U.S. patent application number 12/563585 was filed with the patent office on 2010-01-21 for signal peptides, nucleic acid molecules and methods for treatment of caries.
This patent application is currently assigned to The Governing Council of the University of Toronto. Invention is credited to Dennis G. Cvitkovitch, Yi-Chen Cathy Huang, Celine Levesque.
Application Number | 20100016234 12/563585 |
Document ID | / |
Family ID | 36099404 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100016234 |
Kind Code |
A1 |
Huang; Yi-Chen Cathy ; et
al. |
January 21, 2010 |
SIGNAL PEPTIDES, NUCLEIC ACID MOLECULES AND METHODS FOR TREATMENT
OF CARIES
Abstract
Compounds that competitively inhibit binding of CSP to S. mutans
histidine kinase are provided. The compounds are preferably a
peptide or an antibody, and are preferably a derivative of [SEQ ID
NO:2], a fragment of [SEQ ID NO:2] or a derivative of a fragment of
[SEQ ID NO:2]. Methods of making these compounds and their use for
inhibiting the growth of S. mutans, for inhibiting dental caries,
and for improving dental health are also disclosed.
Inventors: |
Huang; Yi-Chen Cathy;
(Toronto, CA) ; Levesque; Celine; (Toronto,
CA) ; Cvitkovitch; Dennis G.; (Oakville, CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
The Governing Council of the
University of Toronto
Toronto
CA
|
Family ID: |
36099404 |
Appl. No.: |
12/563585 |
Filed: |
September 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11005636 |
Dec 6, 2004 |
7597895 |
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12563585 |
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09833017 |
Apr 10, 2001 |
6923962 |
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11005636 |
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60269949 |
Feb 20, 2001 |
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Current U.S.
Class: |
514/1.1 ;
435/320.1; 435/325; 435/69.1; 530/324; 536/23.7 |
Current CPC
Class: |
C07K 14/315 20130101;
A61K 38/00 20130101; A61K 8/64 20130101; C07K 14/3156 20130101;
A61Q 11/00 20130101; A61K 2039/505 20130101; A61K 8/606
20130101 |
Class at
Publication: |
514/12 ; 530/324;
536/23.7; 435/320.1; 435/325; 435/69.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/315 20060101 C07K014/315; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 5/06 20060101
C12N005/06; C12P 21/02 20060101 C12P021/02 |
Claims
1-20. (canceled)
21. A CSP analog comprising a polypeptide having 95% sequence
identity to SEQ ID NO: 11, wherein the polypeptide inhibits genetic
competence.
22. The CSP analog of claim 21, wherein the polypeptide comprises a
single amino acid deletion of SEQ ID NO: 11.
23. The CSP analog of claim 21, wherein the polypeptide comprises a
single amino acid substitution of SEQ ID NO: 11.
24. The CSP analog of claim 21, wherein the polypeptide comprises a
single amino acid addition of SEQ ID NO: 11.
25. The CSP analog of claim 21, wherein the polypeptide is selected
from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID
NO: 46, SEQ ID NO: 48, and SEQ ID NO: 51.
26. A pharmaceutical composition comprising a CSP analog of claim
21 and pharmaceutically acceptable carrier.
27. A method of inhibiting a biofilm comprising applying a
pharmaceutical composition according to claim 25 to the
biofilm.
28. A method of claim 27, further comprising administering an
antibiotic.
29. A nucleic acid comprising an oligonucleotide encoding a CSP
analog of claim 21.
30. An expression vector comprising the nucleic acid of claim
29.
31. A host cell comprising the expression vector of claim 30.
32. A method of expressing a CSP analog comprising culturing the
host cell of claim 31 in culture medium.
33. A fusion protein comprising the CSP analog of claim 21 and a
second peptide.
34. A fusion protein of claim 33, wherein the CSP analog is
selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43,
SEQ ID NO: 46, SEQ ID NO: 48, and SEQ ID NO: 51.
35. A nucleic acid molecule encoding the fusion protein of claim
33.
36. A pharmaceutical composition comprising the fusion protein of
claim 33 and a pharmaceutically acceptable carrier.
37. A method of inhibiting a biofilm comprising administering the
pharmaceutical composition of claim 36.
38. The method of claim 36 further comprising administering an
antibiotic.
39. An expression vector comprising the nucleic acid of claim
35.
40. A host cell comprising the expression vector of claim 39.
41. A method of producing a fusion protein comprising culturing the
host cell of claim 40 in culture medium
42. An antimicrobial composition comprising a) the CSP analog of
claim 21, and b) a surfactant, a disinfectant, or both a surfactant
and a disinfectant.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/005,636 filed Dec. 6, 2004, which is
a continuation-in-part of U.S. patent application Ser. No.
09/833,017, now U.S. Pat. No. 6,923,962, filed Apr. 10, 2001, which
application claims the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Patent Application No. 60/269,949 filed Feb. 20, 2001.
The disclosures of said applications are hereby incorporated herein
by reference.
BACKGROUND OF INVENTION
[0002] 1. Field of the Invention
[0003] The invention generally relates to compounds and methods
that inhibit or disrupt microbial biofilms involved in infections
in man and animals and in biofouling of surfaces susceptible to
microbial accumulation.
[0004] 2. Description of the Related Art
[0005] Bacteria often attach and accumulate on surfaces, enabling
them to resist removal and killing by mechanical and chemical
means. This can result in persistent and chronic infections and
fouling of devices that are in contact with liquids containing the
colonizing bacteria. Bacteria respond to signals resulting from the
proximity, density, and identity of microbial neighbors. Through
the process of quorum sensing (QS), bacteria can indirectly
determine population density by sensing concentration of a secreted
signal molecule (Bassler, 2002). The ability of bacteria to
communicate with one another by QS and behave collectively as a
group confers significant advantages, including more efficient
proliferation, better access to resources and niches, and a
stronger defense against competitors (Jefferson, 2004). Many QS
systems having various effects on bacterial cell physiology have
been studied. Examples include biofilm differentiation in
Pseudomonas aeruginosa (Davies et al., 1998), swarming motility in
Serratia liquefaciens (Eberl et al., 1999), competence development
in Streptococcus pneumoniae (Lee and Morrison, 1999) and
Streptococcus mutans (Li et al., 2001), and induction of virulence
factors in Staphylococcus aureus (Ji et al., 1995).
[0006] Controlling bacterial biofilms is desirable for almost every
human enterprise in which solid surfaces are introduced into
non-sterile aqueous environments. U.S. Pat. No. 6,024,958 describes
peptides that attempt to control biofilm formation by preventing
bacterial adherence to teeth. In addition to occurrence in dental
caries, medical examples of biofilm growth include cases involving
indwelling medical devices, joint implants, prostatitis,
endocarditis, and respiratory infections. In fact, the Centers for
Disease Control and Prevention (CDC; Atlanta, Ga.) estimate that
65% of human bacterial infections involve biofilms. Non-medical
examples of biofilm colonization are water and beverage lines,
cooling towers, radiators, aquaculture contamination, submerged
pumps and impellers, hulls of commercial, fishing and military
vessels and literally every situation where biofouling occurs. The
potential benefits of basic research focused at biofilm physiology
and genetics with the ultimate goal of controlling surface-mediated
microbial growth are limitless.
[0007] Interest in the study of biofilm-grown cells has increased
partly because biofilm growth provides a microenvironment for cells
to exist in a physical and physiological state that can increase
their resistance to antimicrobial compounds and mechanical forces
(reviewed in Costerton and Lewandowski, Adv Dent Res, 11: 192-195).
Growth in biofilms can also facilitate the transfer of genetic
information between different species (Christensen et al. Appl
Environ Microbiol, 64:2247-2255). Recent evidence suggests that
biofilm-grown cells may display a dramatically different phenotype
when compared with their siblings grown in liquid culture. In some,
this altered physiological state has been shown to result from gene
activation initiated by contact with surfaces (Finlay and Falkow.
Microbiol Molec Rev, 61:136-169) or from signal molecules produced
by the bacteria allowing them to sense the cell density (quorum
sensing) (Davies et al. Appl Environ Microbiol, 61:860-867).
Biofilms may also act as `genotypic reservoirs`, allowing
persistence, transfer and selection of genetic elements conferring
resistance to antimicrobial compounds.
[0008] Streptococcus mutans is the principal etiological agent of
dental caries in humans. None of the known types of S. mutans
antibiotics has satisfactorily controlled caries. There is a need
to identify new ways to control S. mutans induced caries.
SUMMARY OF THE INVENTION
[0009] In accordance with certain embodiments of the present
invention a compound is provided that competitively inhibits
binding of CSP [SEQ ID NO:1] to S. mutans histidine kinase [SEQ ID
NO:2]. In certain embodiments the compound is a peptide or an
antibody. In some embodiments the compound is a derivative of SEQ
ID NO:5, a fragment of SEQ ID NO:5 or a derivative of a fragment of
SEQ ID NO:5.
[0010] In accordance with certain embodiments of the present
invention methods of making an above-described compound are
provided. In still other embodiments of the invention methods are
provided in which an above-described compound is used for
inhibiting the growth of S. mutans, for inhibiting dental caries,
or for improving dental health. These and other embodiments,
features and advantages of the invention will be apparent with
reference to the following description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the schematic layout of the arrangement of the
genetic locus encoding the signal peptide precursor (ComC) [SEQ ID
NO:4], the histidine kinase (ComD) [SEQ ID NO:2] and the response
regulator (ComE) [SEQ ID NO:3]. Note that this arrangement is
different from other loci in related streptococci for the following
reasons: a). The comC gene [SEQ ID NO:4] is transcribed from its
own unique promoter, unlike the genes thus far described in other
streptococci that are arranged in an operon-like cluster with the
comC/DE genes being transcribed from a single promoter, b) The comC
gene [SEQ ID NO:4] is separated by 148 nucleotides from the comD
gene [SEQ ID NO:6].
[0012] FIG. 2 shows the nucleic acid molecule that is SEQ ID NO:4.
In a preferred embodiment, the figure shows a nucleic acid encoding
CSP (competence signal peptide [SEQ ID NO:5]). FIG. 2 also shows
histidine kinase [SEQ ID NO:6] sequences and response regulator
[SEQ ID NO:7] sequences. FIG. 2A. S. mutans comC gene [SEQ ID
NO:4]. Encodes a precursor to a signal peptide [SEQ ID NO:1]. FIG.
2B. S. mutans CSP encoding sequence [SEQ ID NO:5]. Encodes a
Competence Signal Peptide [SEQ ID NO:11]. FIG. 2C. S. mutans comD
gene [SEQ ID NO:6]. Encodes a response regulator that activates
transcription of a number of genes. FIG. 2D. S. mutans comE gene
[SEQ ID NO:7].
[0013] FIG. 3. Sequence of the deduced amino acid sequence of the
signal peptide [SEQ ID NO:1], histidine kinase [SEQ ID NO:2], and
response regulator [SEQ ID NO:3]. FIG. 3A. S. mutans ComC protein
(CSP Precursor) [SEQ ID NO:1]. FIG. 3B. S. mutans ComD protein
(Histidine Kindase) [SEQ ID NO:2]. FIG. 3C. S. mutans ComE protein
(Response Regulator) [SEQ ID NO:3].
[0014] FIG. 4. The deduced amino acid sequence of the signal
peptide precursor in various strains and its predicted cleavage
site. The original peptide is expressed as a 46 amino acid peptide
that is cleaved after the glycine-glycine residues to generate an
active signal peptide.
[0015] FIG. 5 shows the peptide that is SEQ ID NO:11. The synthetic
signal peptide is effective at inducing competence, biofilm
formation and acid tolerance in Streptococcus mutans.
[0016] FIG. 6 shows the natural activity of the signal/receptor
system functioning in vitro in model biofilms as determined by the
ability of various strains of S. mutans to accept donor plasmid DNA
conferring erythromycin resistance.
[0017] FIG. 7 is a table illustrating the effect of synthetic
peptide on genetic competence in S. mutans cells. Induction of
genetic transformation in Streptococcus mutans by synthetic
competence stimulating peptide (SCSP).
[0018] FIG. 8 is a list of the primers used to amplify the genes or
internal regions of the target genes by polymerase chain reaction
(PCR) for subsequent sequencing or inactivation.
[0019] FIG. 9 shows the ComCDE local region [SEQ ID NO:18 and SEQ
ID NO:19]. The ComC (first highlighted region; nucleotide 101 to
241), ComD (second highlighted region; nucleotides 383 to 1708) and
ComE (third highlighted region; nucleotides 1705 to 2457) proteins
are highlighted.
[0020] FIG. 10 shows the comX DNA sequence [SEQ ID NO:28], protein
sequence [SEQ ID NO:29], and the comX gene local region [SEQ ID
NO:30] with 100 bp included both upstream and downstream (promoter
is upstream). FIG. 10A. S. mutans comX gene [SEQ ID NO:28]. FIG.
10B. S. mutans ComX protein [SEQ ID NO:29]. FIG. 10C. S. mutans
comX gene local region [SEQ ID NO:30].
[0021] FIG. 11 shows the comA and comB nucleotide [SEQ ID NO:31]
and [SEQ ID NO:33] and amino acid sequences [SEQ ID NO:32] and [SEQ
ID NO:34]. ComA and ComB are the components of the CSP exporter.
FIG. 11A. S. mutans comA gene [SEQ ID NO:31]. FIG. 11B. S. mutans
ComA protein [SEQ ID NO:32]. FIG. 11C. S. mutans comB gene [SEQ ID
NO:33] FIG. 11D. S. mutans ComB protein [SEQ ID NO:34].
[0022] FIG. 12 illustrates the effect of synthetic peptide on acid
resistance tolerance in S. mutans comC deficient cells. Addition of
synthetic signal peptide (CSP) [SEQ ID NO:11] into the culture of
the comC mutant restored the ability of the mutant to survive a low
pH challenge when compared to the parent strain NG8.
[0023] FIG. 13 is a schematic representation of quorum sensing
circuit in S. mutans.
[0024] FIG. 14 shows the effect of different concentrations of H1
on genetic transformation of S. mutans wild-type UA159. Results are
expressed as the mean.+-.SE of three independent experiments.
[0025] FIG. 15 shows the effect of different concentrations of H1
on genetic transformation of S. mutans comD null mutant.
[0026] FIG. 16 shows the effect of different concentrations
(.mu.g/ml) of CSP and H1 on cell growth ofS. mutans wild-type UA159
in THYE at pH 5.5. Means OD.sub.600 values.+-.SE, Results represent
the average of three independent experiments.
[0027] FIG. 17 effect of different concentrations (.mu.g/ml) of CSP
and H1 on cell growth of S. mutans wild-type UA159 in THYE at pH
7.5. Means OD.sub.600 values.+-.SE. Results represent the average
of three independent experiments.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] In some Gram-positive bacteria (including Streptococcus
mutans), when a specific histidine kinase receptor located in the
cell membrane is disrupted, the cells become ineffective at
developing a biofilm. The cells growing in this biofilm environment
use a small peptide signal molecule to activate the receptor in
surrounding cells, thereby communicating the message to form a
biofilm. This same signal peptide and histidine kinase are also
involved in the induction of genetic competence, the cell's ability
to take up and incorporate DNA from its extracellular environment,
as well as that of acid tolerance, the cell's ability to survive pH
levels as low as pH 3.0. A mechanism that blocks the signal
molecule from activating the histidine kinase receptor molecule
provides a novel method for controlling microbial biofilms, either
alone or in combination with chemical or physical means.
[0029] We have identified a genetic locus in S. mutans consisting
of three genes that encode: 1) a peptide precursor [SEQ ID NO:1]
that is processed during export into a secreted 21-amino acid
peptide (CSP) [SEQ ID NO:11]; 2) a histidine kinase [SEQ ID NO:2]
that acts as a cell surface receptor activated by the peptide; 3) a
response regulator [SEQ ID NO:3] that activates a number of other
genes involved in genetic competence, biofilm formation, and acid
tolerance of S. mutans. These properties have been attributed to
the bacterium's ability to cause dental caries. Inactivation of any
of these three genes or impairment of interaction or activity of
any of their encoded proteins will disrupt the bacterium's ability
to take up foreign DNA, form biofilms, and tolerate acidic pH.
[0030] Streptococcus mutans is a resident of the biofilm
environment of dental plaque, a matrix of bacteria and
extracellular material that adheres to the tooth surface. Under
appropriate environmental conditions populations of S. mutans and
the pH of the surrounding plaque will drop. S. mutans, being among
the most acid tolerant organisms residing in dental plaque, will
increase its numbers in this acidic environment and eventually
become a dominant member of the plaque community. This situation
eventually leads to dissolution of the tooth enamel, resulting in
the development of dental caries. We control the accumulation and
acid tolerance of this bacterium to make it less able to cause
caries. We accomplish this by using inhibitors of an extracellular
signal peptide that promotes the expression of genes involved in S.
mutans biofilm formation and acid tolerance. Compounds are
disclosed that inhibit the action of the peptide. These inhibitors
can include peptides, antibodies, or other agents that specifically
inhibit the activation of the histidine kinase and the family of
genes activated as a result of the histidine kinase activation by
the signal molecule. Inhibitors include: modified structures of the
peptide where amino acids are removed from the N-- and/or COOH
terminal of the peptide and/or substitutions of internal amino acid
residues. We delete, one, two to 5, 6 to 10 and 10 to 15 amino
acids from the peptide (for example at either terminal) and measure
competitive inhibition of signal peptide binding to histidine
kinase (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids are
deleted and inhibition measured). Inhibitors also include
antibodies raised against the 21-amino acid CSP [SEQ ID NO:11]
alone or coupled to a larger molecule to increase immunogenicity.
