Note: Descriptions are shown in the official language in which they were submitted.
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SIGNAL PEPTIDES, NUCLEIC ACID MOLECULES AND
METHODS OF TREATMENT
BACKGROUND OF INVENTION
Field of the Invention
[0001] 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.
Description of the Related Art
[0002] 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
cornmunicate 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 inutans (Li et al., 2001), and induction of virulence factors in
Staphylococcus aureus (Ji et aL, 1995).
[0003] Controlling bacterial biofihns is desirable for almost every human
enterprise in which solid surfaces are introduced into non-sterile aqueous
environments. U.S. Patent 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
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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
biofilrn
physiology and genetics with the ultimate goal of controlling surface-mediated
microbial growth are lim.itless.
[0004] 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.
[0005] Caries and periodontal diseases are two of the most common chronic
irifectious diseases affecting mankind, and are always associated with dental
plaque
formed as a biof'ilm on tooth surfaces. Thus the prevention of dental caries
and
periodontal diseases is targeted at the control of dental plaque.
[0006] Dental plaque is produced by sequential attachment of a variety of
bacteria, which is dependent on both species involved and the surface
composition
(Kawashima et al., Oral. Microbiol. Iminunol. 18: 220-225, 2003). Oral
streptococci
and Actinonlyces spp. are the first to appear on the surface of the teeth.
Streptococci
account for approximately 20% of the salivary bacteria, which include
Streptococcus
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spp. such as Streptococcus mutans, Streptococcus sobrinus, Streptococcus
sanguis,
Streptococcus gordonii, Streptococcus oralis and Streptococcus initis. Two
"mutans
group" streptococci, S. mutans and S. sobrinus, are directly involved in the
initiation
of dental caries, (Devulapalle et al., Carbohydr. Res.339:029-1034, 2004). As
S.
rnutans has evolved to depend on a biofihn lifestyle for survival and
persistence in the
oral cavity combined with its role as an opportunistic pathogen, it has become
the
best-studied example of a biofilm-fonning, disease-causing Streptococcus
(Burne,
R.A., J. Dent. Res. 77:445-452, 1998).
[0007] Many Streptococci use quorum-sensing systems to regulate several
physiological processes, including the ability to incorporate foreign DNA,
tolerate
acid, fonn biofilm, and become virulent. These quorum-sensing systems are
primarily
made of a small competence-stimulating peptide (CSP) that is detected by
neighbouring cells via a histidine kinase/response regulator pair. It has been
demonstrated that analogues of quorum-sensing peptides (analogues of CSP) can
competitively inhibit biofilm fonnation in S. mutans (Cvitkovitch, et al., US
Patent
Application No. 20020081302, 2002).
[0008] 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:30]
to
S. mutans histidine kinase [SEQ ID NO:4]. In certain embodiments the compound
is
a peptide or an antibody. In some embodiments the compound is 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].
[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
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compound is used for inhibiting the growth of S. mutans, for inhibiting dental
caries,
or for improving dental health.
[0011] In an aspect of the invention, provided is an isolated polypeptide
comprising a fragment of SEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting
S. mutans genetic competence.
[0012] In an aspect of the invention, provided is an isolated polypeptide that
is
at least 95% identical to SEQ ID NO:2 or SEQ ID NO:30, and capable of
inhibiting S.
mutans genetic competence.
[0013] In an embodiment of the invention, 1-5 amino acids have been
removed from the COOH terminal of SEQ ID NO:2 or SEQ ID NO:30.
[0014] In an aspect of the invention, provided is an isolated polypeptide
comprising a fragment of SEQ ID NO:2 or SEQ ID NO:30 and capable of inhibiting
S. mutans biofilm formation.
[0015] In an aspect of the invention, provided is an isolated polypeptide that
is
at least 95% identical to SEQ ID NO:2 or SEQ ID NO:30 and capable of
inhibiting S.
mutans biofilm formation.
[0016] In an embodiment of the invention, 1-5 amino acids of the aniino acid
sequence of SEQ ID NO:2 or SEQ ID NO:30 have been modified to include up to 1
amino acid substitution per each 10 amino acids.
[0017] In an aspect of the invention, provided is a composition for reducing
S.
mutans biofilm formation comprising a peptide derivative of CSP.
[0018] In an aspect of the invention, provided is a composition for reducing
S.
mutans transformation efficiency comprising a peptide derivative of CSP.
[0019] In an embodiment of the invention, the peptide analog is a peptide
having an amino acid sequence selected from a group consisting of: SEQ ID No:
37,
SEQ ID No: 39, SEQ ID No: 42,SEQ ID No: 44 and SEQ ID No: 47.
[0020] In an embodiment of the invention, the peptide analog is a peptide
having amino acid sequnece SEQ ID No:44.
[0021] In an aspect of the invention, provided is a cell genetically
engineered
to produce the any one of the compositions of the invention set out above.
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[0022] In an embodiment of the invention, the cell is bacterial.
[0023] In an embodiment of the invention, the cell is S. mutans.
