by a Xhol restriction site
(5'-
ccgctcaaaTTACTCAT'TTGATTCACCTAC-3') (SEQ ID NO: 8). The initiation
codon used to amplified STAAU-R4 (SEQ ID NO: 1 ) corresponds to the predicted
starfi codon (Fig. 9B) based on the sequence alignment of STAAU-R4 with other
PIsX polypeptides, exemplified for B. subtilis PIsX in Fig. 9A. The digested
PCR
product was purified using the Qiagen PCR purification kit, ligated in frame
at the
3'-end of the HMK labeling site into BamHl and Sall digested pGEX-6PK vector
and used to transform E. coli strain BL21 (Amersham-Pharmacia). The sequence
integrity of STAAU R4 polypeptides fused to GST was verified directly by DNA
sequencing.
B. Purification and radiolabelina of GST-fusion proteins
Expression of the GSTl3A ORF 33 and GST/STAAU-R4
recombinant proteins from the plasmid pGEX-6P was induced by the addition of
1 mM of isopropyl-1-thio-~-D-galactosidase (IPTG) to a culture at OD6oo ~0.5.
The
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protein was expressed at 37°C for 3h and the cells were harvested by
centrifugation at 5,000 rpm in a JA-10 rotor (Beckman) for 15 min at
4°C. The
bacterial pellet was resuspended in 100 ml of ice-cold phosphate buffer saline
(PBS), divided into 4 aliquots and centrifuged as above. Each aliquot was
resuspended in 7 ml of STE (10 mM Tris pH 8.0, 1 mM EDTA, 150 mM NaCI and
0.1 mg/ml lysozyme). After incubation for 15 min, 10 mM dithiothreithol (Gibco
BRL) and 1.4% Sarkosyl (Sigma) were added and the lysed cells were submitted
to three bursts of sonication of 20 seconds on ice.
The cell lysate was centrifuged at 16,000 rpm in a JA-20 rotor
(Beckman) for 20 min at 4°C and the supernatant was treated with 2%
Triton X-
100 (Sigma) in a total volume of 20 ml for 30 min at room temperature with end-
over-end rotation. The lysate was centrifuged at 16,000 rpm in an JA-20 rotor
for
min at 4C° and the supernatant was incubated with 1 ml of glutathione
Sepharose-4B beads (Amersham-Pharmacia) for 60 min at 4C°. Beads
were
15 washed extensively with PBS, transferred to an eppendorf tube and the pure
proteins were cleaved from the GST portion by digestion with 40 Units of
PreScission protease (Pharmacia -Amersham) in 500 p1 of 50 mM Tris pH 7.0,
150 mM NaCI, 1 mM EDTA and 1 mM DTT. After 5 hrs incubation at 4C° with
end-over-end rotation, samples were centrifuged for 5 min at 13,000 g in a
20 microfuge and the supernatant was collected. Protein concentrations were
determined using the Biorad reagent according to the manufacturer's
instructions
(Biorad) and the proteins were stored at -80°C until use. Proteins were
analyzed
by 12% SDS-PAGE and visualized by Coommassie Brilliant Blue R-250 staining.
For radiolabeling with [32P]-ATP, 4 p,g of GST-cleaved
STAAU_R4 polypeptide were incubated with 50 Units of catalytic sub-unit of
cAMP dependent protein kinase "Heart Muscle Kinase" (Sigma) in a total volume
of 100 p,1 containing 200 mM Tris pH 7.5, 1 M NaCI, 120 mM MgCl2, 10 mM DTT
and 50 p.Ci of [y32P]-ATP (3000 ci/mmol) (NEN/Mandel) for 30 min at room
temperature. To remove free nucleotides, the proteins were applied to Sephadex
G50 NICK columns (Amersham-Pharmacia) and eluted with Z-buffer (25 mM
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Hepes pH 7.7, 12.5 mM MgCh, 20% Glycerol, 100 mM KCI & 1 mM DTT and the
incorporation of y32P-ATP was determined by counting on a liquid scintillation
counter.
C. Affinity blotting assay:
Increasing amounts (0.125, 0.250, 0.5 and 1.0 ~.g respectively
lanes 1, 2, 3 and 4 in Fig 1 OD) of GST-cleaved 3A ORF 33 were resolved on a
sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) gels
and blotted onto nitrocellulose membrane (Millipore). Immobilized proteins
were
denatured by incubating the membrane with 6M urea in HBB buffer (25 mM
Hepes-KOH pH 7.7, 25 mM NaCI, 5 mM MgCl2, 1 mM DTT) for 20 min at
4°C. The
proteins were renatured in situ by a progressive dilution of urea in HBB
buffer.
The membrane was blocked for at least 1 hr at 4°C with 5% milk in
HBB
supplemented with 0.05% NP-40 and for 45 min in 1 % milk in HBB supplemented
with 0.05% NP-40. The hybridization was performed for overnight at 4°C
in
hybridization buffer (20 mM Hepes-KOH pH 7.7, 75 mK KCI, 0.1 mM EDTA 2.5
mM MgCh, 0.05% NP-40 and 1 % milk) containing 250,000 cpm/ml of [32P]-ATP
.labelled STAAU_R4 polypeptide as a probe. The membrane was washed 3 times
for 10 min with hybridization buffer and exposed to x-ray film (Fig 10D). A
band
corresponding to the [32P]-ATP labelled STAAU_R4 polypeptide was detected at
, a position on the blot that corresponds to 3A ORF 33 (18 kDa).
CONCLUSION
By virtue of the interaction between the inhibitory
bacteriophage 3A ORF 33 and the STAAU R4, the STAAU R4 gene and its
gene product have thus been identified as novel bacterial targets for the
screening and identification of anti-bacterial agents and more particularly
for anti
S. aureus agents. The present invention also provides novel diagnosis,
prognosis
and therapeutic methods based on STAAU_R4, and/or bacteriophage 3A ORF
33 and/or a compound identified in accordance with the present invention.
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Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified
without
departing from the spirit and nature of the subject invention as defined in
the
appended claims.