We also test inhibitors described in Barrett et al. Proc. Natl.
Acad. Sci USA 95:5317-5322) and measure competitive inhibition of
signal peptide binding to histidine kinase.
[0031] In addition to identifying the genes encoding this
signaling/sensing system, we have identified and chemically
synthesized a 21-amino acid peptide [SEQ ID NO:11] that promotes
biofilm formation and acid tolerance of S. mutans. A survey of the
literature and genome databases reveals that genes similar to this
signal-receptor system are present in most Gram-positive bacteria,
and therefore an inhibitor, or family of related inhibitors may be
effective at inhibiting biofilm formation among a large group of
bacteria.
[0032] Treatment or prevention of dental caries comprises addition
of compounds that inhibit the stimulatory action of the 21-amino
acid peptide [SEQ ID NO:11] on biofilm formation and acid tolerance
of S. mutans. This is accomplished by delivery of these compounds
to the biofilm and/or to incorporate these inhibitors into
materials to control growth on surfaces. This includes delivery by
topical application, alone or in combination with other compounds
including toothpaste, mouthwash, food or food additives.
[0033] Streptococcus mutans is also implicated in causing infective
endocarditis. Inhibitors of biofilm formation, and hence
aggregation are useful in the treatment of these bacterial
infections as well.
Identification and Characterization of Competence Signal Peptide
(CSP), Histidine Kinase (UK) and Response Regulator (RR)
Competence Signal Peptide
[0034] An isolated CSP from S. mutans is provided in accordance
with certain embodiments of the present invention. Also provided in
accordance with certain embodiments of the present invention is a
recombinant isolated CSP [SEQ ID SEQ 11] peptide produced by a cell
including a nucleic acid molecule encoding CSP [SEQ ID NO:5]
operably linked to a promoter. Further provided in accordance with
certain embodiments of the present invention is an isolated nucleic
acid molecule encoding a CSP [SEQ ID NO:5]. The peptide we work
with is preferably chemically synthesized.
[0035] CSP-encoding nucleic acid molecules [SEQ ID NO:5] and
molecules having sequence identity or which hybridize to the
CSP-encoding sequence and which encode a peptide having CSP
activity (preferred percentages for sequence identity are described
below) as well as vectors including these molecules are provided in
accordance with various embodiments of the present invention. In
certain embodiments of the invention, CSP [SEQ ID NO:11] or
peptides having sequence identity (preferred percentages described
below) or which have CSP activity are provided. The nucleic acid
molecules and peptides disclosed herein may be from S. mutans and
they may be isolated from a native source, synthetic or
recombinant. CSP [SEQ ID NO:11] or peptides having sequence
identity, which have CSP activity, as prepared by the processes
described in this application, are also provided in accordance with
the present invention.
Histidine Kinase
[0036] In accordance with certain embodiments of the present
invention, an isolated HK [SEQ ID NO:2] from S. mutans is
disclosed. Also disclosed is a recombinant isolated HK polypeptide
produced by a cell including a nucleic acid molecule encoding HK
[SEQ ID NO:6] operably linked to a promoter. In another embodiment
of the invention an isolated nucleic acid molecule encoding a HK
polypeptide [SEQ ID NO:2] is disclosed.
[0037] HK-encoding nucleic acid molecules and molecules having
sequence identity or which hybridize to the HK-encoding sequence
[SEQ ID NO:6] and which encode a protein having HK activity
(preferred percentages for sequence identity are described below)
as well as vectors including these molecules are disclosed as part
of the present invention. In accordance with some embodiments of
the present invention, HK [SEQ ID NO:2] or polypeptides having
sequence identity (preferred percentages described below) or which
have HK activity are disclosed. The nucleic acid molecules and
polypeptides disclosed herein may be from S. mutans and they may be
isolated from a native source, synthetic or recombinant. Also
provided according to certain embodiments of the present invention
is HK [SEQ ID NO:2] or polypeptides having sequence identity, which
have HK activity, as prepared by the processes described in this
application.
Response Regulator
[0038] In accordance with certain embodiments of the present
invention an isolated RR [SEQ ID NO:3] from S. mutans is disclosed.
A recombinant isolated RR [SEQ ID NO:3] polypeptide produced by a
cell including a nucleic acid molecule encoding RR [SEQ ID NO:7]
operably linked to a promoter is provided according to certain
other embodiments of the present invention. Still other embodiments
of the invention include an isolated nucleic acid molecule encoding
a RR polypeptide.
[0039] Certain embodiments of the invention include RR-encoding
nucleic acid molecules and molecules having sequence identity or
which hybridize to the RR-encoding sequence [SEQ ID NO:7] and which
encode a polypeptide having RR activity (preferred percentages for
sequence identity are described below) as well as vectors including
these molecules. Some embodiments of the invention also include RR
[SEQ ID NO:3] or polypeptides having sequence identity (preferred
percentages described below) or which have RR activity. The nucleic
acid molecules and polypeptides of the invention may be from S.
mutans and they may be isolated from a native source, synthetic or
recombinant. Certain embodiments of the invention include RR [SEQ
ID NO:3] or polypeptides having sequence identity, which have RR
activity, as prepared by the processes described in this
application.
[0040] The comA and comB nucleotide [SEQ ID NO:31 and SEQ ID NO:33]
and amino acid sequences [SEQ ID NO:32 and SEQ ID NO:34] are also
aspects of certain embodiments of the invention. ComA and ComB are
components of the CSP exporter. The discussion of variants,
sequence identity etc. for CSP, HK, RR applies to both the full
sequences shown in the figures as well as bracketed portions of
sequences (coding regions). The peptides and polypeptides may be
natural, recombinantly produced or synthetic.
Functionally Equivalent Nucleic Acid Molecules
[0041] Certain embodiments of the invention include nucleic acid
molecules that are functional equivalents of all or part of the CSP
sequence in SEQ ID NO:5. (A nucleic acid molecule may also be
referred to as a DNA sequence or nucleotide sequence in this
application. All these terms have the same meaning as nucleic acid
molecule). Functionally equivalent nucleic acid molecules are DNA
and RNA (such as genomic DNA, complementary DNA, synthetic DNA, and
messenger RNA molecules) that encode peptides having the same or
similar CSP activity as the CSP peptide shown in SEQ ID NO:11.
Functionally equivalent nucleic acid molecules can encode peptides
that contain a region having sequence identity to a region of a CSP
peptide [SEQ ID NO:11] or more preferably to the entire CSP
peptide. Identity is calculated according to methods known in the
art. The ClustalW program (preferably using default parameters)
[Thompson, J D et al., Nucleic Acid Res. 22:4673-4680.], described
below, is most preferred. For example, if a nucleic acid molecule
(called "Sequence A") has 90% identity to a portion of the nucleic
acid molecule in SEQ ID NO:5, then Sequence A will preferably be
identical to the referenced portion of the nucleic acid molecule in
SEQ ID NO:5, except that Sequence A may include up to 10 point
mutations, such as substitutions with other nucleotides, per each
100 nucleotides of the referenced portion of the nucleic acid
molecule in SEQ ID NO:5. Mutations described in this application
preferably do not disrupt the reading frame of the coding sequence.
Nucleic acid molecules functionally equivalent to the CSP sequences
can occur in a variety of forms as described below.
[0042] Nucleic acid molecules may encode conservative amino acid
changes in CSP peptide [SEQ ID NO:11]. Certain embodiments of the
invention include functionally equivalent nucleic acid molecules
that encode conservative amino acid changes within a CSP amino acid
sequence and produce silent amino acid changes in CSP.
[0043] Nucleic acid molecules may encode non-conservative amino
acid substitutions, additions or deletions in CSP peptide. Some
embodiments of the invention include functionally equivalent
nucleic acid molecules that make non-conservative amino acid
changes within the CSP amino acid sequence in SEQ ID NO:11.
Functionally equivalent nucleic acid molecules include DNA and RNA
that encode peptides, peptides and proteins having non-conservative
amino acid substitutions (preferably substitution of a chemically
similar amino acid), additions, or deletions but which also retain
the same or similar CSP activity as the CSP peptide shown in SEQ ID
NO:11. The DNA or RNA can encode fragments or variants of CSP.
Fragments are useful as immunogens and in immunogenic compositions
(U.S. Pat. No. 5,837,472). The CSP or CSP-like activity of such
fragments and variants is identified by assays as described below.
Fragments and variants of CSP encompassed by the present invention
should preferably have at least about 40%, 60%, 80% or 95% sequence
identity to the naturally occurring CSP nucleic acid molecule, or a
region of the sequence, such as the coding sequence or one of the
conserved domains of the nucleic acid molecule, without being
identical to the sequence in SEQ ID NO:11. Sequence identity is
preferably measured with the ClustalW program (preferably using
default parameters) (Thompson, J D et al., Nucleic Acid Res.
22:4673-4680).
[0044] Nucleic acid molecules functionally equivalent to the CSP
nucleic acid molecule in SEQ ID NO:5 will be apparent from the
following description. For example, the sequence shown in SEQ ID
NO:5 may have its length altered by natural or artificial mutations
such as partial nucleotide insertion or deletion, so that when the
entire length of the coding sequence within SEQ ID NO:5, is taken
as 100%, the functional equivalent nucleic acid molecule preferably
has a length of about 60-120% thereof, more preferably about
80-110% thereof. Fragments may be less than 60%.
[0045] Nucleic acid molecules containing partial (usually 80% or
less, preferably 60% or less, more preferably 40% or less of the
entire length) natural or artificial mutations so that some codons
in these sequences code for different amino acids, but wherein the
resulting peptide retains the same or similar CSP activity as that
of a naturally occurring CSP peptide [SEQ ID NO:11]. The mutated
DNAs created in this manner should preferably encode a peptide
having at least about 40%, preferably at least about 60%, at least
about 80%, and more preferably at least about 90% or 95% sequence
identity to the amino acid sequence of the CSP peptide in SEQ ID
NO:11. The ClustalW program preferably assesses sequence
identity.
[0046] Since the genetic code is degenerate, the nucleic acid
sequence in SEQ ID NO:5 is not the only sequence which may code for
a peptide having CSP activity. This invention includes nucleic acid
molecules that have the same essential genetic information as the
nucleic acid molecule described in SEQ ID NO:5. Nucleic acid
molecules (including RNA) having one or more nucleic acid changes
compared to the sequences described in this application and which
result in production of a peptide shown in SEQ ID NO:11 are within
the scope of various embodiments of the invention.
[0047] Other functional equivalent forms of CSP-encoding nucleic
acids can be isolated using conventional DNA-DNA or DNA-RNA
hybridization techniques. Thus, certain embodiments of the present
invention also include nucleic acid molecules that hybridize to one
or more of the sequences in SEQ ID NO:5 or its complementary
sequence, and that encode expression for peptides, peptides and
proteins exhibiting the same or similar activity as that of the CSP
peptide produced by the DNA in SEQ ID NO:5 or its variants. Such
nucleic acid molecules preferably hybridize to the sequence in SEQ
ID NO:5 under moderate to high stringency conditions (see Sambrook
et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
High stringency washes have low salt (preferably about 0.2% SSC),
and low stringency washes have high salt (preferably about 2% SSC).
A temperature of about 37.degree. C. or about 42.degree. C. is
considered low stringency, and a temperature of about 50-65.degree.
C. is high stringency. Some embodiments of the invention also
include a method of identifying nucleic acid molecules encoding a
CSP activator peptide (preferably a mammalian peptide), including
contacting a sample containing nucleic acid molecules including all
or part of SEQ ID NO:5 (preferably at least about 15 or 20
nucleotides of SEQ ID NO:5) under moderate or high stringency
hybridization conditions and identifying nucleic acid molecules
which hybridize to the nucleic acid molecules including all or part
of SEQ ID NO:5. Similar methods are described in U.S. Pat. No.
5,851,788, which is incorporated by reference in its entirety.
[0048] Certain embodiments of the present invention also include
methods of using all or part of the nucleic acid molecules which
hybridize to all or part of SEQ ID NO:5, for example as probes or
in assays to identify antagonists or inhibitors of the peptides
produced by the nucleic acid molecules (described below). Some
embodiments of the present invention include methods of using
nucleic acid molecules having sequence identity to the CSP nucleic
acid molecule (as described below) in similar methods.
[0049] Certain embodiments of the invention also include a nucleic
acid molecule detection kit including, preferably in a suitable
container means or attached to a surface, a nucleic acid molecule
as disclosed herein encoding CSP [SEQ ID NO:5] or a peptide having
CSP activity and a detection reagent (such as a detectable label).
Other variants of kits will be apparent from this description and
teachings in patents such as U.S. Pat. Nos. 5,837,472 and
5,801,233, which are incorporated by reference in their
entirety.
[0050] A nucleic acid molecule described above is considered to
have a function substantially equivalent to the CSP nucleic acid
molecules [SEQ ID NO:5] of the present invention if the peptide
[SEQ ID NO:11] produced by the nucleic acid molecule has CSP
activity. A peptide has CSP activity if it can stimulate genetic
competence and acid tolerance in S. mutans. Activation of the HK
[SEQ ID NO:2]/RR [SEQ ID NO:3] is shown where a peptide is capable
of stimulating the uptake and incorporation of foreign DNA. We
describe below how the activity of these peptide-mediated processes
can be measured by determining the efficiency of plasmid uptake,
which is a measure of genetic competence. Since the ability to
transport and incorporate foreign DNA relies on activation of the
HK [SEQ ID NO:2]/RR [SEQ ID NO:3] and subsequent genes activated by
the signal cascade initiated by the signal peptide, measurement of
the conferment of erythromycin resistance by cells exposed to the
peptide and plasmid DNA conferring erythromycin resistance
indicates its level of function. Conversely if an inhibitor is
capable of interfering with the action of the peptide the
competence assay will indicate this by a corresponding decrease in
the number of cells that acquire erythromycin resistance as
described in the assays below (assays of genetic competence and
assay of transformation of biofilms). Activation of the HK [SEQ ID
NO:2]/RR [SEQ ID NO:3] is also shown where a peptide is capable of
stimulating an acid tolerance response. We describe below how the
activity of these peptide-mediated processes can be measured by
determining the survival rate of cells in acidic pH conditions.
Since the ability to survive exposure to acidic pH depends on the
activation of the HK/RR and subsequent genes activated by the
signal peptide, measurement of the survival of S. mutans in low pH
conditions indicates the level of function of the signal peptide.
Conversely, if an inhibitor is capable of interfering with the
signal peptide sensing system the assay for acid adaptation will
indicate this by a corresponding decrease in the survival rate of
cells grown in acidic pH conditions as described in the assay below
(assay of acid adaptation).
Production of CSP in Eukaryotic and Prokaryotic Cells
[0051] The nucleic acid molecules disclosed herein may be obtained
from a cDNA library. The nucleotide molecules can also be obtained
from other sources known in the art such as expressed sequence tag
analysis or in vitro synthesis. The DNA described in this
application (including variants that are functional equivalents)
can be introduced into and expressed in a variety of eukaryotic and
prokaryotic host cells. A recombinant nucleic acid molecule for the
CSP contains suitable operatively linked transcriptional or
translational regulatory elements. Suitable regulatory elements are
derived from a variety of sources, and they may be readily selected
by one with ordinary skill in the art (Sambrook, J, Fritsch, E. E.
& Maniatis, T. (Most Recent Edition). Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press. New York;
Ausubel et al. (Most Recent Edition). Current Protocols in
Molecular Biology, John Wiley & Sons, Inc.). For example, if
one were to upregulate the expression of the nucleic acid molecule,
one could insert a sense sequence and the appropriate promoter into
the vector. Promoters can be inducible or constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific. Transcription is enhanced with promoters known in
the art for expression. The CMV and SV40 promoters are commonly
used to express desired peptide in cells. Other promoters known in
the art may also be used (many suitable promoters and vectors are
described in the applications and patents referenced in this
application).
[0052] If one were to downregulate the expression of the nucleic
acid molecule, one could insert the antisense sequence and the
appropriate promoter into the vehicle. The nucleic acid molecule
may be either isolated from a native source (in sense or antisense
orientations), synthesized, or it may be a mutated native or
synthetic sequence or a combination of these.
[0053] Examples of regulatory elements include a transcriptional
promoter and enhancer or RNA polymerase binding sequence, a
ribosomal binding sequence, including a translation initiation
signal. Additionally, depending on the vector employed, other
genetic elements, such as selectable markers, may be incorporated
into the recombinant molecule. Other regulatory regions that may be
used include an enhancer domain and a termination region. The
regulatory elements may bacterial, fungal, viral or avian in
origin. Likewise the regulatory elements may originate from animal,
plant, yeast, insect or other sources, including synthetically
produced elements and mutated elements.
[0054] In addition to using the expression vectors described above,
the peptide may be expressed by inserting a recombinant nucleic
acid molecule in a known expression system derived from bacteria,
viruses, yeast, mammals, insects, fungi or birds. The recombinant
molecule may be introduced into the cells by techniques such as
Agrobacterium tumefaciens-mediated transformation,
particle-bombardment-mediated transformation, direct uptake,
microinjection, coprecipitation, transfection and electroporation
depending on the cell type. Retroviral vectors, adenoviral vectors,
Adeno Associated Virus (AAV) vectors, DNA virus vectors and
liposomes may be used. Suitable constructs are inserted in an
expression vector, which may also include markers for selection of
transformed cells. The construct may be inserted at a site created
by restriction enzymes.