[0024] In an aspect of the invention, provided is use of the cell according to
the invention to produce any one of the compositions of the invention set out
above.
[0025] In an aspect of the invention, provided is a composition comprising the
cell according to the invention and a pharmaceutically acceptable adjuvant.
[0026] In an aspect of the irivention, provided is a method of inhibiting the
growth of S. nzutans comprising administering a composition according to any
one of
claims 9 to 12 or 17.
[0027] In an aspect of the invention, provided is a method of inhibiting
dental
caries comprising administering any one of the coinpositions of the invention
set out
above.
[0028] In an aspect of the invention, provided is a method of improving dental
health comprising administering any one of the compositions of the invention
set out
above.
[0029] In an aspect of the invention, provided is an isolated polypeptide
having
the amino acid sequence selected from a group consiting of SEQ ID No: 37, SEQ
ID
No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0030] In an aspect of the invention, provided is a peptide analog of S.mutans
competence stimulating peptide (CSP) which inhibits biofilm formation.
[0031] In an aspect of the invention, provided is a composition for inhibiting
biofilm forxnation comprising a polypeptide having an amino acid sequence
selected
from a group consisting of: SEQ II.ID No: 37, SEQ ID No: 39, SEQ ID No: 42,
SEQ ID
No: 44, and SEQ ID No: 47 and an orally acceptable excipient.
[0032] In an aspect of the invention, provided is a pharmaceutical composition
comprising at least one CSP inhibitor and a pharmaceutically acceptable
carrier.
[0033] In an aspect of the invention, provided is a method of treating or
preventing a bacterial infection caused by a biofilm forming bacterium
comprising
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administering a therapeutically effective amount of a pharmaceutical
composition
according to the invention.
[0034] In an aspect of the invention, provided is a method of preventing
dental
plaque formation comprising administering a therapeutically effective amount
of a
pharmaceutical composition according to the invention.
[0035] In an aspect of the invention, provided is a method of treating or
preventing a condition caused by a dental plaque associated bacterium
comprising
administrering a therapeutically effective amount of a pharmaceutical
composition
according to the invention.
[0036] In an aspect of the invention, provided is a use of a CSP inhibitor for
the preparation of a medicament for treatment and prevention of an infection
caused
by a biofilm forming bacterium.
[0037] In an embodiment of the invention, the CSP inhibitor is is a peptide
analog of S.mutans competence stimulating peptide (CSP) which inhibits biofilm
formation.
[0038] In another embodiment of the invention, the CSP inhibtior is a
polypeptide having the amino acid sequence selected from a group consisting of
:
SEQ ID No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0039] In an embodiment of the invention, the CSP inhibitor is a polypeptide
having the amino acid sequence of SEQ ID No: 44.
[0040] In an embodiment of the invention, the CSP inhibitor is an antibody
specific for CSP or a fragment thereof.
[0041] In an embodiment of the invention, the CSP inhibitor is an antisense
oligonucleotide which inhibits CSP expression or transcription.
[0042] In an embodiment of the invention, the CSP inhibitor is an antisense
oligonucleotide which inhibits CSP peptide export.
[0043] In an aspect of the present invention, provided is a composition for
inhibiting biofilm formation in Streptococcus spp, said composition comprising
at
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least one peptide having an amino acid sequence selected from a group
consisting of:
No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0044] In an embodiment of the invention, the composition further comprises
one or more ingredients selected from a group consisting of: a surfactant, an
antiseptic, and an antibiotic.
[0045] In an embodiment of the invention, the composition comprises
between 1 gg/ml and 100 g/ml of the peptide.
[0046] In an aspect of the present invention, provided is a use of at least
one
peptide having an amino acid sequence selected from a group consisting of: No:
37,
SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47., for the
preparation of a medicament for inhibition of biofilm formation in
Streptococcus spp.
[0047] In an aspect of the present invention, provided is a use of at least
one
peptide having an amino acid sequence selected from a group consisting of No:
37,
SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47, for prevention
or inhibition of biofilm formation in Streptococcus spp.
[0048] In an aspect of the present invention, provided is a use of at least
one
peptide having an amino acid sequence selected from a group consisting of: No:
37,
SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47, for treatment
or prevention of a condition caused by dental plaque associated Streptococcus
spp.
[0049] In an aspect of the present invention, provided is a method of treating
or preventing a Streptococcus spp infection in a patient in need thereof,
comprising
administering a therapeutically effective amount of at least one peptide
having an
amino acid sequence selected from a group consisting of: No: 37, SEQ ID No:
39,
SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0050] In an aspect of the present invention, provided is a method of
inhibiting or preventing biofihn formation in Streptococcus spp in a patient
in need thereof, comprising administering a therapeutically effective amount
of at least one
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peptide having an amino acid sequence selected from a group consisting of: No:
37,
SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0051] In an aspect of the present invention, provided is a method of treating
or preventing a condition caused by dental plaque associated Streptococcus spp
in a
patient in need thereof comprising administering a therapeutically effective
amount of
at least one peptide having an amino acid sequence selected from a group
consisting
of: No: 37, SEQ ID No: 39, SEQ ID No: 42, SEQ ID No: 44, and SEQ ID No: 47.