[0055] In one embodiment of the invention, a cell is transfected
with a nucleic acid molecule of the invention inserted in an
expression vector to produce cells expressing a peptide encoded by
the nucleic acid molecule.
[0056] Another embodiment of the invention relates to a method of
transfecting a cell with a nucleic acid molecule disclosed herein,
inserted in an expression vector to produce a cell expressing the
CSP peptide [SEQ ID NO:11] or other peptide of the invention. In
accordance with certain embodiments of the invention a method is
provided for expressing the disclosed peptides in a cell. A
preferred process would include culturing a cell including a
recombinant DNA vector including a nucleic acid molecule encoding
CSP [SEQ ID NO:5] (or another nucleic acid molecule of the
invention) in a culture medium so that the peptide is expressed.
The process preferably further includes recovering the peptide from
the cells or culture medium.
Probes
[0057] Certain embodiments of the present invention include
oligonucleotide probes made from the cloned CSP nucleic acid
molecules described in this application or other nucleic acid
molecules disclosed herein (see Materials and Methods section). The
probes may be 15 to 20 nucleotides in length. A preferred probe is
at least 15 nucleotides of SEQ ID NO:5. Certain embodiments of the
invention also include at least 15 consecutive nucleotides of SEQ
ID NO:5. The probes are useful to identify nucleic acids encoding
CSP peptides as well as peptides functionally equivalent to CSP.
The oligonucleotide probes are capable of hybridizing to the
sequence shown in SEQ ID NO:5 under stringent hybridization
conditions. A nucleic acid molecule encoding a peptide disclosed
herein may be isolated from other organisms by screening a library
under moderate to high stringency hybridization conditions with a
labeled probe. The activity of the peptide encoded by the nucleic
acid molecule is assessed by cloning and expression of the DNA.
After the expression product is isolated, the peptide is assayed
for CSP activity as described in this application.
[0058] Functionally equivalent CSP nucleic acid molecules from
other cells, or equivalent CSP-encoding cDNAs or synthetic DNAs,
can also be isolated by amplification using Polymerase Chain
Reaction (PCR) methods. Oligonucleotide primers, such as degenerate
primers, based on SEQ ID NO:5 can be prepared and used with PCR and
reverse transcriptase (E. S. Kawasaki (1990), In Innis et al.,
Eds., PCR Protocols, Academic Press, San Diego, Chapter 3, p. 21)
to amplify functional equivalent DNAs from genomic or cDNA
libraries of other organisms. The oligonucleotides can also be used
as probes to screen cDNA libraries.
Functionally Equivalent Peptides, Peptides and Proteins
[0059] The present invention includes not only the peptides encoded
by the sequences disclosed herein, but also functionally equivalent
peptides, peptides and proteins that exhibit the same or similar
CSP peptide activity.
[0060] We designed and synthesized peptide analogs based on the
native sequence of the S. mutans CSP and assayed their ability to
interfere with competence development, acid tolerance response, and
biofilm formation.
Peptide Analogs were Altered Based on the Amino Acid Sequence of
Native CSP.
[0061] A panel of 17 peptide analogs with modification in length
and hydrophobicity were designed and synthesized. The first set of
peptide analogs were generated by deleting the 1.sup.st, 2.sup.nd,
3.sup.rd, 4.sup.th, or 5.sup.th residues from the N- and C-termini
of the mature CSP sequence. The second set included peptide analogs
with substitutions of charged internal residues with neutral
(valine) or hydrophobic (alanine) residues. The peptide analogs
synthesized and tested in this study are listed at Table 1.
Peptide Analog H1 is Capable of Inhibiting Genetic Competence.
[0062] All 17 peptide analogs designed and synthesized based on the
sequence of the native S. mutans CSP were first screened for their
ability to hinder transformation efficiency in the S. mutans
wild-type UA159 strain. Among them, analog H1 caused a significant
decrease (18-fold) in transformation efficiency compared to that of
the natural transformation (without addition of exogenous CSP) of
the S. mutans UA159 strain (Table 2 and FIG. 2). These results
demonstrate that H1 inhibited the S. mutans natural genetic
transformation. The competence regulon identified and characterized
by our laboratory indicated that transformation in S. mutans is a
comD-dependent process. To test the hypothesis that the peptide
analog H1 is able to compete with the natural CSP produced by S.
mutans for occupying the ComD histidine kinase receptor, we tested
the ability of H1 to induce genetic competence in an S. mutans comD
null mutant. As expected, the results showed that the effect of
peptide analog H1 is indeed accomplished via the comD ComD
receptor, and therefore is a ComD-dependent process (FIG. 3).
[0063] In contrast, the peptide analogs IH-1, IH-2, B1, and C1
showed no significant effect on transformation efficiency compared
to the wild-type CSP (Table 2). These results suggested that these
peptide analogs have retained the native CSP activity despite the
sequence modifications. However, the transformation efficiency of
S. mutans UA159 in the presence of the peptide analogs D1, E1, F1,
G1, A2, B2, C2, D2, E2, F2, G2, or B3 is diminished compared to the
wild-type CSP (5 .mu.g CSP). This suggested that these peptide
analogs behave similarly to CSP in terms of competence stimulation
but may not have the same affinity for the comD receptor as the
native wild-type CSP.
TABLE-US-00001 TABLE 1 Modified versions of the mature S. mutans
CSP peptide Peptide Analog Amino acid sequence Modification CSP
SGSLSTFFRLFNRSFTQALGK mature wild-type CSP sequence [SEQ ID NO: 11]
IH-1 GSLSTFFRLFNRSFTQALGK 1st residue removed from N' [SEQ ID NO:
35] IH-2 SGSLSTFFRLFNRSFTQALG 1st residue removed from C' [SEQ ID
NO: 36] B1 S SLSTFFRLFNRSFTQALGK 2nd residue removed from N' [SEQ
ID NO: 37] C1 SG LSTFFRLFNRSFTQALGK 3rd residue removed from N'
[SEQ ID NO: 38] D1 SGS STFFRLFNRSFTQALGK 4th residue removed from
N' [SEQ ID NO: 39] E1 SGSL TFFRLFNRSFTQALGK 5th residue removed
from N' [SEQ ID NO: 40] F1 SGSLSTFFRLFNRSFTQAL K 2nd residue
removed from C' [SEQ ID NO: 41] G1 SGSLSTFFRLFNRSFTQA GK 3rd
residue removed from C' [SEQ ID NO: 42] H1 SGSLSTFFRLFNRSFTQ LGK
4th residue removed from C' [SEQ ID NO: 43] A2 SGSLSTFFRLFNRSFT
ALGK 5th residue removed from C' [SEQ ID NO: 44] B2
SGSLSTFFVLFNRSFTQALGK Substitution of 1st R residue with V [SEQ ID
NO: 45] C2 SGSLSTFFALFNRSFTQALGK Substitution of 1st R residue with
A [SEQ ID NO: 46] D2 SGSLSTFFRLFNVSFTQALGK Substitution of 2nd R
residue with V [SEQ ID NO: 47] E2 SGSLSTFFRLFNASFTQALGK
Substitution of 2nd R residue with A [SEQ ID NO: 48] F2
SGSLSTFFRLFNRSFTQALGV Substitution of K residue with V [SEQ ID NO:
49] G2 SGSLSTFFRLFNRSFTQALGA Substitution of K residue with A [SEQ
ID NO: 50] B3 SGTLSTFFRLFNRSFTQA JH1005 CSP sequence [SEQ ID NO:
51]
TABLE-US-00002 TABLE 2 Effect of 5 .mu.g/ml of peptide analogs on
competence of S. mutans wild-type UA159 Peptide Transformation
Transformation Analog efficiency (vs no CSP) efficiency (vs 5 .mu.g
CSP) CSP 1554-fold increase -- IH-1 no effect.sup.a no effect IH-2
no effect no effect B1 no effect no effect C1 no effect no effect
D1 275-fold increase 6-fold decrease E1 791-fold increase 2-fold
decrease F1 541-fold increase 3-fold decrease G1 848-fold increase
2-fold decrease H1 18-fold decrease 28,000-fold decrease A2
125-fold increase 7-fold decrease B2 4-fold increase 414-fold
decrease C2 32-fold increase 48-fold decrease D2 99-fold increase
16-fold decrease E2 252-fold increase 6-fold decrease F2 543-fold
increase 3-fold decrease G2 56-fold increase 28-fold decrease B3
195-fold increase 8-fold decrease .sup.aNo effect: no significant
difference by comparison with CSP.
TABLE-US-00003 TABLE 3 Effect of 5 .mu.g/ml of peptide analogs on
growth at pH 7.5, acid resistance, and biofilm formation of S.
mutans wild-type UA159 Peptide Growth Acid resistance Biofilm
formation analog (pH 7.5) (pH 5.5) (SDM-glucose) CSP .dwnarw.
growth growth no effect IH-1 .dwnarw. growth growth no effect IH-2
.dwnarw. growth growth no effect B1 .dwnarw. growth growth no
effect C1 .dwnarw. growth growth no effect D1 .dwnarw. growth
growth no effect E1 .dwnarw. growth growth no effect F1 .dwnarw.
growth .dwnarw. growth .dwnarw. 36.7% biomass G1 .dwnarw. growth
growth .dwnarw. 24.4% biomass H1 no effect .dwnarw. growth no
effect A2 no effect .dwnarw. growth no effect B2 no effect .dwnarw.
growth no effect C2 no effect .dwnarw. growth no effect D2 no
effect .dwnarw. growth no effect E2 no effect .dwnarw. growth
.dwnarw. 38.9% biomass F2 .dwnarw. growth .dwnarw. growth .dwnarw.
38.7% biomass G2 .dwnarw. growth .dwnarw. growth .dwnarw. 35.6%
biomass B3 no effect .dwnarw. growth .dwnarw. 34.4% biomass
Multiple Peptide Analogs Affect Cell Growth in an Acidic
Medium.
[0064] In order to determine if the peptide analogs were capable of
inhibiting the acid tolerance mechanisms of S. mutans, the cells'
ability to withstand acid challenge typically encountered in dental
plaque, the S. mutans UA159 cells were grown in THYE medium at pH
7.5 and pH 5.5 in the presence of various concentrations of peptide
analogs. The results presented at Table 3 showed that the peptide
analogs F1, F2, and G2 caused a diminution of cell growth at pH 7.5
and 5.5. The peptide analogs H1, A2, B2, C2, D2, E2, and B3 have no
effect on S. mutans cell growth at pH 7.5. Moreover, when the same
peptide analogs were tested at pH 5.5, the results showed that
there was a significant decrease in cell growth. Interestingly, the
peptide analog H1 involved in the inhibition of genetic competence
is also able to inhibit the S. mutans cell growth in an acidic
medium (FIG. 4), while the growth at neutral pH is unaffected (FIG.
5).
Peptide Analogs Inhibit of S, mutans Biofilm Formation.
[0065] It has been demonstrated that the S. mutans comC null mutant
unable to produce the CSP signal peptide forms a biofilm lacking
the wild-type architecture. Moreover, the exogenous addition of
synthetic CSP restores the wild-type phenotype in the comC
defective mutant (Li et al., 2002). Therefore, CSP seems to play an
integral part in S. mutans biofilm formation. Consequently, the
peptide analogs were tested for their ability to inhibit the
formation of S. mutans biofilms. Results presented at Table 3
indicated that the peptide analogs F1, G1, E2, F2, G2 and B3 had a
significantly reduced biomass ranging from 24.4% to 38.9% compared
to the S. mutans biofilm grown in the presence of wild-type CSP
suggesting that these peptide analogs are able to hinder the signal
pathway regulating the formation of biofilm by S. mutans. Peptide
analog E2 could be a potent S. mutans QS inhibitor as it elicited a
significant decrease in biofilm formation as well as inhibited cell
growth at pH 5.5 without affecting the cell growth at neutral
pH.
[0066] A peptide is considered to possess a function substantially
equivalent to that of the CSP peptide [SEQ ID NO:11] if it has CSP
activity. CSP activity means that it is able to confer genetic
competence to S. mutans, as measured by an increased ability to
incorporate and express foreign genetic material, when added to
cells as described in the assay of genetic competence below. CSP
activity also means that the peptide is able to confer an acid
tolerance response in S. mutans as measured by an increase in cell
survival under acidic pH conditions when added to cells as
described in the assay for acid adaptation below. Functionally
equivalent peptides, peptides and proteins include peptides,
peptides and proteins that have the same or similar protein
activity as CSP when assayed, i.e. they are able to stimulate
genetic competence and low pH tolerance (the ability to withstand
acid challenges of pH 3.5-pH 3.0 for up to 3 hours) in S. mutans. A
peptide has CSP activity if it is capable of increasing the
frequency of uptake and expression of foreign DNA as described in
the following assay for genetic competence and if the peptide can
promote an acid tolerance response as described in the assay for
acid adaptation.
[0067] Identity refers to the similarity of two peptides or
proteins that are aligned so that the highest order match is
obtained. Identity is calculated according to methods known in the
art, such as the ClustalW program. For example, if a peptide
(called "Sequence A") has 90% identity to a portion of the peptide
in SEQ ID NO:3, then Sequence A will be identical to the referenced
portion of the peptide in SEQ ID NO:3, except that Sequence A may
include up to 1 point mutations, such as substitutions with other
amino acids, per each 10 amino acids of the referenced portion of
the peptide in SEQ ID NO:3. Peptides, peptides and proteins
functional equivalent to the CSP peptides can occur in a variety of
forms as described below.
[0068] Peptides biologically equivalent in function to CSP peptide
include amino acid sequences containing amino acid changes in the
CSP sequence [SEQ ID NO:11]. The functional equivalent peptides
have at least about 40% sequence identity, preferably at least
about 60%, at least about 75%, at least about 80%, at least about
90% or at least about 95% sequence identity, to the natural CSP
peptide [SEQ ID NO:11] or a corresponding region. The ClustalW
program preferably determines sequence identity. Most preferably,
1, 2, 3, 4, 5, 5-10, 10-15 amino acids are modified.
[0069] Variants of the CSP peptide may also be created by splicing.
A combination of techniques known in the art may be used to
substitute, delete or add amino acids. For example, a hydrophobic
residue such as methionine can be substituted for another
hydrophobic residue such as alanine. An alanine residue may be
substituted with a more hydrophobic residue such as leucine, valine
or isoleucine. An aromatic residue such as phenylalanine may be
substituted for tyrosine. An acidic, negatively-charged amino acid
such as aspartic acid may be substituted for glutamic acid. A
positively-charged amino acid such as lysine may be substituted for
another positively-charged amino acid such as arginine.
Modifications of the peptides disclosed herein may also be made by
treating such peptide with an agent that chemically alters a side
group, for example, by converting a hydrogen group to another group
such as a hydroxy or amino group.
[0070] Peptides having one or more D-amino acids are contemplated
in certain embodiments of the present invention. Also contemplated
are peptides where one or more amino acids are acetylated at the
N-terminus. Those skilled in the art recognize that a variety of
techniques are available for constructing peptide mimetics (i.e., a
modified peptide or peptide or protein) with the same or similar
desired biological activity as the corresponding disclosed peptide
but with more favorable activity than the peptide with respect to
characteristics such as solubility, stability, and/or
susceptibility to hydrolysis and proteolysis. See for example,
Morgan and Gainor, Ann. Rep. Med. Chem., 24:243-252(1989).
[0071] Certain embodiments of the invention also include hybrid
nucleic acid molecules and peptides, for example where a nucleic
acid molecule from the nucleic acid molecule disclosed herein is
combined with another nucleic acid molecule to produce a nucleic
acid molecule which expresses a fusion peptide. One or more of the
other domains of CSP described in this application could also be
used to make fusion peptides. For example, a nucleotide domain from
a molecule of interest may be ligated to all or part of a nucleic
acid molecule encoding CSP peptide (or a molecule having sequence
identity) described in this application. Fusion nucleic acid
molecules and peptides can also be chemically synthesized or
produced using other known techniques. Certain embodiments of the
invention include a nucleic acid molecule encoding a fusion peptide
or a recombinant vector including the nucleic acid molecule.
[0072] The variants preferably retain the same or similar CSP
activity as the naturally occurring CSP [SEQ ID NO:11]. The CSP
activity of such variants can be assayed by techniques described in
this application and known in the art.
[0073] Variants produced by combinations of the techniques
described above but which retain the same or similar CSP activity
as naturally occurring CSP [SEQ ID NO:11] are also included in
certain embodiments of the invention (for example, combinations of
amino acid additions, and substitutions).
[0074] Variants of CSP produced by techniques described above which
competitively inhibit CSP activity are also included in certain
embodiments of the invention (for example, combinations of amino
acid additions, and substitutions).
[0075] Variants of CSP produced by techniques described above which
decrease transformation efficiency of bacteria are also included in
the invention (for example, combinations of amino acid additions,
and substitutions).
[0076] Variants of CSP produced by techniques described above which
decrease biofilm formation are also included in certain embodiments
of the invention (for example, combinations of amino acid
additions, and substitutions).
[0077] Variants of CSP encompassed by the present invention
preferably have at least about 40% sequence identity, preferably at
least about 60%, 75%, 80%, 90% or 95% sequence identity, to the
naturally occurring peptide, or corresponding region or moiety of
the peptide, or corresponding region. Sequence identity is
preferably measured with the ClustalW.
Histidine Kinase & Response Regulator
[0078] Certain embodiments of the invention also include sequences
having identity with the histidine kinase, response regulator of
the invention and comA and comB. Preferred percentages of identity
(nucleic acid molecule and polypeptide) are the same as those
described for the CSP.