[0052] In an embodiment of the invention, the condition caused by dental
plaque associated Streptococcus spp is selected from a group consisting of:
dental
caries, gingivitis, and endocarditis.
[0053] In an embodiment of the invention, the peptide has the amino acid
sequence of SEQ ID NO. 44.
[0054] In an embodiment of the invention, the Streptococcus spp is selected
from a group consisting o~ Streptococcus sobrinus, Streptococcus sanguis,
Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis,
Streptococcus
salivarius, Streptococcus cristatus, Streptococcus pneumoniae, Streptococcus
pyogenes, and Streptococcus agalactiae.
[0055] In an embodiment of the invention, the Streptococcus spp is
Streptococcus pneumoniae.
[0056] 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
[0057] Figure 1 shows the schematic layout of the arrangement of the genetic
locus encoding the signal peptide precursor (ComC) [SEQ ID NO: 1], the
histidine
kinase (ComD) [SEQ ID NO:3] and the response regulator (ComE) [SEQ ID NO:5].
This arrangement is different from other loci in related streptococci since
the comC
gene [SEQ ID NO:1] is transcribed from its own unique promoter, unlike the
genes
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thus far described in other streptococci that are arranged in an operon-like
cluster with
the comC/DE genes being transcribed from a single promoterand the comC gene
[SEQ ID NO:1] is separated by 148 nucleotides from the comD gene [SEQ ID
NO:3].
[0058] Figure 2 shows the nucleic acid molecules SEQ ID Nos. 1, 3, 5, and
29. Figure 2A. S. mutans comC gene [SEQ ID NO:1]. Encodes a precursor to a
signal peptide [SEQ ID NO:2]. Figure 2B. S. mutans CSP encoding sequence [SEQ
ID NO:29]. Encodes a Competence Stimulating Peptide [SEQ ID NO:30]. Figure
2C. S. mutans comD gene [SEQ ID NO:3]. Figure 2D. S. mutans comE gene [SEQ
ID NO:5]. Encodes a response regulator that activates transcription of a
number of
genes [SEQ ID NO:6].
[0059] Figure 3. Sequence of the deduced amino acid sequence of the signal
peptide [SEQ ID NO:2], histidine kinase [SEQ ID NO:4], and response regulator
[SEQ ID NO:6]. Figure 3A. S. mutans ComC protein (CSP Precursor) [SEQ ID
NO:2]. Figure 3B. S. mutans ComD protein (Histidine Kinase) [SEQ ID NO:4].
Figure 3C. S. tnutans ComE protein (Response Regulator) [SEQ ID NO:6].
[0060] Figure 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 peptidel'.
[0061] Figure 5 shows the synthetic signal peptide [SEQ ID NO:14] that is
effective at inducing competence, biofilm formation . and acid tolerance in
Streptococcus mutans.
[0062] Figure 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.
[0063] Figure 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).
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[0064] Figure 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.
[0065] Figure 9 shows the ComCDE local region [SEQ ID NO:21]. 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.
[0066] Figure 10 shows The comX DNA sequence [SEQ ID NO:22], protein
sequence [SEQ ID NO:23], and the comX gene local region [SEQ ID NO:24] with
100bp included both upstream and downstream (promoter is upstream). Figure
10A.
S. mutans comX gene [SEQ ID NO:22]. Figure lOB. S. mutans ComX protein [SEQ
ID NO:23]. Figure 10C. S. mutans comX gene local region [SEQ ID NO:24].
[0067] Figure 11 shows the coinA and comB nucleotide [SEQ ID NO:25] and
[SEQ ID NO:27] and amino acid sequences [SEQ ID NO:26] and [SEQ ID NO:28].
ComA and ComB are the components of the CSP exporter. Figure 11A. S. mutans
comA gene [SEQ ID NO:25]. Figure 11B. S. mutans ComA protein [SEQ ID
NO:26]. Figure 11C. S. mutans comb gene [SEQ ID NO:27] Figure 11D. S. mutans
ComB protein [SEQ ID NO:28].
[0068] Figure 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:14] 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.
[0069] Figure 13 is a schematic representation of quorum sensing circuit in S.
mutans.
[0070] Figure 14 shows the effect of different concentrations of Hl on
genetic transformation of S. mutans wild-type UA159. Results are expressed as
the
mean SE of three independent experiments.
[0071] Figure 15 shows the effect of different concentrations of H1 on
genetic transformation of S. nautans comD null mutant.
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[0072] Figure 16 shows the effect of,different concentrations ( g/ml) of CSP
and Hl on cell growth of S. mutans wild-type UA159 in THYE at pH 5.5. Means
GD600 values SE. Results represent the average of three independent
experiments.
[0073] Figure 17 shows the effect of different concentrations ( g/ml) of CSP
and H1 on cell growth of S. mutans wild-type UA159 in THYE at pH 7.5. Means
GD600 values SE. Results represent the average of three independent
experiments.
[0074] Figure 18 shows the effects of synthetic CSP analogues (B2, B3, C2,
E2, Fl and H1 peptides) on Streptococcus nautans biofilm formation.
[0075] Figure 19 shows the effect of E2 peptide on Streptococcus sobrinus
biofilm formation.
[0076] Figure 20 shows the effect of E2 peptide on Streptococcus oralis
biofihn formation.
[0077] Figure 21 shows the effect of E2 peptide on Streptococcus sanguis
biofilm formation.
[0078] Figure 22 shows the effect of E2 peptide on Streptococcus mitis
biofilm formation.
[0079] Figure 23 shows the effect of E2 peptide on Streptococcus gordonii
biofilm formation.
[0080] Figure 24 shows the effect of E2 peptide on Streptococcus
pneumoniae biofilm formation
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0081] 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 biof lm. The cells growing in
this biofilm
environment use a small peptide signal rriolecule 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
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environment, as well as that of acid tolerance, the cell's ability to survive
pH levels as
low as pH 3Ø 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.
[0082] We have identified a genetic locus in S. mutans consisting of three
genes that encode: 1) a peptide precursor [SEQ ID NO:2] that is processed
during
export into a secreted 21-amino acid peptide (CSP) [SEQ ID NO:30]; 2) a
histidine
kinase [SEQ ID NO:4] that acts as a cell surface receptor activated by the
peptide; 3)
a response regulator [SEQ ID NO:6] 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.
[0083] Streptococcus mutans is a resident of the biofiln 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 it 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 mature wild type CSP peptide
where
amino acids are removed from the N- and/or COOH terminal of the peptide andlor
substitutions of internal amino acid residues. We delete, one, two to 5, 6 to
10 and 10
to 15 amino acids from the mature wild type CSP peptide (for example at either
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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:30] 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.
[0084] 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:14] 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.
[0085] Treatment or prevention of dental caries comprises addition of
compounds that inhibit the stimulatory action of the 21-amino acid peptide
[SEQ.ID
NO:30] 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.
[0086] Streptococcus mutans is also implicated in causing infective
endocarditis. Jnhibitors of biofilm formation, and hence aggregation are
useful in the
treatment of these bacterial infections as well.
[0087] We have also identified compounds which inhibit biofilm formation
for other dental plaque baterial species such as Actinomyces spp. and other
types of
streptococci such as Streptococcus sObrinus, Streptococcus sanguis,
Streptococcus
gordonii, Streptococcus or=alis and Streptococcus mitis. Streptococci account
for
approximately 20% of the salivary bacteria. The compounds comprise the
modified
structures of the S. mutans mature wild type CSP peptide having amino acids
removed
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from the N- and/or COOH terminal of the peptide and/or substitutions of
internal
amino acid residues.
Identification and Characterization of Competence Stimulating Peptide (CSP),
Histidine Kinase (HIi) and Response Regulator (RR)
Competence Stimulating Peptide
[0088] 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
NO:2]
peptide produced by a cell including a nucleic acid molecule encoding CSP [SEQ
ID
NO:l] 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 IDNO:1]. The peptide we work with is preferably chemically
synthesized
[SEQ ID NO:14].
[0089] CSP-encoding nucleic acid molecules [SEQ ID NO:1] 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:2] 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:2] 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
[0090] In accordance with certain embodiments of the present invention, an
isolated HK [SEQ ID NO:4] 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:3] operably linked to a promoter. In another
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embodiment of the invention an isolated, nucleic acid molecule encoding a HK
polypeptide [SEQ IDNO:4] is disclosed.
[0091] HK-encoding nucleic acid molecules and molecules having sequence
identity or which hybridize to the HK-encoding sequence [SEQ ID NO:3] 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:4] 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:4]
or
polypeptides having sequence identity, which have HK activity, as prepared by
the
processes described in this application.
Response Regulator
[0092] In accordance with certain embodiments of the present invention an
isolated RR [SEQ ID NO:6] from S. mutans is disclosed. A recombinant isolated
RR
[SEQ ID NO:6] polypeptide produced by a cell including a nucleic acid molecule
encoding RR [SEQ ID NO:5] 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.
[0093] 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:5] 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:6] 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
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16
include RR [SE ID NO:6] or polypeptides having sequence identity, which have
RR
activity, as prepared by the processes described in this application.
[0094] The comA and comB nucleotide [SEQ ID NO:25 and SEQ ID NO:27]
and amino acid sequences [SEQ ID NO:26 and SEQ ID NO:28] are also aspects of
certain embodiments of the invention. ConiA 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
[0095] 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:1].
(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:2]. 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:2] 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, JD 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:1], then Sequence A will preferably be identical to the referenced portion
of the
nucleic acid molecule in [SEQ ID NO: 1], except that Sequence A may include up
to
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:1].
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.
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17
[0096] Nucleic acid molecules may encode conservative amino acid changes
in CSP peptide [SEQ ID NO:2]. 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.