[0079] As well, probes and antibodies for a histidine kinase [SEQ
ID NO:2 and SEQ ID NO:6], response regulator [SEQ ID NO:3 and SEQ
ID NO:7] comA [SEQ ID NO:31 and SEQ ID NO:32] or comB [SEQ ID NO:33
and SEQ ID NO:34] may be prepared using the description in this
application and techniques known in the art. The description for
preparation of CSP variants and mutants is also applicable to the
histidine kinase [SEQ ID NO:2 and SEQ ID NO:6], response regulator
[SEQ ID NO:3 and SEQ ID NO:7] or comA [SEQ ID NO:31 and SEQ ID
NO:32] and comB [SEQ ID NO:33 and SEQ ID NO:34] of the invention.
Certain embodiments of the invention also include fragments of HK
having HK activity, fragments of RR [SEQ ID NO:3 and SEQ ID NO:7]
having RR activity and fragments of comA [SEQ ID NO:31 and SEQ ID
NO:32] or comB [SEQ ID NO:33 and SEQ ID NO:34] having activity.
Design of CSP Peptide Competitive Inhibitors
[0080] The activity of the CSP peptide [SEQ ID NO:11] may be varied
by carrying out selective site-directed mutagenesis. We
characterize the binding domain and other critical amino acid
residues in the peptide that are candidates for mutation, insertion
and/or deletion. Sequence variants may be synthesized. A DNA
plasmid or expression vector containing the CSP nucleic acid
molecule [SEQ ID NO:5] or a nucleic acid molecule having sequence
identity may be used for these studies using the U.S.E. (Unique
site elimination) mutagenesis kit from Pharmacia Biotech or other
mutagenesis kits that are commercially available, or using PCR.
Once the mutation is created and confirmed by DNA sequence
analysis, the mutant peptide is expressed using an expression
system and its activity is monitored. This approach is useful to
identify CSP inhibitors. All these modifications of the CSP DNA
sequences presented in this application and the peptides produced
by the modified sequences are encompassed by the present
invention.
Pharmaceutical Compositions
[0081] The CSP inhibitors are also useful when combined with a
carrier in a pharmaceutical composition. The compositions are
useful when administered in methods of medical treatment or
prophylaxis of a disease, disorder or abnormal physical state
caused by S. mutans. Certain embodiments of the invention also
include methods of medical treatment of a disease, disorder or
abnormal physical state characterized by excessive S. mutans or
levels or activity of CSP peptide [SEQ ID NO:11], for example by
administering a pharmaceutical composition including a carrier and
a CSP inhibitor. Caries is one example of a disease, which can be
treated or prevented by antagonizing CSP [SEQ ID NO:11].
[0082] The pharmaceutical compositions can be administered to
humans or animals by methods such as food, food additives, gel,
toothpaste, mouthwash, dental floss or chewing gum in methods of
medical treatment. The peptides of the invention may be coupled to
lipids or carbohydrates. This increases their ability to adhere to
teeth, either by prolonging the duration of the adhesion or by
increasing its affinity, or both. They may also be coupled to
polymers, for example in dental work (eg. crowns, braces, fillings)
or dental floss. The pharmaceutical compositions can be
administered to humans or animals. Dosages to be administered
depend on individual patient condition, indication of the drug,
physical and chemical stability of the drug, toxicity of the
desired effect and the chosen route of administration (Robert
Rakel, ed., Conn's Current Therapy (1995, W.B. Saunders Company,
USA)). The pharmaceutical compositions are used to treat diseases
caused by streptococcal infections such as caries and
endocarditis.
[0083] CSP activity could be blocked by antisense mRNA or by
inhibiting the activity of the exporter that secretes it from the
cell. We have the sequence of these exporters. There are two copies
of the genes (comAB) [SEQ ID NO:31 and SEQ ID NO:33] that are
involved in export.
[0084] Nucleic acid molecules (antisense inhibitors of CSP) and
competitive inhibitors of CSP may be introduced into cells using in
vivo delivery vehicles such as liposomes. They may also be
introduced into these cells using physical techniques such as
microinjection and electroporation or chemical methods such as
coprecipitation or using liposomes.
[0085] The pharmaceutical compositions can be prepared by known
methods for the preparation of pharmaceutically acceptable
compositions which can be administered to patients, and such that
an effective quantity of the nucleic acid molecule or peptide is
combined in a mixture with a pharmaceutically acceptable vehicle.
Suitable carriers are described, for example in Remington's
Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., USA). Carriers include saline and
D5W (5% dextrose and water). Excipients include additives such as a
buffer, solubilizer, suspending agent, emulsifying agent, viscosity
controlling agent, flavor, lactose filler, antioxidant,
preservative or dye. There are preferred excipients for stabilizing
peptides for parenteral and other administration. The excipients
include serum albumin, glutamic or aspartic acid, phospholipids and
fatty acids.
[0086] On this basis, the pharmaceutical compositions could include
an active compound or substance, such as a CSP inhibitor, in
association with one or more pharmaceutically acceptable vehicles
or diluents, and contained in buffered solutions with a suitable pH
and isoosmotic with the physiological fluids. The methods of
combining the active molecules with the vehicles or combining them
with diluents is well known to those skilled in the art. The
compositions may also contain additives such as antioxidants,
buffers, bacteriostatis, bactericidal antibiotics and solutes which
render the formulation isotonic in the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The composition could
include a targeting agent for the transport of the active compound
to specified sites.
[0087] According to another aspect of the present invention, there
is provided a process of producing cells genetically modified to
produce a CSP derivative which inhibits transformation efficiency.
Another aspect comprises administering to a patient S. mutans
genetically modified to produce a CSP derivative which inhibits
transformation efficiency. Methods of producing and administering
genetically engineered cells are known in the art, see, for
example, WO002/44230.
[0088] A further aspect of the present invention provides the use
in the preparation of a medicament for administration to a
mammalian patient to alleviate dental caries, of viable,
transfected S. mutans genetically modified to produce a CSP
derivative which inhibits transformation efficiency.
[0089] According to another aspect of the present invention, there
is provided a process of producing cells genetically modified to
produce a CSP derivative which inhibits biofilm formation. Another
aspect comprises administering to a patient cells genetically
modified to produce a CSP derivative which inhibits biofilm
formation.
[0090] A further aspect of the present invention provides the use
in the preparation of a medicament for administration to a
mammalian patient to improve oral health or to alleviate dental
caries, of viable, transfected cells genetically modified to
produce a CSP derivative which inhibits biofilm formation.
Vaccines
[0091] Antibodies directed against CSP [SEQ ID NO:11] would provide
protection against caries. Antibodies may be manufactured as
described below. Alternatively, a disclosed peptide [SEQ ID NO:11]
or a fragment thereof may be used with a carrier to make a vaccine.
The peptide or fragment may also be conjugated to another molecule
to increase its antigenicity. Antibodies can also be coupled to the
peptide (Brady, L J. et al., "Monoclonal Antibody-Mediated
Modulation of the Humoral Immune Response against Mucosally Applied
Streptococcus mutans" (in press). In order to enhance the immune
response the peptide can be coupled to KLH, ovalbumin, or
thyroglobulin prior to immunization. The vaccine composition will
trigger the mammal's immune system to produce antibodies. Certain
embodiments of the invention include vaccine compositions and
methods of vaccinating a mammal, preferably a human, against dental
caries by administering to the mammal an effective amount of a
vaccine composition. Techniques for preparing and using vaccines
are known in the art. To prepare the vaccine, the peptide, or a
fragment of the peptide, may be mixed with other antigens (of
different immunogenicity), a vehicle or an excipient. Examples of
peptide vaccines are found in U.S. Pat. Nos. 5,679,352, 5,194,254
and 4,950,480. Techniques for preparing vaccines involving
site-directed mutagenesis are described in U.S. Pat. Nos.
5,714,372, 5,543,302, 5,433,945, 5,358,868, 5,332,583, 5,244,657,
5,221,618, 5,147,643, 5,085,862 and 5,073,494. Vaccines may be
administered by known techniques, such as topical or parenteral
administration. Vast changes are taking place in vaccinology
consequent to the introduction of new technologies. Acellular
purified fractions devoid of side effects, non-pathogenic but
immunogenic mutants, recombinant technology, conjugated vaccines,
combination vaccines (to limit the number of injections). Vaccine
delivery systems can deliver multiple doses of the vaccine at a
single contact point. A genetically engineered oral vaccine is
useful to impart better and longer duration of immunity. Oral
vaccines are useful. The nose as a route for immunization is also
useful. DNA alone can constitute the vaccines, inducing both
humoral and cell-mediated immune responses. Live recombinant
vaccines are also useful. Potent adjuvants add to the efficacy of
the vaccines. One can also `humanize` mouse monoclonals by genetic
engineering and express these efficiently in plants. These
recombinant antibodies are opening out an era of highly specific
and safe therapeutic interventions. An advantage of preformed
antibodies directed at a defined target and given in adequate
amounts is the certainty of efficacy in every recipient, in
contrast to vaccines, where the quality and quantum of immune
response varies from individual to individual. For example, nasal
immunization may be done as described in C. Jespersgaard et al.
"Protective Immunity against Streptococcus mutans Infection in Mice
after Intranasal Immunization with the Glucan-Binding Region of S.
mutans Glucosyltransferase" Infection and Immunity, December 1999,
p. 6543-6549, Vol. 67, No. 12. Vaccine compositions may comprise
solid or liquid formulations such as gels, sprays, inhalants,
tablets, toothpastes, mouthwashes or chewing gum.
[0092] For vaccine application, cholera toxin can be used by
coupling the peptide to its B-subunit to stimulate production of
secretory antibody i.e., Coupling to CTB. Screening for inhibitors
of CSP.
[0093] Inhibitors are preferably directed towards CSP [SEQ ID
NO:11] to block S. mutans competence, low pH tolerance and biofilm
formation.
[0094] A method of identifying a compound which reduces the
interaction of CSP [SEQ ID NO:11] with HK [SEQ ID NO:2], can
include: contacting (i) CSP [SEQ ID NO:11] with (ii) HK [SEQ ID
NO:2], a CSP-binding fragment of HK or a derivative of either of
the foregoing in the presence of the compound; and b) determining
whether the interaction between (i) and (ii) is reduced, thereby
indicating that the compound reduces the interaction of CSP [SEQ ID
NO:11] and HK [SEQ ID NO:2]. A CSP inhibitor (caries treating or
preventing compound) inhibits the interaction between (i) and (ii).
By way of example, one can screen a synthetic peptide library. One
could also screen small non-peptide organic molecules.
[0095] In one embodiment, the invention includes an assay for
evaluating whether test compounds are capable of acting as agonists
or antagonists for CSP, or a peptide having CSP functional
activity, including culturing cells containing DNA which expresses
CSP [SEQ ID NO:5], or a peptide having CSP activity so that the
culturing is carried out in the presence of at least one compound
whose ability to modulate CSP activity is sought to be determined
and thereafter monitoring the cells for either an increase or
decrease in the level of CSP [SEQ ID NO:11] or CSP activity. Other
assays (as well as variations of the above assay) will be apparent
from the description of this invention and techniques such as those
disclosed in U.S. Pat. Nos. 5,851,788, 5,736,337 and 5,767,075
which are incorporated by reference in their entirety. For example,
the test compound levels may be either fixed or variable.
Preparation of Antibodies
[0096] The CSP peptide [SEQ ID NO:11] is also useful as an antigen
for the preparation of antibodies that can be used to purify or
detect other CSP-like peptides. Antibodies may also block CSP [SEQ
ID NO:11] binding to HK [SEQ ID NO:2]. Antibodies are preferably
targeted to the entire CSP [SEQ ID NO:11] sequence. The CSP peptide
[SEQ ID NO:11 ] may be conjugated to other compounds, in order to
increase immunogenicity.
[0097] We generate polyclonal antibodies against CSP [SEQ ID
NO:11], which is a unique sequence. Monoclonal and polyclonal
antibodies are prepared according to the description in this
application and techniques known in the art. For examples of
methods of preparation and uses of monoclonal antibodies, see U.S.
Pat. Nos. 5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356,
5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and
5,496,705, which are incorporated by reference in their entirety.
Examples of the preparation and uses of polyclonal antibodies are
disclosed in U.S. Pat. Nos. 5,512,282, 4,828,985, 5,225,331 and
5,124,147 which are incorporated by reference in their entirety.
Antibodies recognizing CSP can be employed to screen organisms or
tissues containing CSP peptide [SEQ ID NO:11] or CSP-like peptides.
The antibodies are also valuable for immuno-purification of CSP or
CSP-like peptides from crude extracts.
[0098] An antibody (preferably the antibody described above) may be
used to detect CSP [SEQ ID NO:11] or a similar peptide, for
example, by contacting a biological sample with the antibody under
conditions allowing the formation of an immunological complex
between the antibody and a peptide recognized by the antibody and
detecting the presence or absence of the immunological complex
whereby the presence of CSP [SEQ ID NO:11] or a similar peptide is
detected in the sample. Certain embodiments of the invention also
include compositions preferably including the antibody, a medium
suitable for the formation of an immunological complex between the
antibody and a peptide recognized by the antibody and a reagent
capable of detecting the immunological complex to ascertain the
presence of CSP [SEQ ID NO:11] or a similar peptide. Certain
embodiments of the invention also include a kit for the in vitro
detection of the presence or absence of CSP [SEQ ID NO:11] or a
similar peptide in a biological sample, wherein the kit preferably
includes an antibody, a medium suitable for the formation of an
immunological complex between the antibody and a peptide recognized
by the antibody and a reagent capable of detecting the
immunological complex to ascertain the presence of CSP [SEQ ID
NO:11] or a similar peptide in a biological sample. Further
background on the use of antibodies is provided, for example in
U.S. Pat. Nos. 5,695,931 and 5,837,472, which are incorporated by
reference in their entirety.
Assay of Genetic Competence
[0099] The ability of the peptide to activate the HK [SEQ ID NO:2]
and RR [SEQ ID NO:3] and the subsequent genes involved in the
conferral of the properties of genetic competence, acid tolerance
and biofilm formation can be determined by measuring the efficiency
of uptake and expression of DNA (preferably plasmid DNA) in S.
mutans when exposed to signal peptide and/or inhibitor. Two methods
modified based on the protocols described by Perry et al. Infect
Immun, 41:722-727 and Lindler and Macrina J Bacteriol, 166:658-665
are used to assay genetic competence. The method involves adding
DNA and CSP DNA [SEQ ID NO:5] (preferably plasmid DNA) to a S.
mutans culture (or culture of a bacteria expressing CSP [SEQ ID
NO:11] or a variant thereof). The rate of transformation is then
determined. S. mutans is preferably grown in THYE plus 5% horse
serum (THYE-HS). After 2-hr incubation, 1 .mu.g/ml plasmid DNA or
10 .mu.g/ml of chromosomal DNA is added to the culture. To assay
induction of competence, competence signal peptide, (SCSP) [SEQ ID
NO:11] is then added to the cultures, incubation continued for 30
minutes with a final concentration of 500 ng/ml of SCSP added to
each sample. After the 30-minute incubation equal amounts of DNA is
added to each well (1 .mu.g/ml plasmid or 10 .mu.g/ml of
chromosomal DNA) and incubation continued for another 2 hrs. Cell
dilutions were immediately spread on THYE agar plates plus
appropriate antibiotics. Transformation frequency was expressed as
the number of transformants (antibiotic resistant cells) per number
of viable recipients. This is determined by comparing the number of
cells able to grow in the presence of antibiotic (conferred by the
applied plasmid or chromosomal DNA) relative to the total number of
cells present (i.e., that grow in the absence of antibiotic). A
higher value indicates a higher rate of transformation and thus is
reflective of a stimulatory effect by the peptide. Consequently,
addition of a molecule that successfully acts as an inhibitor
results in a lower ratio of transformants/recipients, indicating
that the inhibitor is effective at blocking activity of the CSP
[SEQ ID NO:11]. CSP deficient cells may also be used in a variation
of these assays. One can identify compounds that inhibit CSP or
variants thereof by adding a test compound to the mixture to
determine if the rate of transformation is decreased by the
addition of the test compound.
[0100] The activity of the system can also be measured by an in
vitro assay that relies on the measurement of marker protein
expression (such as green fluorescent protein (GFP)) via expression
from a fusion to a promoter controlled by the signal cascade
initiated by CSP [SEQ ID NO:11]/HK [SEQ ID NO:2] /RR [SEQ ID NO:3].
One such promoter occurs immediately 5' proximal to the S. mutans
comX gene. S. mutans cells grown in microtiter wells are exposed to
the CSP [SEQ ID NO:11] and/or inhibitor and the level of
fluorescence of the comX::GFP strain is measured to give a
quantitative measure of CSP [SEQ ID NO:11] stimulation (and
conversely inhibitor activity). One can identify compounds that
inhibit CSP [SEQ ID NO:11] or variants thereof by adding a test
compound to the mixture to determine if the quantitative, measure
of CSP [SEQ ID NO:11] stimulation is decreased by the addition of
the test compound.
Assay of Acid Resistance Tolerance
[0101] The ability of CSP [SEQ ID NO:11] to promote acid resistance
tolerance is determined by measuring the cell survival rate of S.
mutans when exposed to acidic pH. In one example, S. mutans are
first grown in batch culture to assay acid tolerance response in
`standard` log- and stationary-phase cells by using a modification
of methods described previously by Svensater et al. Oral Microbiol.