[0097] 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:2]. 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:2]. The DNA or RNA can encode fragments or variants of
CSP. Fragments are useful as immunogens and in iinmunogenic compositions (U.S.
Patent 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:1]. Sequence identity is preferably measured with the
ClustalW program (preferably using default parameters) (Thompson, JD et al.,
Nucleic Acid Res. 22:4673-4680).
[0098] Nucleic acid molecules functionally equivalent to the CSP nucleic acid
molecule in [SEQ ID NO:1] will be apparent from the following description. For
example, the sequence shown in [SEQ ID NO:1] 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:1], 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%.
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18
[0099] 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:2]. 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:2]. The ClustalW program preferably assesses sequence identity.
[00100] Since the genetic code is degenerate, the nucleic acid sequence in
[SEQ ID NO: 1] 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:1].
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:2] are within the scope of various
embodiments of the invention.
[00101] 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: 1] 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:1 ] or its variants. Such nucleic acid molecules
preferably
hybridize to the sequence in [SEQ ID NO:1] 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 C or about 42 C is considered low stringency, and a teinperature of about
50-65
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
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19
mammalian peptide), including contacting a sample containing nucleic acid
molecules
including all or part of [SEQ ID NO:1] (preferably at least about 15 or 20
nucleotides
of [SEQ ID NO:1]) 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:1].). Similar methods are described in
U.S. Patent
No. 5,851,788, which is incorporated by reference in its entirety.
[00102] 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: 1], for example as probes or in assays to identify antagonists or
iiiliibitors 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.
[00103] Certain einbodiments 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:2] 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. Patent Nos. 5,837,472 and 5,801,233, which are
incorporated by
reference in their entirety.
[00104] A nucleic acid molecule described above is considered to have a
function substantially equivalent to the CSP nucleic acid molecules [SEQ ID
NO:1] of
the present invention if the peptide [SEQ ID NO:2] produced by the nucleic
acid
molecule has CSP activity. A peptide has CSP activity if it can stimulate
genetic
conipetence and acid tolerance in S. inutans. Activation of the HK [SEQ ID
NO:4]IRR [SEQ ID NO:6] 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
plasniid 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:4]/RR [SEQ ID NO:6] and subsequent genes activated by the signal cascade
initiated by the signal peptide, measurement of the conferment of erythromycin
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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 biofihns). Activation of the HK [SEQ ID NO:4]/R.R [SEQ ID
NO:6] 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
[00105] 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
elenients are derived from a variety of sources, and they may be readily
selected by
one with ,ordinary skill in the art (Sainbrook, 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-
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21
specific. Transcription is enhanced with promoters known in the art for
expression.
The CMV and SV40 promoters are commonly iised 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).
[00106] 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.
[00107] 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.
[00108] 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 turnefaciens-mediated transformation, particle-bombardment-
mediated transformation, direct uptake, microinj ection, 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.
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22
[00109] 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.
[00110] 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:2]
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: 1] (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
[00111] 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 CSP in [SEQ ID NO:1]. Certain embodiments of the invention also
include at least 15 consecutive nucleotides of [SEQ ID NO:1]. 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:1] 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.
[00112] Functionally equivalent CSP nucleic acid molecules from other cells,
or equivalent CSP-encoding cDNAs or synthetic DNAs, can also be isolated by
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23
amplification using Polymerase Chain Reaction (PCR) methods. Oligonucleotide
primers, such as degenerate primers, based on [SEQ ID NO:1] 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 eDNA libraries of other organisms. The
oligonucleotides can also be used as probes to screen cDNA libraries.
Functionally equivalent peptides, peptides and proteins
[00113] 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.
[00114] 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 S. nautans
native CSP.
[00115] 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 1st, 2nd, 3rd, 4th' or 5th residues from the N- and
C-termini of
the mature S. mutans CSP sequence [SEQ ID NO:30]. 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.
[00116] 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 [SEQ ID NO:39] 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
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24
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 Hl 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 receptor, and therefore is a ConiD-dependent process (Fig. 3).
[00117] In contrast; the peptide analogs IH-1 [SEQ ID NO:31], 1H-2 [SEQ ID
NO:32], B1 [SEQ ID NO:33], and Cl [SEQ ID NO:34] showed no significant effect
on transformation efficiency compared to the wild-type S. mutans 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 Dl [SEQ ID NO:35], El
[SEQ ID NO:36], F1 [SEQ ID NO:37], Gl [SEQ ID NO:38], A2 [SEQ ID NO:40],
B2 [SEQ ID NO:41], C2 [SEQ ID NO:42], D2 [SEQ ID NO:43], E2 [SEQ ID
NO:44], F2 [SEQ ID NO:45], G2 [SEQ ID NO:46], or B3 [SEQ ID NO:47] is
diminished compared to the wild-type S. fnutans CSP (5 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
S.
mutans CSP.
[00118] TABLE 1. Modified versions of the mature S. mutans CSP peptide
Peptide Amino acid sequence Modification
analog
CSP SGSLSTFFRLFNRSFTQALGK mature wild-type CSP sequence
IH-1 SGSLSTFFRLFNRSFTQALGK lst residue removed from N'
IH-2 SGSLSTFFRLFNRSFTQALGK lst residue removed from C'
B1 SGSLSTFFRLFNRSFTQALGK 2"d residue removed from N'
C 1 SGSLSTFFRLFNRSFTQALGK 3ra residue removed from N'
Dl SGSLSTFFRLFNRSFTQALGK 4th residue removed from N'
El SGSLSTFFRLFNRSFTQALGK 5th residue removed from N'
Fl SGSLSTFFRLFNRSFTQALGK 2"a residue removed from C'
Gl SGSLSTFFRLFNRSFTQAL-GK 3rd residue removed from C'
H1 SGSLSTFFRLFNRSFTQALGK 4th residue removed from C'
A2 SGSLSTFFRLFNRSFTQALGK 5th residue removed from C'
B2 SGSLSTFFVLFNRSFTQALGK Substitution of 1st R residue with V
C2 SGSLSTFFALFNRSFTQALGK Substitution of lst R residue with A
D2 SGSLSTFFRLFNVSFTQALGK Substitution of 2"a R residue with V
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E2 SGSLSTFFRLFNASFTQALGK Substitution of 2"d R residue with A
F2 SGSLSTFFRLFNRSFTQALGV Substitution of K residue with V
G2 SGSLSTFFRLFNRSFTQALGA Substitution of K residue with A
B3 SGTLSTFFRLFNRSFTQAD;K_ JH1005 CSP sequence
[00119] TABLE 2. Effect of 5 g/ml of peptide analogs on competence of S.
mutans wild-type UA159
Peptide Transformation Transformation
analog efficiency (vs no CSP) efficiency (vs 5 g CSP)
CSP 1554-fold increase -
IH-1 no effecta no effect
IH-2 no effect no effect
B 1 no effect no effect
C1 no effect no effect
Dl 275-fold increase 6-fold decrease
El 791-fold increase 2-fold decrease
Fl 541-fold increase 3-fold decrease
G1 848-fold increase 2-fold decrease
Hl 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
aNo effect: no significant difference by comparison with CSP.
[00120] TABLE 3. Effect of 5 g/ml of peptide analogs on growth at pH 7.5,
acid resistance, and biofilrn formation of S. mutans wild-type UA159
Peptide Growth Acid resistance Biofilm formation
analog (pH 7.5) (pH 5.5) (SDM-glucose)
CSP V growth growth no effect
IH-1 y growth growth no effect
IH-2 y growth growth no effect
B 1 y growth growth no effect
C1 y growth growth no effect
Dl y growth growth no effect
El y growth growth no effect
Fl y growth y growth 13 6.7% biomass
Gl y growth growth 124.4% biomass
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26
H1 no effect y growth no effect
A2 no effect I growth no effect
B2 no effect y growth no effect
C2 no effect y growth no effect
D2 no effect y growth no effect
E2 no effect y growth 138.9% biomass
F2 y growth y growth 138.7% biomass
G2 4 growth I growth 135.6% biomass
B3 no effect y growth 134.4% biomass
Multiple peptide analogs affect S. mutans cell growth in an acidic medium.
[00121] 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 Fl, 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. rnutans cell growth in an acidic medium (Fig. 4), while the
growth at
neutral pH is unaffected (Fig. 5).
S. mutans peptide analogs inhibit Streptococci biofilm formation.
[00122] 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 S. rnutarzs
peptide
analogs were tested for their ability to inhibit the formation of S. mutans
biofilms. In a
first study, the results of which are presented at Table 3, S. mutans peptide
analogs Fl
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27
[SEQ ID NO:37], Gl [SEQ ID NO:38], E2 [SEQ ID NO:44], F2 [SEQ ID NO:45],
G2 [SEQ ID NO:46] and B3 [SEQ ID NO:47] 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. mutan
[00123] In a second study, the anti-biofilm activity of B2 [SEQ ID NO:41], B3
[SEQ ID NO:47], C2 [SEQ ID NO:42], E2 [SEQ ID NO:44], Fl [SEQ ID NO":37],
and H1 [SEQ ID NO:39] was investigated. The anti-biofilm activity of the
peptide
analogues against S. mutans in terms of percentage inhibition varied from 0 to
80%
(Figure 18). Peptide analog E2 [SEQ ID NO:44] showed the highest anti-biofilm
activity (80% inhibition) among the six synthetic CSP analogues tested.
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.