Immunol., 12:266-73. Mid-log-phase cells are obtained by
transferring one volume of overnight culture into nine volumes
(1:10) of fresh TYG medium (pH 7.5) and incubated at 37.degree. C.
with 5% CO.sub.2 for 2 hours. These cells are then collected by
centrifugation at 8,000.times.g for 10 min and resuspended in 2 ml
of fresh TYG (pH 5.5) at various cell densities as determined by
O.D.sub.600. The cells are induced for acid adaptation by
incubation at pH 5.5 for 2 h at 37.degree. C. with 5% CO.sub.2. The
adapted log-phase cells are then exposed to the killing pH. Killing
pH is pre-determined by incubating unadapted, mid-log phase cells
in TYG medium at pH values from 6.0 to 2.0. Stationary-phase cells
are prepared by re-suspending late-log phase cells in TY medium
(tryptone-yeast extract) without glucose. The culture is incubated
at 37.degree. C. for 2 h to allow the cells to fully enter into
stationary phase. Induction of acid adaptation in stationary-phase
cells follows a similar procedure to that for log-phase cells.
Adaptation of both log- and stationary-phase cells to acidic pH is
determined by measuring the ability of bacterial cells to survive a
killing pH for 3 h. Acid killing is initiated by resuspending cells
in the same volume of fresh TYG (pH 3.5) and an aliquot of cell
suspension is taken immediately from each sample to determine total
viable cell number at zero time. The cells are then incubated for 3
h at 37.degree. C. with 5% CO.sub.2 and an aliquot of sample is
taken to determine survival rate by viable cell counts. Addition of
a molecule that successfully acts as an inhibitor results in a
decrease in the acid resistance tolerance of S mutans resulting in
a corresponding decrease in cell survival indicating that the
inhibitor is effective at blocking activity of CSP. CSP deficient
cells may also be used in a variation of these assays wherein
addition of the signal peptide can complement the
acid-adaptation-defective phenotype of a comC deficient cell. One
can identify compounds that inhibit CSP or variants thereof by
adding a test compound to the mixture to determine if the survival
rate of cells is decreased by the addition of the test
compound.
[0102] Cells transformed with a nucleic acid molecule disclosed
herein (histidine kinase [SEQ ID NO:6], CSP [SEQ ID NO:5] or
response regulator [SEQ ID NO:7]) are useful as research tools. For
example, one may obtain a cell (or a cell line, such as an
immortalized cell culture or a primary cell culture) that does not
express histidine kinase [SEQ ID NO:2], CSP [SEQ ID NO:11] or
response regulator [SEQ ID NO:3], insert a histidine kinase [SEQ ID
NO:6], CSP [SEQ ID NO:5] or response regulator [SEQ ID NO:7]
nucleic acid molecule in the cell, and assess the level of
expression and activity. Alternatively, histidine kinase [SEQ ID
NO:6], CSP [SEQ ID NO:5] or response regulator [SEQ ID NO:7]
nucleic acid molecules may be over-expressed in a cell that
expresses a histidine kinase [SEQ ID NO:2], CSP [SEQ ID NO:11] or
response regulator [SEQ ID NO:3] nucleic acid molecule. In another
example, experimental groups of cells may be transformed with
vectors containing different types of histidine kinase, CSP or
response regulator nucleic acid molecules to assess the levels of
polypeptides and peptides produced, its functionality and the
phenotype of the cells. The polypeptides and peptides are also
useful for in vitro analysis of histidine kinase, CSP [SEQ ID
NO:11] or response regulator [SEQ ID NO:3] activity or structure.
For example, the polypeptides and peptides produced can be used for
microscopy or X-ray crystallography studies.
[0103] The histidine kinase [SEQ ID NO:2 and SEQ ID NO:6], CSP [SEQ
ID NO:5 and SEQ ID NO:11] or response regulator [SEQ ID NO:3 and
SEQ ID NO:7] nucleic acid molecules and polypeptides are also
useful in assays for the identification and development of
compounds to inhibit and/or enhance polypeptide or peptide function
directly. For example, they are useful in an assay for evaluating
whether test compounds are capable of acting as antagonists for
histidine kinase [SEQ ID NO:2], CSP [SEQ ID NO:11] or response
regulator [SEQ ID NO:3] by: (a) culturing cells containing a
nucleic acid molecule which expresses histidine kinase [SEQ ID
NO:2], CSP [SEQ ID NO:11] or response regulator peptides [SEQ ID
NO:3] (or fragments or variants thereof having histidine kinase
[SEQ ID NO:2], CSP or response regulator activity) wherein the
culturing is carried out in the presence of increasing
concentrations of at least one test compound whose ability to
inhibit histidine kinase [SEQ ID NO:2], CSP [SEQ ID NO:11] or
response regulator [SEQ ID NO:3] is sought to be determined; and
(b) monitoring in the cells the level of inhibition as a function
of the concentration of the test compound, thereby indicating the
ability of the test compound to inhibit histidine kinase [SEQ ID
NO:2], CSP [SEQ ID NO:11] or response regulator [SEQ ID NO:3]
activity.
[0104] Suitable assays may be adapted from, for example, U.S. Pat.
No. 5,851,788.
Materials and Methods:
Growth Conditions of Cells
[0105] Cells are grown in Todd Hewitt yeast extract medium at
various dilutions with and without 5% horse serum and 0.01% hog
gastric mucin.
Protocol for Transformation of Biofilm-Grown Cells
[0106] Biofilms are developed on polystyrene microtiter plates to
provide a rapid and simple method for assaying biofilm formation,
and hence activity of the peptide [SEQ ID NO:11]/receptor [SEQ ID
NO3]/kinase [SEQ ID NO:2] system. Formation of biofilms is
initiated by inoculating 20 .mu.l of cell suspension into each well
containing 2 ml of biofilm medium (4.times. diluted Todd-Hewitt
Yeast Extract supplemented with final concentration of 0.01% hog
gastric mucin) for overnight incubation at 37.degree. C. under an
anaerobic condition. After 20-h incubation, fluid medium is removed
and added with 2 ml of pre-warmed, fresh THYE plus 5% horse serum.
The cultures are incubated for 30 minutes and each well is
supplemented with a final concentration of 200 ng/ml of synthetic
competence stimulating peptide (SCSP) and varying concentrations of
the inhibitor and the incubation is continued. After 30 minutes,
plasmid DNA (1 mg/ml) or chromosomal DNA (10 mg/ml) is added to
each well and the cultures are incubated for an additional 2 hr.
Planktonic cells are then removed and the wells are washed once
with PBS buffer. Biofilm cells are collected into 2 ml fresh medium
by a gentle sonication or washing the wells using a pipette. The
samples are centrifuged at 12,000.times.g for 5 min. Both biofilm
and planktonic cells are resuspended into 200 .mu.l of fresh medium
and are immediately spread on THYE agar plus appropriate
antibiotics. Transformation frequency is determined after 48-h of
incubation.
Genome Database Analysis
[0107] Homologues of the Streptococcus pneumoniae comD/comE genes
encoding a histidine kinase/response regulator system were
identified. This sequence was used to design primers to amplify the
region from a number of S. mutans isolates. An open reading frame
consisting of 138 nucleotides was located 148 nucleotides 5'
proximal from the end of the comD homolog in the opposite
orientation (FIG. 1). This ORF was found to encode a peptide of
46-amino acid [SEQ ID NO:1] in length, the precursor of the
21-amino acid CSP [SEQ ID NO:11].
PCR Amplification and Nucleotide Sequencing
[0108] The comCDE genes [SEQ ID NO:18 and SEQ ID NO:19] were
amplified from the genomes of several S. mutans isolates by PCR
using primers designed based on the genome database sequence and
their nucleotide sequences determined. The deduced amino acid
sequences are compared among the isolates by sequence alignment to
confirm identity.
Gene Inactivations
[0109] Genes are inactivated by integration of internal homologous
fragments into the suicide vector pVA8912. Mutants defective in
each of the individual genes (comC, comD, comE) are inactivated and
their phenotypes are compared to the parent strain NG8 for their
abilities to form biofilms, tolerate acidic pH (pH 2-4), and
transport and incorporate DNA. The knockout mutants of com D and
comE were constructed by insertion-duplication mutagenesis, whereas
the knockout comC mutant was created by allelic exchange via
insertion of an erythromycin resistance determinant into the comC
locus (Li et al, 2001). All mutant strains were therefore resistant
to erythromycin. The wild-type strain was subcultured routinely on
Todd-Hewitt-Yeast Extract (THYE) agar plates (BBL; Becton
Dickinson, Cockeysville, Md.), whereas the mutants were maintained
on THYE agar plus 10 .mu.g/ml of erythromycin. A minimal medium
(DMM) was prepared to grow biofilms by a modification of the method
described previously (Loo et al, 2000). The medium contained 58 mM
K.sub.2HPO.sub.4, 15 mM KH.sub.2PO.sub.4, 10 mM
(NH.sub.4).sub.2SO.sub.4, 35 mM NaCl, 2 mM MgSO.sub.2 7H.sub.2O,
0.2% (wt/vol) Casamino Acids and was supplemented with
filter-sterilized vitamins, (0.04 mM nicotinic acid, 0.1 mM
pyridoxine HCl, 0.01 mM pantothenic acid, 1 .mu.M riboflavin, 0.3
.mu.M thiamin HCl, and 0.05 .mu.M D-biotin), amino acids (4 mM
L-glutamic acid, 1 mM L-arginine HCl, 1.3 mM L-cysteine HCl, and
0.1 mM L-tryptophan) and 20 mM glucose.
Creation of comD Deletion Mutant.
[0110] An S. mutans UA159 comD null mutant was constructed by a
PCR-based deletion strategy involving restriction-ligation and
allelic replacement as described previously (Lau et al., 2002). The
primers used to construct and confirm the S. mutans comD deletion
mutant are
TABLE-US-00004 [SEQ ID NO: 52] P1-HK13
(5'-CACAACAACTTATTGACGCTATCCC-3'), [SEQ ID NO: 53] P2-HK13
(5'-GGCGCGCCAACTGGCAACAGGCAGCAGACC-3'), [SEQ ID NO: 54] P3-HK13
(5'-GGCCGGCCTCAAAACGATGCTGTCAAGGG-3'), [SEQ ID NO: 55] P4-HK13
(5'-AGATTATCATTGGCGGAAGCG-3'), [SEQ ID NO: 56] Erm-19
(5'-GGCGCGCCCCGGGCCCAAAATTTGTTTGAT-3'), and [SEQ ID NO: 57] Erm-20
(5'-GGCCGGCCAGTCGGCAGCGACTCATAGAAT-3').
Synthesis of Synthetic Peptide
[0111] The sequence of the processed peptide was deduced by
determining the cleavage site to be located beside the gly-gly
amino acid residues (numbers 24 and 25) (FIG. 4). A peptide was
synthesized corresponding to amino acid sequence of residues 26-46
inclusive.
Synthesis of Peptide Analogs;
[0112] The sequences of the peptide analogs used in this study are
listed in Table 1. The peptides were synthesized by methods known
in the art. The peptides were dissolved to 1 mg per ml in sterile
distilled deionized water. To any insoluble peptides, 10% (vol/vol)
acetic acid, 20% (vol/vol) acetonitrile or 100% (vol/vol)
dimethylformamide (DMF) was subsequently added. Peptides were
stored at -20.degree. C. until used.
Restoration of Phenotypic Defects by Addition of CSP
[0113] To determine if the synthetic peptide could restore
defective phenotypes of the comC mutants, a chemically synthesized
21-amino acid competence-stimulating peptide (CSP) [SEQ ID NO:11]
(Li et al., 2001) was used in complementary experiments. The
peptide was freshly dissolved in sterile distilled water to a
concentration of 1 mg/ml. The CSP solution was then added to the
cultures at a final concentration of 2 .mu.g/ml 2 h after
inoculation of bacterial cells.
Growth Rates
[0114] The parent and mutant strains were grown in THYE medium for
assaying their growth curves using a Bioscreen Microbiology Reader
incorporating a multi-well disposable microtiter plate (Bioscreen
C, Helsinki, Finland). The Bioscreen Reader was equipped with
Biolink software program that allowed us to record and display the
growth curves and growth rate calculations automatically. The
growth of the strains was initiated by inoculating 5 .mu.l of cell
suspension into each well containing 200 .mu.l of fresh THYE
medium. The cell suspensions were pre-adjusted to the same optical
density at O.D.sub.600 before inoculation. The plates were then
placed in the Bioscreen system, which was set up to read optical
density automatically every 15 minutes with shaking. The readings
of optical density were automatically recorded and converted into
growth curves. Each assay was performed in quadruplicate.
Bacterial Strains and Growth Conditions
[0115] Seven strains of S. mutans were used in this study (strains
include: BM71, GB14, H7, JH1005, LT11, NG8, and UAB159. All the
strains were cultured from freeze-dried ampoules and routinely
maintained on Todd-Hewitt Yeast Extract (THYE) plates. For
selection of antibiotic resistant colonies following
transformation, the medium was supplemented with either
erythromycin (Em) (10 .mu.g/ml) or kanamycin (Km) (500
.mu.g/ml).
[0116] S. mutans strain wild-type UA159 and its comD null mutant
were routinely grown on Todd-Hewitt supplemented with 0.3% (wt/vol)
yeast extract (THYE) agar plates and incubated at 37.degree. C. in
air with 5% CO.sub.2. For biofilm experiments, S. mutans strains
were grown in a semidefined minimal medium (SDM) supplemented with
5 mM glucose as described previously (Li et al., 2002). The
replicative plasmid pDL289 (Buckley et al., 1995) was used as donor
DNA for genetic transformation experiments. Plasmid DNA was
prepared from Escherichia coli cultures by using a commercial
plasmid preparation kit (Qiagen). When needed, antibiotics were
added as follows: 10 .mu.g erythromycin per ml or 500 .mu.g
kanamycin per ml for S. mutans, and 50 .mu.g kanamycin per ml for
E. coli.
Assay for Biofilms Formed on Polystyrene Microtiter Plates (a)
[0117] Biofilms were developed on polystyrene microtiter plates to
provide a rapid and simple method for assaying genetic
transformation. A 4.times. diluted THYE medium supplemented with
final concentration of 0.01% hog gastric mucin was used as biofilm
medium (BM). Formation of biofilms was initiated by inoculating 20
.mu.l of cell suspension into each well containing 2 ml of BM and
four wells were set up: two for assaying transformation and two for
quantification of biofilms. After cultures were incubated at
37.degree. C. for 20 h under an anaerobic condition, fluid medium
was removed for viable cell counts. The wells were rinsed once with
10 mM PBS buffer (pH 7.2) and biofilm cells were collected in 2 ml
PBS by a gentle sonication for 15 seconds. Both biofilm and the
planktonic cells were immediately spread on THYE plates using a
spiral system (Spriral Plater, Model D, Cincinnati, Ohio) and
incubated at 37.degree. C. under an anaerobic condition. Formation
of biofilms was quantified by viable cell counts after 48 h of
incubation.
Biofilm Assay. (b)
[0118] Biofilms were developed in 96-well polystyrene microtiter
plates. The growth of the biofilm was initiated by inoculating 10
.mu.l of an overnight S. mutans UA159 culture into 300 .mu.l of
SDM-glucose containing different concentrations (0, 0.1, 0.5, 2,
and 5 .mu.g per ml) of peptide analogs in the individual wells of a
96-well microtiter plate. Wells without cells were used as blank
controls. The microtiter plates were then incubated at 37.degree.
C. in air with 5% CO.sub.2 for 16 h without agitation. After the
incubation, the planktonic cells were carefully removed and the
plates were air dried overnight. The plates were then stained with
0.01% (wt/vol) safranin for 10 min, rinsed with sterile distilled
water and air dried. Biofilms were quantified by measuring the
absorbance of stained biofilms at 490 nm with a microplate reader
(model 3550; Bio-Rad Laboratories, Richmond, Calif.).
Competence Assay;
[0119] To determine if the peptide analogs had any impact on the
development of genetic competence, S. mutans UA159 wild-type cells
were assayed for genetic transformation. Overnight cultures of S.
mutans UA159 were diluted (1:20) with prewarmed THYE broth and
incubated at 37.degree. C. in air with 5% CO.sub.2 until an optical
density (OD) of approximately 0.1 at 600 nm was reached. The
culture was then divided into six aliquots containing 1 .mu.g/ml of
plasmid pDL289 and different concentrations (0, 0.1, 0.5, 2, and 5
.mu.g per ml) of peptide analogs. The cultures were incubated at
37.degree. C. in air with 5% CO.sub.2 for 2.5 h, gently sonicated
for 10 s to disperse the streptococcal chains, and spread on THYE
plates containing kanamycin. Plates were incubated at 37.degree. C.
in air with 5% CO.sub.2 for 48 h. Total recipient cells were
counted by spreading serial dilutions on THYE agar plates without
antibiotic. Transformation efficiency was expressed as the
percentage of kanamycin resistant transformants over the total
number of recipient cells.
Acid resistance Assay.
[0120] The effect of peptides on acid tolerance was evaluated by
assessment of growth in THYE at pH 7.5 and pH 5.5. Overnight S.
mutans wild-type UA159 cells were diluted (1:20) with prewarmed
THYE broth and incubated at 37.degree. C. in air with 5% CO.sub.2
until an OD.sub.600 of approximately 0.4 was reached. A 20-fold
dilution was made into 400 .mu.l of either THYE pH 7.5 or THYE pH
5.5 broth containing different concentrations (0, 0.1, 0.5, 2, and
5 .mu.g per ml) of peptide analogs and added in the individual
wells of a 100-well Bioscreen C plate in triplicate. Wells without
cells were used as blank controls. A Bioscreen microbiology reader
(Labsystems, Helsinki, Finland) was employed to continuously grow
cells and measure cell growth for 16 h at 37.degree. C.