[00124] In additional studies, the S. mutans peptide analogs are further
tested
for their ability to inhibit the forination of biofilms by other types of
bacteria, and in
particular, other dental plaque associated bacteria. The peptide analogs B3
[SEQ ID
NO:47], C2 [SEQ ID NO:42], E2 [SEQ ID NO:44] and Fl [SEQ ID NO:37], are
found to signifantly reduce biofilm formation in dental plaque associated
streptococci
including S. sobrinus, S. sanguis, S. gordonii, S. oralis, S. mitis and non-
dental plaque
associated Streptococci such as S. pneumoniae
[00125] The S. mutans derived E2 [SEQ ID NO:44] peptide at a concentration
as low as 5 g/mi showed inhibitory effects on both growth and biofilm
formation in
S. sobrinus, S. sanguis, S. gordonii, S. oralis, S. mitis and S. pneumoniae..
The percent
inhibition of biofilm formation in these organisms varied from 40 to 75%
(Figures 19,
20, 21, 22, 23, 24 and 25). Furthermore, the anti-biofilm activity of E2 [SEQ
ID
NO:44] peptide was tested against mixed culture of the above Streptococcus
spp. It
also showed a significant inhibitory effect on the mixed culture biofilm
formation
(data not shown).
[00126] A peptide is considered to possess a function substantially equivalent
to that of the CSP peptide [SEQ ID NO:2] if it has CSP activity. CSP activity
means
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28
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.
[00127] 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:30], then Sequence A will be identical to the referenced portion of the
peptide in
[SEQ ID NO:30], except that Sequence A may include up to 1 point niutations,
such
as substitutions with other amino acids, per each 10 amino acids of the
referenced
portion of the peptide in [SEQ ID NO:30]. Peptides, peptides and proteins
functional
equivalent to the CSP peptides can occur in a variety of forms as described
below.
[00128] Peptides biologically equivalent in function to CSP peptide include
amino acid sequences containing amino acid changes in the CSP sequence [SEQ ID
NO:2]. 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:2] 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.
[00129] 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
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29
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.
[00130] 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).
[00131] 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
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[00132] The variants preferably retain the same or similar CSP activity as the
naturally occurring CSP [SEQ ID NO:2]. The CSP activity of such variants can
be
assayed by techniques described in this application and known in the art.
[00133] Variants produced by combinations of the tech.niques described above
but which retain the same or similar CSP activity as naturally occurring CSP
[SEQ ID
NO:2] are also included in certain embodiments of the invention (for example,
combinations of amino acid additions, and substitutions).
[00134] 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).
[00135] 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).
[00136] Variants of CSP produced by techniques described above wh.ich
decrease biofilm formation are also included hi certain embodiments of the
invention
(for example, combinations of amino acid additions, and substitutions).
[00137] 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
[00138] 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.
[00139] As well, probes and antibodies for a histidine kinase [SEQ ID NO:3
and SEQ ID NO:4], response regulator [SEQ ID NO:5 and SEQ ID NO:6] comA
[SEQ IDNO:25 and SEQ ID NO:26] or comb [SEQ ID NO:27 and SEQ ID NO:28]
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31
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:3 and SEQ ID NO:4], response regulator [SEQ ID
NO:5 and SEQ ID NO:6] or comA [SEQ ID NO:25 and SEQ ID NO:26] and comB
[SEQ ID NO:27 and SEQ ID NO:28] of the invention. Certain embodiments of the
invention also include fragments of HK having HK activity, fragments of RR
[SEQ
ID NO:5 and SEQ ID NO:6] having RR activity and fragments of comA [SEQ ID
NO:25 and SEQ ID NO:26] or comB [SEQ ID NO:27 and SEQ ID NO:28] having
activity.
Design of CSP peptide competitive inhibitors
[00140] The activity of the CSP peptide [SEQ ID NO:2] may be varied by
carrying out selective site-directed mutagenesis. We characterize the binding
domain
and other critical arnino 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:1] 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. Peptide
analogs of S.
mutans CSP peptide can be prepared by deleting and/or substitituting amino
acids at
the C' or N' terminus of the CSP peptides using mutagenesis methods known in
the
art. 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 [SEQ ID NO:1] presented in this application and the peptides
produced by the modified sequences are encompassed by the present invention.
[00141] Peptide analogs of S. mutans CSP peptide prepared by deleting and/or
substitituting amino acids of the CSP peptides can be screened for biolfilm
formation
inhibitions. Screening assays are described below.
Pharmaceutical compositions
[00142] The CSP inhibitors are also useful when combined with a carrier in a
pharmaceutical composition. The compositions are useful when administered in
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32
methods of nledical 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:2], 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:2]. The compositions are also
useful
when administered in methods of medical treatment or prophylaxis of a disease,
disorder or abnormal physical state caused by other dental plaque causing
bacteria
including but not limited to Actinomyces spp. and other Streptococci spp.
[00143] The pharmaceutical compositions can be administered to humans or
animals by methods such as food, food additives, dentrifice gels, toothpaste,
mouthwash, dental floss, denture wash, denture adhesives, chewing gum,
candies,
biscuits, soft drinks or sports drinks in methods of medical treatment. The
CSP
inhibitors 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 dental caries, peridontal disease and
endocarditis.
In a preferred embodiment, the pharmaceutical compositions are used to treat
diseases
caused by Actinomyces spp. and Streptococci spp. In fiirther preferred
embodiment,
the pharmaceutical compositions are used to treat Streptococci infections
caused by
but not limited to: S. mutans, S. sobrinus, S. oralis, S. sanguis, S. rnitis,
S. gordonii, S.
pneumoniae, S. pyogenes, and S. agalactiae.