Measurements were taken every 20 min with shaking to prevent cell
aggregation.
Assay for "Steady-State" Biofilms
[0121] Biofilms were also grown in a chemostat-based biofilm
fermentor to define and optimize the conditions for genetic
competence of biofilm-grown cells. The biofilm fermentor was
modified in the Mechanical Engineering and Glass Blowing Shops,
University of Toronto, based on a similar system described
previously (Li and Bowden, 1994). The vessel was made of glass with
a working volume of 400 ml. The vessel lip was constructed of
stainless steel with 10 sampling ports, which allowed sterile
insertion and retrieval of glass rods (0.5 cm in diameter,
approximately 4.0 cm.sup.2 area immersed in fluid medium),
providing abiotic surfaces for accumulation of biofilms.
Temperature in the chemostat vessel was maintained at 37.degree.
C..+-.0.1 by a temperature controller (Model R-600F, Cole Farmer
Instrument Cop., Vernon Hill, Ill.). The culture pH was controlled
by a pH control unit (Digital pH Meter/Controller, Model 501-3400,
Barnant Corp. Barrington, Ill.) through the addition of 1M KOH or
1M HCl. The vessel was placed on a magnetic stirrer (Fisher
Scientific) and the culture was stirred at 200 rpm by a
polypropylene coated magnetic stirrer bar (3 cm in length).
Continuous cultures were obtained by pumping fresh 4.times. diluted
THYE medium supplemented with a final concentration of 0.01% hog
gastric mucin (Type III, Sigma) into the vessel (400 ml) at the
desired dilution rates. Daily maintenance of the chemostat included
optical density reading, viable cell counts and pH measurement in
fluid cultures. When the cultures reached "steady-state" (at least
10 mean generation times), glass rods were aseptically inserted
into the chemostat for the initiation of biofilm formation. Then,
biofilms of different ages were removed from the cultures for both
genetic transformation and quantification of biofilms using viable
cell counts.
Scanning Electron Microscopy (SEM)
[0122] To examine spatial distribution and biofilm thickness by
scanning electron microscopy, biofilms of different ages were
removed by slicing off the bottom of the microtiter wells that were
then washed once with 10 mM KPO.sub.4 and fixed with 2 ml of 3.7%
formaldehyde in 10 mM KPO.sub.4 buffer overnight. The samples were
then dehydrated with a series of alcohol baths (30%, 50%, 70%, 95%
and 100%), critical point dried with liquid CO.sub.2, mounted and
sputter coated with gold. The samples were then examined using a
scanning electron microscope (Model S-2500, Hitachi Instruments,
San Jose, Calif.).
Transformation Protocol
[0123] Two methods modified based on the protocols described by
Perry et al (Infect Immun, 41:722-727) and Lindler and Macrina (J
Bacteriol, 166:658-665) were used to assay natural transformation
of biofilm cells. Biofilms formed on polystyrene microtiter plates
were added with 2 ml of pre-warmed, fresh THYE plus 5% horse serum
(THYE-HS) immediately following removal of the BM medium, and the
incubation continued at 37.degree. C. After 2 h incubation, a final
concentration of 1 .mu.g/ml plasmid DNA or 10 .mu.g/ml of
chromosomal DNA was added to each well. The cultures were incubated
for an additional 2 h before collection of the cells for plating.
To assay induction of competence by synthetic competence
stimulating peptide (SCSP) [SEQ ID NO:11], the cultures were
incubated for 30 min and a final concentration of 500 ng/ml of SCSP
[SEQ ID NO:11] was added to each well. After a 30 min incubation,
equal amounts of DNA was added to each well (1 .mu.g/ml plasmid or
10 .mu.g/ml of chromosomal DNA) and incubation continued for
another 2 h. Fluid medium was then removed from individual wells
and the wells were washed once with PBS buffer. Biofilm cells were
collected into 2 ml PBS buffer by gentle sonication or by washing
the wells using a pipette. The samples were centrifuged at
12,000.times.g for 5 min. Both biofilm and planktonic cells were
resuspended into 200 .mu.l of fresh medium and were immediately
spread on THYE agar plates plus appropriate antibiotics. For the
biofilms developed in the chemostat, rods with biofilm cells were
removed and placed into 2 ml of pre-warmed, fresh THYE-HS medium
for 30 min incubation. Transformation was then initiated by using
the same methods as described above. The planktonic cells were also
removed to compare the transformation frequency. After completion
of the transformation procedures, both biofilm and planktonic cells
were spread on THYE agar plus appropriate antibiotic.
Transformation frequency was assessed after 48-h incubation.
Transformation frequency was expressed as the number of
transformants per .mu.g DNA per viable recipient at the time of DNA
added.
Donor DNA
[0124] Both plasmid and chromosomal DNA were used as donor DNA to
assay genetic transformation in this study. Plasmid DNA included an
integrative plasmid, pVAGTFA carrying an erythromycin resistance
(Em.sup.r) determinant and a fragment of the S. mutans gtfA gene.
The replicative plasmid, pDL289 carrying a kanamycin resistance
gene (Km.sup.r) was also used. Chromosomal DNA harboring an
Em.sup.r gene was prepared from a recombinant S. mutans strain
harboring a chromosomally integrated copy of pVAGTFA.
[0125] The present invention has been described in detail and with
particular reference to the preferred embodiments; however, it will
be understood by one having ordinary skill in the art that changes
can be made without departing from the spirit and scope thereof.
For example, where the application refers to peptides, it is clear
that polypeptides may often be used. Likewise, where a gene is
described in the application, it is clear that nucleic acid
molecules or gene fragments may often be used.
[0126] All publications (including GenBank entries), patents and
patent applications are incorporated by reference in their entirety
to the same extent as if each individual publication, patent or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety.
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Morrison. 1999. Identification of a new regulator in Streptococcus
pneumonias linking quorum sensing to competence for genetic
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Riddle of biofilm resistance. Antimicrob. Agents Chemother.
45:999-1007. [0143] Li, Y.-H., P. C. Y. Lau, J. H. Lee, R. P.
Ellen, and D. G. Cvitkovitch. 2001. Natural genetic transformation
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Sequence CWU 1
1
57146PRTStreptococcus mutans 1Met Lys Lys Thr Leu Ser Leu Lys Asn
Asp Phe Lys Glu Ile Lys Thr1 5 10 15Asp Glu Leu Glu Ile Ile Ile Gly
Gly Ser Gly Ser Leu Ser Thr Phe 20 25 30Phe Arg Leu Phe Asn Arg Ser
Phe Thr Gln Ala Leu Gly Lys 35 40 452441PRTStaphylococcus mutans
2Met Asn Glu Ala Leu Met Ile Leu Ser Asn Gly Leu Leu Thr Tyr Leu1 5
10 15Thr Val Leu Phe Leu Leu Phe Leu Phe Ser Lys Val Ser Asn Val
Thr 20 25 30Leu Ser Lys Lys Glu Leu Thr Leu Phe Ser Ile Ser Asn Phe
Leu Ile 35 40 45Met Ile Ala Val Thr Met Val Asn Val Asn Leu Phe Tyr
Pro Ala Glu 50 55 60Pro Leu Tyr Phe Ile Ala Leu Ser Ile Tyr Leu Asn
Arg Gln Asn Ser65 70 75 80Leu Ser Leu Asn Ile Phe Tyr Gly Leu Leu
Pro Val Ala Ser Ser Asp 85 90 95Leu Phe Arg Arg Ala Ile Ile Phe Phe
Ile Leu Asp Gly Thr Gln Gly 100 105 110Ile Val Met Gly Ser Ser Ile
Ile Thr Thr Tyr Met Ile Glu Phe Ala 115 120 125Gly Ile Ala Leu Ser
Tyr Leu Phe Leu Ser Val Phe Asn Val Asp Ile 130 135 140Gly Arg Leu
Lys Asp Ser Leu Thr Lys Met Lys Val Lys Lys Arg Leu145 150 155
160Ile Pro Met Asn Ile Thr Met Leu Leu Tyr Tyr Leu Leu Ile Gln Val
165 170 175Leu Tyr Val Ile Glu Ser Tyr Asn Val Ile Pro Thr Leu Lys
Phe Arg 180 185 190Lys Phe Val Val Ile Val Tyr Leu Ile Leu Phe Leu
Ile Leu Ile Ser 195 200 205Phe Leu Ser Gln Tyr Thr Lys Gln Lys Val
Gln Asn Glu Ile Met Ala 210 215 220Gln Lys Glu Ala Gln Ile Arg Asn
Ile Thr Gln Tyr Ser Gln Gln Ile225 230 235 240Glu Ser Leu Tyr Lys
Asp Ile Arg Ser Phe Arg His Asp Tyr Leu Asn 245 250 255Ile Leu Thr
Ser Leu Arg Leu Gly Ile Glu Asn Lys Asp Leu Ala Ser 260 265 270Ile
Glu Lys Ile Tyr His Gln Ile Leu Glu Lys Thr Gly His Gln Leu 275 280
285Gln Asp Thr Arg Tyr Asn Ile Gly His Leu Ala Asn Ile Gln Asn Asp
290 295 300Ala Val Lys Gly Ile Leu Ser Ala Lys Ile Leu Glu Ala Gly
Asn Lys305 310 315 320Lys Ile Ala Val Asn Val Glu Val Ser Ser Lys
Ile Gln Leu Pro Glu 325 330 335 Met Glu Leu Leu Asp Phe Ile Thr Ile
Leu Ser Ile Leu Cys Asp Asn 340 345 350Ala Ile Glu Ala Ala Phe Glu
Ser Leu Asn Pro Glu Ile Gln Leu Ala 355 360 365Phe Phe Lys Lys Asn
Gly Ser Ile Val Phe Ile Ile Gln Asn Ser Thr 370 375 380Lys Glu Lys
Gln Ile Asp Val Ser Lys Ile Phe Lys Glu Asn Tyr Ser385 390 395
400Thr Lys Gly Ser Asn Arg Gly Ile Gly Leu Ala Lys Val Asn His Ile
405 410 415Leu Glu His Tyr Pro Lys Thr Ser Leu Gln Thr Ser Asn His
His His 420 425 430Leu Phe Lys Gln Leu Leu Ile Ile Lys 435
4403250PRTStaphylococcus mutans 3Met Ile Ser Ile Phe Val Leu Glu
Asp Asp Phe Leu Gln Gln Gly Arg1 5 10 15Leu Glu Thr Thr Ile Ala Ala
Ile Met Lys Glu Lys Asn Trp Ser Tyr 20 25 30Lys Glu Leu Thr Ile Phe
Gly Lys Pro Gln Gln Leu Ile Asp Ala Ile 35 40 45Pro Glu Lys Gly Asn
His Gln Ile Phe Phe Leu Asp Ile Glu Ile Lys 50 55 60Lys Glu Glu Lys
Lys Gly Leu Glu Val Ala Asn Gln Ile Arg Gln His65 70 75 80Asn Pro
Ser Ala Val Ile Val Phe Val Thr Thr His Ser Glu Phe Met 85 90 95Pro
Leu Thr Phe Gln Tyr Gln Val Ser Ala Leu Asp Phe Ile Asp Lys 100 105
110Ser Leu Asn Pro Glu Glu Phe Ser His Arg Ile Glu Ser Ala Leu Tyr
115 120 125Tyr Ala Met Glu Asn Ser Gln Lys Asn Gly Gln Ser Glu Glu
Leu Phe 130 135 140Ile Phe His Ser Ser Glu Thr Gln Phe Gln Val Pro
Phe Ala Glu Ile145 150 155 160Leu Tyr Phe Glu Thr Ser Ser Thr Ala
His Lys Leu Cys Leu Tyr Thr 165 170 175Tyr Asp Glu Arg Ile Glu Phe
Tyr Gly Ser Met Thr Asp Ile Val Lys 180 185 190Met Asp Lys Arg Leu
Phe Gln Cys His Arg Ser Phe Ile Val Asn Pro 195 200 205Ala Asn Ile
Thr Arg Ile Asp Arg Lys Lys Arg Leu Ala Tyr Phe Arg 210 215 220Asn
Asn Lys Ser Cys Leu Ile Ser Arg Thr Lys Leu Thr Lys Leu Arg225 230
235 240Ala Val Ile Ala Asp Gln Arg Arg Ala Lys 245
2504141DNAStaphylococcus mutans 4atgaaaaaaa cactatcatt aaaaaatgac
tttaaagaaa ttaagactga tgaattagag 60attatcattg gcggaagcgg aagcctatca
acatttttcc ggctgtttaa cagaagtttt 120acacaagctt tgggaaaata a
141563DNAStaphylococcus mutans 5agcggaagcc tatcaacatt tttccggctg
tttaacagaa gttttacaca agctttggga 60aaa 6361326DNAStaphylococcus
mutans 6atgaatgaag ccttaatgat actttcaaat ggtttattaa cttatctaac
cgttctattt 60ctcttgtttc tattttctaa ggtaagtaat gtcactttat cgaaaaagga
attaactctt 120ttttcgataa gcaattttct gataatgatt gctgttacga
tggtgaacgt aaacctgttt 180tatcctgcag agcctcttta ttttatagct
ttatcaattt atcttaatag acagaatagt 240ctttctctaa atatatttta
tggtctgctg cctgttgcca gttctgactt gtttaggcgg 300gcaatcatat
tctttatctt ggatggaact caaggaattg taatgggcag tagcattata
360accacctata tgatcgagtt tgcaggaata gcgctaagtt acctctttct
cagtgtgttc 420aatgttgata ttggtcgact taaagatagt ttgaccaaga
tgaaggtcaa aaaacgcttg 480attccaatga atattactat gcttctatac
taccttttaa tacaggtatt gtatgttata 540gagagttata atgtgatacc
gactttaaaa tttcgtaaat ttgtcgttat tgtctatctt 600attttatttt
tgattctgat ctcattttta agccaatata ccaaacaaaa ggttcaaaat
660gagataatgg cacaaaagga agctcagatt cgaaatatca cccagtatag
tcagcaaata 720gaatctcttt acaaggatat tcgaagtttc cgccatgatt
atctgaatat tttaactagc 780ctcagattag gcattgaaaa taaagattta
gctagtattg aaaagattta ccatcaaatc 840ttagaaaaaa caggacatca
attgcaggat acccgttata atatcggcca tctagctaat 900attcaaaacg
atgctgtcaa gggtatcttg tcagcaaaaa tcttagaagc tcagaataaa
960aagattgctg tcaatgtaga agtctcaagt aaaatacaac tgcctgagat
ggagttgctt 1020gatttcatta ccatactttc tatcttgtgt gataatgcca
ttgaggctgc tttcgaatca 1080ttaaatcctg aaattcagtt agcctttttt
aagaaaaatg gcagtatagt ctttatcatt 1140cagaattcca ccaaagaaaa
acaaatagat gtgagtaaaa tttttaaaga aaactattcc 1200actaaaggct
ccaatcgcgg tattggttta gcaaaggtga atcatattct tgaacattat
1260cccaaaacca gtttacaaac aagcaatcat catcatttat tcaagcaact
cctaataata 1320aaatag 13267753DNAStaphylococcus mutans 7atgatttcta
tttttgtatt ggaagatgat tttttacaac aaggacgtct tgaaaccacc 60attgcagcta
tcatgaaaga aaaaaattgg tcttataaag aattgactat ttttggaaaa
120ccacaacaac ttattgacgc tatccctgaa aagggcaatc accagatttt
ctttttggat 180attgaaatca aaaaagagga aaagaaagga ctggaagtag
ccaatcagat tagacagcat 240aatcctagtg cagttattgt ctttgtcacg
acacattctg agtttatgcc cctcactttt 300cagtatcagg tatctgcttt
ggattttatt gataaatctt tgaatcctga ggagttctcc 360caccgcattg
aatcagcgct gtattatgct atggaaaaca gccagaagaa tggtcaatca
420gaggaacttt ttattttcca ttcatctgaa actcagtttc aggtcccttt
tgctgagatt 480ctgtattttg aaacatcttc aacagcccat aagctctgcc
tttatactta tgatgaacgg 540attgaattct acggcagtat gactgacatt
gttaaaatgg ataagagact ttttcagtgc 600catcgctctt ttattgtcaa
tcctgccaat attacccgta ttgatcggaa aaaacgcttg 660gcctattttc
gaaataataa gtcttgtctt atttcacgaa ctaagttaac aaaactgaga
720gctgtgattg ctgatcaaag gagagcaaaa tga 753846PRTStaphylococcus
mutans 8Met Lys Lys Thr Pro Ser Leu Lys Asn Asp Phe Lys Glu Ile Lys
Thr1 5 10 15Asp Glu Leu Glu Ile Ile Ile Gly Gly Ser Gly Ser Leu Ser
Thr Phe 20 25 30Phe Arg Leu Phe Asn Arg Ser Phe Thr Gln Ala Leu Gly
Lys 35 40 45946PRTStaphylococcus mutans 9Met Lys Lys Thr Leu Ser
Leu Lys Asn Asp Phe Lys Glu Ile Lys Thr1 5 10 15Asp Glu Leu Glu Ile
Ile Ile Gly Gly Ser Gly Ser Leu Ser Thr Phe 20 25 30Phe Arg Leu Phe
Asn Arg Ser Phe Thr Gln Ala Leu Gly Lys 35 40
451043PRTStaphylococcus mutans 10Met Lys Lys Thr Leu Ser Leu Lys
Asn Asp Phe Lys Glu Ile Lys Thr1 5 10 15Asp Glu Leu Glu Ile Ile Ile
Gly Gly Ser Gly Thr Leu Ser Thr Phe 20 25 30Phe Arg Leu Phe Asn Arg
Ser Phe Thr Gln Ala 35 401121PRTArtificialSynthetic signal peptide
11Ser Gly Ser Leu Ser Thr Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1
5 10 15Gln Ala Leu Gly Lys 201219DNAArtificialSynthetic ComC
Forward Primer 12agttttttgt ctggctgcg 191320DNAArtificialSynthetic
ComC Backward Primer 13tccactaaag gctccaatcg
201424DNAArtificialSynthetic ComD Forward Primer 14cgctaagtta
cctctttctc agtg 241521DNAArtificialSynthetic ComD Backward Primer
15gcttcctttt gtgccattat c 211621DNAArtificialSynthetic ComE Forward
Primer 16cctgaaaagg gcaatcacca g 211722DNAArtificialSynthetic ComE
Backward Primer 17gcgatggcac tgaaaaagtc tc
22182557DNAStaphylococcus mutans 18acattatgtg tcctaaggaa aatattactt
tttcaagaaa atccatgatt ttttcataaa 60aaatagtata ctaattataa tcaaaaaaag
gagatataaa atgaaaaaaa cactatcatt 120aaaaaatgac tttaaagaaa
ttaagactga tgaattagag attatcattg gcggaagcgg 180aagcctatca
acatttttcc ggctgtttaa cagaagtttt acacaagctt tgggaaaata
240agataggcta acattggaat aaaacaaggc tggatttatt attccagcct
ttttaaatgt 300aaaataaaaa tacagggtta aataatcaag tgtgctgtcg
tggatgagaa gataaaacta 360tctcttagag aataggcctc ctctatttta
ttattaggag ttgcttgaat aaatgatgat 420gattgcttgt ttgtaaactg
gttttgggat aatgttcaag aatatgattc acctttgcta 480aaccaatacc
gcgattggag cctttagtgg aatagttttc tttaaaaatt ttactcacat
540ctatttgttt ttctttggtg gaattctgaa tgataaagac tatactgcca
tttttcttaa 600aaaaggctaa ctgaatttca ggatttaatg attcgaaagc
agcctcaatg gcattatcac 660acaagataga aagtatggta atgaaatcaa
gcaactccat ctcaggcagt tgtattttac 720ttgagacttc tacattgaca
gcaatctttt tattctgagc ttctaagatt tttgctgaca 780agataccctt
gacagcatcg ttttgaatat tagctagatg gccgatatta taacgggtat
840cctgcaattg atgtcctgtt ttttctaaga tttgatggta aatcttttca
atactagcta 900aatctttatt ttcaatgcct aatctgaggc tagttaaaat
attcagataa tcatggcgga 960aacttcgaat atccttgtaa agagattcta
tttgctgact atactgggtg atatttcgaa 1020tctgagcttc cttttgtgcc
attatctcat tttgaacctt ttgtttggta tattggctta 1080aaaatgagat
cagaatcaaa aataaaataa gatagacaat aacgacaaat ttacgaaatt
1140ttaaagtcgg tatcacatta taactctcta taacatacaa tacctgtatt
aaaaggtagt 1200atagaagcat agtaatattc attggaatca agcgtttttt
gaccttcatc ttggtcaaac 1260tatctttaag tcgaccaata tcaacattga
acacactgag aaagaggtaa cttagcgcta 1320ttcctgcaaa ctcgatcata
taggtggtta taatgctact gcccattaca attccttgag 1380ttccatccaa
gataaagaat atgattgccc gcctaaacaa gtcagaactg gcaacaggca
1440gcagaccata aaatatattt agagaaagac tattctgtct attaagataa
attgataaag 1500ctataaaata aagaggctct gcaggataaa acaggtttac
gttcaccatc gtaacagcaa 1560tcattatcag aaaattgctt atcgaaaaaa
gagttaattc ctttttcgat aaagtgacat 1620tacttacctt agaaaataga
aacaagagaa atagaacggt tagataagtt aataaaccat 1680ttgaaagtat
cattaaggct tcattcattt tgctctcctt tgatcagcaa tcacagctct
1740cagttttgtt aacttagttc gtgaaataag acaagactta ttatttcgaa
aataggccaa 1800gcgttttttc cgatcaatac gggtaatatt ggcaggattg
acaataaaag agcgatggca 1860ctgaaaaagt ctcttatcca ttttaacaat
gtcagtcata ctgccgtaga attcaatccg 1920ttcatcataa gtataaaggc
agagcttatg ggctgttgaa gatgtttcaa aatacagaat 1980ctcagcaaaa
gggacctgaa actgagtttc agatgaatgg aaaataaaaa gttcctctga
2040ttgaccattc ttctggctgt tttccatagc ataatacagc gctgattcaa
tgcggtggga 2100gaactcctca ggattcaaag atttatcaat aaaatccaaa
gcagatacct gatactgaaa 2160agtgaggggc ataaactcag aatgtgtcgt
gacaaagaca ataactgcac taggattatg 2220ctgtctaatc tgattggcta
cttccagtcc tttcttttcc tcttttttga tttcaatatc 2280caaaaagaaa
atctggtgat tgcccttttc agggatagcg tcaataagtt gttgtggttt
2340tccaaaaata gtcaattctt tataagacca atttttttct ttcatgatag
ctgcaatggt 2400ggtttcaaga cgtccttgtt gtaaaaaatc atcttccaat
acaaaaatag aaatcattat 2460ttctccttta atcttctatt taggttagct
gattaacact atacacagaa aaggtataaa 2520acgatatcac tcaataaaat
ctactaactt aataacc 2557192557DNAStaphylococcus mutans 19ggttattaag
ttagtagatt ttattgagtg atatcgtttt ataccttttc tgtgtatagt 60gttaatcagc
taacctaaat agaagattaa aggagaaata atgatttcta tttttgtatt
120ggaagatgat tttttacaac aaggacgtct tgaaaccacc attgcagcta
tcatgaaaga 180aaaaaattgg tcttataaag aattgactat ttttggaaaa
ccacaacaac ttattgacgc 240tatccctgaa aagggcaatc accagatttt
ctttttggat attgaaatca aaaaagagga 300aaagaaagga ctggaagtag
ccaatcagat tagacagcat aatcctagtg cagttattgt 360ctttgtcacg
acacattctg agtttatgcc cctcactttt cagtatcagg tatctgcttt
420ggattttatt gataaatctt tgaatcctga ggagttctcc caccgcattg
aatcagcgct 480gtattatgct atggaaaaca gccagaagaa tggtcaatca
gaggaacttt ttattttcca 540ttcatctgaa actcagtttc aggtcccttt
tgctgagatt ctgtattttg aaacatcttc 600aacagcccat aagctctgcc
tttatactta tgatgaacgg attgaattct acggcagtat 660gactgacatt
gttaaaatgg ataagagact ttttcagtgc catcgctctt ttattgtcaa
720tcctgccaat attacccgta ttgatcggaa aaaacgcttg gcctattttc
gaaataataa 780gtcttgtctt atttcacgaa ctaagttaac aaaactgaga
gctgtgattg ctgatcaaag 840gagagcaaaa tgaatgaagc cttaatgata
ctttcaaatg gtttattaac ttatctaacc 900gttctatttc tcttgtttct
attttctaag gtaagtaatg tcactttatc gaaaaaggaa 960ttaactcttt
tttcgataag caattttctg ataatgattg ctgttacgat ggtgaacgta
1020aacctgtttt atcctgcaga gcctctttat tttatagctt tatcaattta
tcttaataga 1080cagaatagtc tttctctaaa tatattttat ggtctgctgc
ctgttgccag ttctgacttg 1140tttaggcggg caatcatatt ctttatcttg
gatggaactc aaggaattgt aatgggcagt 1200agcattataa ccacctatat
gatcgagttt gcaggaatag cgctaagtta cctctttctc 1260agtgtgttca
atgttgatat tggtcgactt aaagatagtt tgaccaagat gaaggtcaaa
1320aaacgcttga ttccaatgaa tattactatg cttctatact accttttaat
acaggtattg 1380tatgttatag agagttataa tgtgataccg actttaaaat
ttcgtaaatt tgtcgttatt 1440gtctatctta ttttattttt gattctgatc
tcatttttaa gccaatatac caaacaaaag 1500gttcaaaatg agataatggc
acaaaaggaa gctcagattc gaaatatcac ccagtatagt 1560cagcaaatag
aatctcttta caaggatatt cgaagtttcc gccatgatta tctgaatatt
1620ttaactagcc tcagattagg cattgaaaat aaagatttag ctagtattga
aaagatttac 1680catcaaatct tagaaaaaac aggacatcaa ttgcaggata
cccgttataa tatcggccat 1740ctagctaata ttcaaaacga tgctgtcaag
ggtatcttgt cagcaaaaat cttagaagct 1800cagaataaaa agattgctgt
caatgtagaa gtctcaagta aaatacaact gcctgagatg 1860gagttgcttg
atttcattac catactttct atcttgtgtg ataatgccat tgaggctgct
1920ttcgaatcat taaatcctga aattcagtta gcctttttta agaaaaatgg
cagtatagtc 1980tttatcattc agaattccac caaagaaaaa caaatagatg
tgagtaaaat ttttaaagaa 2040aactattcca ctaaaggctc caatcgcggt
attggtttag caaaggtgaa tcatattctt 2100gaacattatc ccaaaaccag
tttacaaaca agcaatcatc atcatttatt caagcaactc 2160ctaataataa
aatagaggag gcctattctc taagagatag ttttatcttc tcatccacga
2220cagcacactt gattatttaa ccctgtattt ttattttaca tttaaaaagg
ctggaataat 2280aaatccagcc ttgttttatt ccaatgttag cctatcttat
tttcccaaag cttgtgtaaa 2340acttctgtta aacagccgga aaaatgttga
taggcttccg cttccgccaa tgataatctc 2400taattcatca gtcttaattt
ctttaaagtc attttttaat gatagtgttt ttttcatttt 2460atatctcctt
tttttgatta taattagtat actatttttt atgaaaaaat catggatttt
2520cttgaaaaag taatattttc cttaggacac ataatgt
25572047PRTStaphylococcus mutans 20Met Ile Ile Ser Asn Ser Ser Val
Leu Ile Ser Leu Lys Ser Phe Phe1 5 10 15Asn Asp Ser Val Phe Phe Ile
Leu Tyr Leu Leu Phe Leu Ile Ile Ile 20 25 30Ser Ile Leu Phe Phe Met
Lys Lys Ser Trp Ile Phe Leu Lys Lys 35 40 452136PRTStaphylococcus
mutans 21Met Ala Leu Ser His Lys Ile Glu Ser Met Val Met Lys Ser
Ser Asn1 5 10 15Ser Ile Ser Gly Ser Cys Ile Leu Leu Glu Thr Ser Thr
Leu Thr Ala 20 25 30Ile Phe Leu Phe 352242PRTStaphylococcus mutans
22Met Ala Glu Thr Ser Asn Ile Leu Val Lys Arg Phe Tyr Leu Leu Thr1
5 10 15Ile Leu Gly Asp Ile Ser Asn Leu Ser Phe Leu Leu Cys His Tyr
Leu 20 25 30Ile Leu Asn Leu Leu Phe Gly Ile Leu Ala 35
402327PRTStreptococcus mutans 23Met Val Cys Cys Leu Leu Pro Val Leu
Thr Cys Leu Gly Gly Gln Ser1
5 10 15Tyr Ser Leu Ser Trp Met Glu Leu Lys Glu Leu 20
252434PRTStaphylococcus mutans 24Met Ala Leu Lys Lys Ser Leu Ile
His Phe Asn Asn Val Ser His Thr1 5 10 15Ala Val Glu Phe Asn Pro Phe
Ile Ile Ser Ile Lys Ala Glu Leu Met 20 25 30Gly
Cys2557PRTStaphylococcus mutans 25Met Leu Trp Lys Thr Ala Arg Arg
Met Val Asn Gln Arg Asn Phe Leu1 5 10 15Phe Ser Ile His Leu Lys Leu
Ser Phe Arg Ser Leu Leu Leu Arg Phe 20 25 30Cys Ile Leu Lys His Leu
Gln Gln Pro Ile Ser Ser Ala Phe Ile Leu 35 40 45Met Met Asn Gly Leu
Asn Ser Thr Ala 50 552680PRTStaphylococcus mutans 26Met Cys Arg Asp
Lys Asp Asn Asn Cys Thr Arg Ile Met Leu Ser Asn1 5 10 15Leu Ile Gly
Tyr Phe Gln Ser Phe Leu Phe Leu Phe Phe Asp Phe Asn 20 25 30Ile Gln
Lys Glu Asn Leu Val Ile Ala Leu Phe Arg Asp Ser Val Asn 35 40 45Lys
Leu Leu Trp Phe Ser Lys Asn Ser Gln Phe Phe Ile Arg Pro Ile 50 55
60Phe Phe Phe His Asp Ser Cys Asn Gly Gly Phe Lys Thr Ser Leu Leu65
70 75 802734PRTStaphylococcus mutans 27Met Ile Ala Ala Met Val Val
Ser Arg Arg Pro Cys Cys Lys Lys Ser1 5 10 15Ser Ser Asn Thr Lys Ile
Glu Ile Ile Ile Ser Pro Leu Ile Phe Tyr 20 25 30Leu
Gly28480DNAStaphylococcus mutans 28atggaagaag attttgaaat tgtttttaat
aaggttaagc caattgtatg gaaattaagc 60cgttattact ttattaaaat gtggactcgt
gaagattggc aacaagaggg aatgttgatt 120ttgcaccaat tattaaggga
acatccagaa ttagaagagg atgatacaaa attgtatatc 180tattttaaga
cacgtttttc taattacatt aaagatgttt tgcgtcagca agaaagtcag
240aaacgtcgtt ttaatagaat gtcttatgaa gaagtcggtg agattgaaca
ctgtttgtca 300agtggcggta tgcaattgga tgaatatatt ttatttcgtg
atagtttgct tgcatataaa 360caaggtctga gtactgaaaa gcaagagctg
tttgagcgct tggtagcagg agagcacttt 420ttgggaaggc aaagtatgct
gaaagattta cgtaaaaaat taagtgattt taaggaaaaa
48029160PRTStaphylococcus mutans 29Met Glu Glu Asp Phe Glu Ile Val
Phe Asn Lys Val Lys Pro Ile Val1 5 10 15Trp Lys Leu Ser Arg Tyr Tyr
Phe Ile Lys Met Trp Thr Arg Glu Asp 20 25 30Trp Gln Gln Glu Gly Met
Leu Ile Leu His Gln Leu Leu Arg Glu His 35 40 45Pro Glu Leu Glu Glu
Asp Asp Thr Lys Leu Tyr Ile Tyr Phe Lys Thr 50 55 60Arg Phe Ser Asn
Tyr Ile Lys Asp Val Leu Arg Gln Gln Glu Ser Gln65 70 75 80Lys Arg
Arg Phe Asn Arg Met Ser Tyr Glu Glu Val Gly Glu Ile Glu 85 90 95His
Cys Leu Ser Ser Gly Gly Met Gln Leu Asp Glu Tyr Ile Leu Phe 100 105
110Arg Asp Ser Leu Leu Ala Tyr Lys Gln Gly Leu Ser Thr Glu Lys Gln
115 120 125Glu Leu Phe Glu Arg Leu Val Ala Gly Glu His Phe Leu Gly
Arg Gln 130 135 140Ser Met Leu Lys Asp Leu Arg Lys Lys Leu Ser Asp
Phe Lys Glu Lys145 150 155 16030680DNAStaphylococcus mutans
30gtaaataaaa cagccagtta agatgggaca tttatgtcct gttcttaaag tctttttcgt
60tttataataa ttttattata aaaggaggtc atcgtaatag atggaagaag attttgaaat
120tgtttttaat aaggttaagc caattgtatg gaaattaagc cgttattact
ttattaaaat 180gtggactcgt gaagattggc aacaagaggg aatgttgatt
ttgcaccaat tattaaggga 240acatccagaa ttagaagagg atgatacaaa
attgtatatc tattttaaga cacgtttttc 300taattacatt aaagatgttt
tgcgtcagca agaaagtcag aaacgtcgtt ttaatagaat 360gtcttatgaa
gaagtcggtg agattgaaca ctgtttgtca agtggcggta tgcaattgga
420tgaatatatt ttatttcgtg atagtttgct tgcatataaa caaggtctga
gtactgaaaa 480gcaagagctg tttgagcgct tggtagcagg agagcacttt
ttgggaaggc aaagtatgct 540gaaagattta cgtaaaaaat taagtgattt
taaggaaaaa tagttaaaaa gggaaagaat 600ggaacatgtg attgtaccat
tctttttggt tgaaaattaa gaaaagttat tataaattat 660tggtttaaca
tgccatatta 680312280DNAStaphylococcus mutans 31atgaaacaag
ttatttatgt tgttttaatc gtcatagccg ttaacattct cttagagatt 60atcaaaagag
taacaaaaag gggagggaca gtttcgtcat ctaatccttt accagatggg
120cagtctaagt tgttttggcg cagacattat aagctagtac ctcagattga
taccagagac 180tgtgggccgg cagtgctggc atctgttgca aagcattacg
gatctaatta ctctatcgct 240tatctgcggg aactctcaaa gactaacaag
cagggaacaa cagctcttgg cattgttgaa 300gctgctaaaa agttaggctt
tgaaacacgc tctatcaagg cggatatgac gctttttgat 360tataatgatt
tgacctatcc ttttatcgtc catgtgatta aaggaaaacg tctgcagcat
420tattatgtcg tctatggcag ccagaataat cagctgatta ttggagatcc
tgatccttca 480gttaaggtga ctaggatgag taaggaacgc tttcaatcag
agtggacagg ccttgcaatt 540ttcctagctc ctcagcctaa ctataagcct
cataaaggtg aaaaaaatgg tttgtctaat 600ttcttcccgt tgatctttaa
gcagaaagct ttgatgactt atattatcat agctagcttg 660attgtgacgc
tcattgatat tgtcggatca tactatctcc aaggaatatt ggacgagtac
720attcctgatc agctgatttc aactttagga atgattacga ttggtctgat
aataacctat 780attatccagc aggtcatggc ttttgcaaaa gaatacctct
tggccgtact cagtttgcgt 840ttagtcattg atgttatcct gtcttatatc
aaacatattt ttacgcttcc tatgtctttc 900tttgcgacaa ggcgaacagg
agaaatcacg tctcgtttta cagatgccaa tcagattatt 960gatgctgtag
cgtcaaccat cttttcaatc tttttagata tgactatggt aattttggtt
1020ggtggggttt tgttggcgca aaacaataac cttttctttc taaccttgct
ctccattccg 1080atttatgcca tcattatttt tgctttcttg aaaccctttg
agaaaatgaa tcacgaagtg 1140atggaaagca atgctgtggt aagttcttct
atcattgaag atatcaatgg gatggaaacc 1200attaaatcac tcacaagtga
gtccgctcgt tatcaaaaca ttgatagtga atttgttgat 1260tatttggaga
aaaactttaa gctacacaag tatagtgcca ttcaaaccgc attaaaaagc
1320ggtgctaagc ttatcctcaa tgttgtcatt ctctggtatg gctctcgtct
agttatggat 1380aataaaatct cagttggtca gcttatcacc tttaatgctt
tgctgtctta tttctcaaat 1440ccaattgaaa atattatcaa tctgcaatcc
aaactgcagt cagctcgcgt tgccaataca 1500cgtcttaatg aggtctatct
tgtcgaatct gaatttgaaa aagacggcga tttatcagaa 1560aatagctttt
tagatggtga tatttcgttt gaaaatcttt cttataaata tggatttggg
1620cgagatacct tatcagatat taatttatca atcaaaaaag gctccaaggt
cagtctagtt 1680ggagccagtg gttctggtaa aacaactttg gctaaactga
ttgtcaattt ctacgagcct 1740aacaagggga ttgttcgaat caatggcaat
gatttaaaag ttattgataa gacagctttg 1800cggcggcata ttagctattt
gccgcaacag gcctatgttt ttagtggctc tattatggat 1860aatctcgttt
taggagctaa agaaggaacg agtcaggaag acattattcg tgcttgtgaa
1920attgctgaaa tccgctcgga cattgaacaa atgcctcagg gctatcagac
agagttatca 1980gatggtgccg gtatttctgg cggtcaaaaa cagcggattg
ctttagctag ggccttatta 2040acacaggcac cggttttgat tctggatgaa
gccaccagca gtcttgatat tttgacagaa 2100aagaaaatta tcagcaatct
cttacagatg acggagaaaa caataatttt tgttgcccac 2160cgcttaagca
tttcacagcg tactgacgaa gtcattgtca tggatcaggg aaaaattgtt
2220gaacaaggca ctcataagga acttttagct aagcaaggtt tctattataa
cctgtttaat 228032760PRTStaphylococcus mutans 32Met Lys Gln Val Ile
Tyr Val Val Leu Ile Val Ile Ala Val Asn Ile1 5 10 15Leu Leu Glu Ile
Ile Lys Arg Val Thr Lys Arg Gly Gly Thr Val Ser 20 25 30Ser Ser Asn
Pro Leu Pro Asp Gly Gln Ser Lys Leu Phe Trp Arg Arg 35 40 45His Tyr
Lys Leu Val Pro Gln Ile Asp Thr Arg Asp Cys Gly Pro Ala 50 55 60Val
Leu Ala Ser Val Ala Lys His Tyr Gly Ser Asn Tyr Ser Ile Ala65 70 75
80Tyr Leu Arg Glu Leu Ser Lys Thr Asn Lys Gln Gly Thr Thr Ala Leu
85 90 95Gly Ile Val Glu Ala Ala Lys Lys Leu Gly Phe Glu Thr Arg Ser
Ile 100 105 110Lys Ala Asp Met Thr Leu Phe Asp Tyr Asn Asp Leu Thr
Tyr Pro Phe 115 120 125Ile Val His Val Ile Lys Gly Lys Arg Leu Gln
His Tyr Tyr Val Val 130 135 140Tyr Gly Ser Gln Asn Asn Gln Leu Ile
Ile Gly Asp Pro Asp Pro Ser145 150 155 160Val Lys Val Thr Arg Met
Ser Lys Glu Arg Phe Gln Ser Glu Trp Thr 165 170 175Gly Leu Ala Ile
Phe Leu Ala Pro Gln Pro Asn Tyr Lys Pro His Lys 180 185 190Gly Glu
Lys Asn Gly Leu Ser Asn Phe Phe Pro Leu Ile Phe Lys Gln 195 200
205Lys Ala Leu Met Thr Tyr Ile Ile Ile Ala Ser Leu Ile Val Thr Leu
210 215 220Ile Asp Ile Val Gly Ser Tyr Tyr Leu Gln Gly Ile Leu Asp
Glu Tyr225 230 235 240Ile Pro Asp Gln Leu Ile Ser Thr Leu Gly Met
Ile Thr Ile Gly Leu 245 250 255Ile Ile Thr Tyr Ile Ile Gln Gln Val
Met Ala Phe Ala Lys Glu Tyr 260 265 270Leu Leu Ala Val Leu Ser Leu
Arg Leu Val Ile Asp Val Ile Leu Ser 275 280 285Tyr Ile Lys His Ile
Phe Thr Leu Pro Met Ser Phe Phe Ala Thr Arg 290 295 300Arg Thr Gly
Glu Ile Thr Ser Arg Phe Thr Asp Ala Asn Gln Ile Ile305 310 315
320Asp Ala Val Ala Ser Thr Ile Phe Ser Ile Phe Leu Asp Met Thr Met
325 330 335Val Ile Leu Val Gly Gly Val Leu Leu Ala Gln Asn Asn Asn
Leu Phe 340 345 350Phe Leu Thr Leu Leu Ser Ile Pro Ile Tyr Ala Ile
Ile Ile Phe Ala 355 360 365Phe Leu Lys Pro Phe Glu Lys Met Asn His
Glu Val Met Glu Ser Asn 370 375 380Ala Val Val Ser Ser Ser Ile Ile
Glu Asp Ile Asn Gly Met Glu Thr385 390 395 400Ile Lys Ser Leu Thr
Ser Glu Ser Ala Arg Tyr Gln Asn Ile Asp Ser 405 410 415Glu Phe Val
Asp Tyr Leu Glu Lys Asn Phe Lys Leu His Lys Tyr Ser 420 425 430Ala
Ile Gln Thr Ala Leu Lys Ser Gly Ala Lys Leu Ile Leu Asn Val 435 440
445Val Ile Leu Trp Tyr Gly Ser Arg Leu Val Met Asp Asn Lys Ile Ser
450 455 460Val Gly Gln Leu Ile Thr Phe Asn Ala Leu Leu Ser Tyr Phe
Ser Asn465 470 475 480Pro Ile Glu Asn Ile Ile Asn Leu Gln Ser Lys
Leu Gln Ser Ala Arg 485 490 495Val Ala Asn Thr Arg Leu Asn Glu Val
Tyr Leu Val Glu Ser Glu Phe 500 505 510Glu Lys Asp Gly Asp Leu Ser
Glu Asn Ser Phe Leu Asp Gly Asp Ile 515 520 525Ser Phe Glu Asn Leu
Ser Tyr Lys Tyr Gly Phe Gly Arg Asp Thr Leu 530 535 540Ser Asp Ile
Asn Leu Ser Ile Lys Lys Gly Ser Lys Val Ser Leu Val545 550 555
560Gly Ala Ser Gly Ser Gly Lys Thr Thr Leu Ala Lys Leu Ile Val Asn
565 570 575Phe Tyr Glu Pro Asn Lys Gly Ile Val Arg Ile Asn Gly Asn
Asp Leu 580 585 590Lys Val Ile Asp Lys Thr Ala Leu Arg Arg His Ile
Ser Tyr Leu Pro 595 600 605Gln Gln Ala Tyr Val Phe Ser Gly Ser Ile
Met Asp Asn Leu Val Leu 610 615 620Gly Ala Lys Glu Gly Thr Ser Gln
Glu Asp Ile Ile Arg Ala Cys Glu625 630 635 640Ile Ala Glu Ile Arg
Ser Asp Ile Glu Gln Met Pro Gln Gly Tyr Gln 645 650 655Thr Glu Leu
Ser Asp Gly Ala Gly Ile Ser Gly Gly Gln Lys Gln Arg 660 665 670Ile
Ala Leu Ala Arg Ala Leu Leu Thr Gln Ala Pro Val Leu Ile Leu 675 680
685Asp Glu Ala Thr Ser Ser Leu Asp Ile Leu Thr Glu Lys Lys Ile Ile
690 695 700Ser Asn Leu Leu Gln Met Thr Glu Lys Thr Ile Ile Phe Val
Ala His705 710 715 720Arg Leu Ser Ile Ser Gln Arg Thr Asp Glu Val
Ile Val Met Asp Gln 725 730 735Gly Lys Ile Val Glu Gln Gly Thr His
Lys Glu Leu Leu Ala Lys Gln 740 745 750Gly Phe Tyr Tyr Asn Leu Phe
Asn 755 76033900DNAStaphylococcus mutans 33atggatccta aatttttaca
aagtgcagaa ttttatagga gacgctatca taattttgcg 60acactattaa ttgttccttt
ggtctgcttg attatcttct tggtcatatt cctttgtttt 120gctaaaaaag
aaattacagt gatttctact ggtgaagttg caccaacaaa ggttgtagat
180gttatccaat cttacagtga cagttcaatc attaaaaata atttagataa
taatgcagct 240gttgagaagg gagacgtttt aattgaatat tcagaaaatg
ccagtccaaa ccgtcagact 300gaacaaaaga atattataaa agaaagacaa
aaacgagaag agaaggaaaa gaaaaaacac 360caaaagagca agaaaaagaa
gaagtctaag agcaagaaag cttccaaaga taagaaaaag 420aaatcgaaag
acaaggaaag cagctctgac gatgaaaatg agacaaaaaa ggtttcgatt
480tttgcttcag aagatggtat tattcatacc aatcccaaat atgatggtgc
caatattatt 540ccgaagcaaa ccgagattgc tcaaatctat cctgatattc
aaaaaacaag aaaagtgtta 600atcacctatt atgcttcttc tgatgatgtt
gtttctatga aaaaggggca aaccgctcgt 660ctttccttgg aaaaaaaggg
aaatgacaag gttgttattg aaggaaaaat taacaatgtc 720gcttcatcag
caactactac taaaaaagga aatctcttta aggttactgc caaagtaaag
780gtttctaaga aaaatagcaa actcatcaag tatggtatga caggcaagac
agtcactgtc 840attgataaaa agacttattt tgattatttc aaagataaat
tactgcataa aatggataat 90034300PRTStaphylococcus mutans 34Met Asp
Pro Lys Phe Leu Gln Ser Ala Glu Phe Tyr Arg Arg Arg Tyr1 5 10 15His
Asn Phe Ala Thr Leu Leu Ile Val Pro Leu Val Cys Leu Ile Ile 20 25
30Phe Leu Val Ile Phe Leu Cys Phe Ala Lys Lys Glu Ile Thr Val Ile
35 40 45Ser Thr Gly Glu Val Ala Pro Thr Lys Val Val Asp Val Ile Gln
Ser 50 55 60Tyr Ser Asp Ser Ser Ile Ile Lys Asn Asn Leu Asp Asn Asn
Ala Ala65 70 75 80Val Glu Lys Gly Asp Val Leu Ile Glu Tyr Ser Glu
Asn Ala Ser Pro 85 90 95Asn Arg Gln Thr Glu Gln Lys Asn Ile Ile Lys
Glu Arg Gln Lys Arg 100 105 110Glu Glu Lys Glu Lys Lys Lys His Gln
Lys Ser Lys Lys Lys Lys Lys 115 120 125Ser Lys Ser Lys Lys Ala Ser
Lys Asp Lys Lys Lys Lys Ser Lys Asp 130 135 140Lys Glu Ser Ser Ser
Asp Asp Glu Asn Glu Thr Lys Lys Val Ser Ile145 150 155 160Phe Ala
Ser Glu Asp Gly Ile Ile His Thr Asn Pro Lys Tyr Asp Gly 165 170
175Ala Asn Ile Ile Pro Lys Gln Thr Glu Ile Ala Gln Ile Tyr Pro Asp
180 185 190Ile Gln Lys Thr Arg Lys Val Leu Ile Thr Tyr Tyr Ala Ser
Ser Asp 195 200 205Asp Val Val Ser Met Lys Lys Gly Gln Thr Ala Arg
Leu Ser Leu Glu 210 215 220Lys Lys Gly Asn Asp Lys Val Val Ile Glu
Gly Lys Ile Asn Asn Val225 230 235 240Ala Ser Ser Ala Thr Thr Thr
Lys Lys Gly Asn Leu Phe Lys Val Thr 245 250 255Ala Lys Val Lys Val
Ser Lys Lys Asn Ser Lys Leu Ile Lys Tyr Gly 260 265 270Met Thr Gly
Lys Thr Val Thr Val Ile Asp Lys Lys Thr Tyr Phe Asp 275 280 285Tyr
Phe Lys Asp Lys Leu Leu His Lys Met Asp Asn 290 295
3003520PRTArtificialSynthetic peptide IH-1 35Gly Ser Leu Ser Thr
Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr Gln1 5 10 15Ala Leu Gly Lys
203620PRTArtificialSynthetic peptide IH-2 36Ser Gly Ser Leu Ser Thr
Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Leu Gly 20
3720PRTArtificialSynthetic peptide B1 37Ser Ser Leu Ser Thr Phe Phe
Arg Leu Phe Asn Arg Ser Phe Thr Gln1 5 10 15Ala Leu Gly Lys
203820PRTArtificialSynthetic peptide C1 38Ser Gly Leu Ser Thr Phe
Phe Arg Leu Phe Asn Arg Ser Phe Thr Gln1 5 10 15Ala Leu Gly Lys
203920PRTArtificialSynthetic peptide D1 39Ser Gly Ser Ser Thr Phe
Phe Arg Leu Phe Asn Arg Ser Phe Thr Gln1 5 10 15Ala Leu Gly Lys
204020PRTArtificialSynthetic peptide E1 40Ser Gly Ser Leu Thr Phe
Phe Arg Leu Phe Asn Arg Ser Phe Thr Gln1 5 10 15Ala Leu Gly Lys
204120PRTArtificialSynthetic peptide F1 41Ser Gly Ser Leu Ser Thr
Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Leu Lys
204220PRTArtificialSynthetic peptide G1 42Ser Gly Ser Leu Ser Thr
Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Gly Lys
204320PRTArtificialSynthetic peptide H1 43Ser Gly Ser Leu Ser Thr
Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Leu Gly Lys
204420PRTArtificialSynthetic peptide A2 44Ser Gly Ser Leu Ser Thr
Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Ala Leu Gly
Lys 204521PRTArtificialSynthetic peptide B2 45Ser Gly Ser Leu Ser
Thr Phe Phe Val Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Leu Gly
Lys 204621PRTArtificialSynthetic peptide C2 46Ser Gly Ser Leu Ser
Thr Phe Phe Ala Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Leu Gly
Lys 204721PRTArtificialSynthetic peptide D2 47Ser Gly Ser Leu Ser
Thr Phe Phe Arg Leu Phe Asn Val Ser Phe Thr1 5 10 15Gln Ala Leu Gly
Lys 204821PRTArtificialSynthetic peptide E2 48Ser Gly Ser Leu Ser
Thr Phe Phe Arg Leu Phe Asn Ala Ser Phe Thr1 5 10 15Gln Ala Leu Gly
Lys 204921PRTArtificialSynthetic peptide F2 49Ser Gly Ser Leu Ser
Thr Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Leu Gly
Val 205021PRTArtificialSynthetic peptide G2 50Ser Gly Ser Leu Ser
Thr Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln Ala Leu Gly
Ala 205118PRTArtificialSynthetic peptide B3 51Ser Gly Thr Leu Ser
Thr Phe Phe Arg Leu Phe Asn Arg Ser Phe Thr1 5 10 15Gln
Ala5225DNAArtificialSynthetic P1-HK13 primer 52cacaacaact
tattgacgct atccc 255330DNAArtificialSynthetic P2-HK13 primer
53ggcgcgccaa ctggcaacag gcagcagacc 305429DNAArtificialSynthetic
P3-HK13 primer 54ggccggcctc aaaacgatgc tgtcaaggg
295521DNAArtificialSynthetic P4-HK13 primer 55agattatcat tggcggaagc
g 215630DNAArtificialSynthetic Erm-19 primer 56ggcgcgcccc
gggcccaaaa tttgtttgat 305730DNAArtificialSynthetic Erm-20 primer
57ggccggccag tcggcagcga ctcatagaat 30
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