[00144] The pharmaceutical compositions according to the invention may be
prepared using a CSP inhibitor which is an antisense oligonucleotide. For
example, CSP
activity could be blocked by antisense mRNA which inhibits CSP expression or
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33
transcription. Alternatively, the antisense oligonucleotide may be one which
inhibits the
activity of the exporter that secretes the CSP from the cell. We have the
sequence of
these exporters. There are two copies of the genes (comAB) [SEQ ID NO:25 and
SEQ
ID NO:27] that are involved in export. The prepartion of antisense technology
is well
known in the art.
[00145] In one embodiment, the CSP inhibitor is an antisense oligonucleotide
which inhibits CSP expression or transcription. Preferrably, the antisense
oligonucleotide is an oligonucleotide complementary to at least 10 consecutive
nucleotides of an oligonucleotide encoding CSP, said oligonucleotide having
the nucleic
acid sequence of SEQ ID NO:1.
[00146] In another embodiment, the CSP inhibitor is an antisense
oligonucleotide
which" inhibits CSP peptide export. Preferably the antisense oligonucleotide
is an
oligonucleotide complementary to at least 10 consecutive nucleotides of an
oligonucleotide encoding a CSP exporter, said oligonucleotide having the
nucleic acid
sequence of SEQ ID. NO: 25 or 27.
[00147] Nucleic acid molecules (antisense inhibitors of CSP) [SEQ ID NO: 1]
and competitive inhibitors of CSP [SEQ ID NO:2] or the peptide analogs of S.
mutans
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. In some instances it will be desirable to employ liposomes
targeted
to the bacteria of interest.
[00148] The pharmaceutical compositions according to the invention may be
prepared using an antibody or a fragment thereof, which selectively inhibits
CSP
activity. A more detailed discussion of the preparation of CSP specific
antibodies is set
out below.
[00149] In a preferred embodiment,'the pharmaceutical compositions according
to the invention are prepared using one or more CSP peptide analogs capable of
inhibiting biofilm formation in dental plaque associated bacteria. The
inhibitory CSP
peptide analog may be a naturally occurring mutant CSP peptide obtained from
Streptococci bacteria having impaired biofilm formation ability. The
inhibitory CSP
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34
peptide analog may be a synthetic peptide prepared using methods known in the
art. In a
more- preferred embodiment, the inhibitory CSP peptide analog is one or more
of the
following modified S. mutans CSP peptides: B3 [SEQ ID No:47], C2 [SEQ ID
No:42],
E2 [SEQ ID No:44], and Fl [SEQ ID No:37]. In further preferred embodiment, the
pharmaceutical compositions are prepared using the E2 [SEQ ID NO:44] peptide.
[00150] Pharmaceutical compositions comprising CSP peptide analogs or
pharmaceutically acceptable salts thereof, and in particular B3 [SEQ ID
No:47], C2
[SEQ ID No:42], E2 [SEQ ID No:44], and Fl [SEQ ID No:37] peptides are
particularly
useful for methods of treatment or prophylaxis of a disease, disorder or
abnormal
physical state caused by Streptococci infection. Such pharmaceutical
compositons are
especially useful for treating infections caused by one or more of one or more
oral
Streptococci bacteria such as S. mutans, S. sobrinus, S. oralis, S. sanguis,
S. mitis, S.
gordonii. The pharmaceutical compositions are also useful for treating and
preventing other types of Streptococci infections such as S. pneumoniae, S.
pyogenes,
and S. agalactiae.
[00151] By;"pharmaceutically acceptable," such as in the recitation of a
"pharmaceutically acceptable carrier," or a"pharmaceutically acceptable salt,"
is
meant(herein a material that is not biologically or otherwise undesirable,
i.e., the
material may be incorporated into a pharmaceutical composition administered to
a
patient without causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the composition in
which it is
contained.
[00152] "Carriers" or "vehicles" as used herein refer to conventional
pharmaceutically acceptable carrier materials suitable for drug
administration, and
include any such materials known in the art that are nontoxic and do not
interact with
other components of a pharmaceutical composition or drug delivery system in a
deleterious manner.
[00153] 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
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described, for example in Remington's Phannaceutical 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.
[00154] On this basis, the pharmaceutical compositions could include an active
compound or substance, such as a CSP inhibitor, in association with one or
more
phamiaceutically acceptable vehicles or diluents, and contained in buffered
solutions
with a suitable pH and isoosmotic with the physiological fluids. The
pharmaceutical
carrier will depend on the intended route of administration. The methods of
combining
the active molecules with the vehicles or combining them with diluents are
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.
[00155] The pharmaceutical compositions of the present invention may be
manufactured in a manner that is itself known, e.g., by means of conventional
mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating,
entrapping or lyophilizing processes.
[00156] Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and auxiliaries
which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Proper formulation is dependent upon the route of
administration
chosen.
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36
[00157] The pharmaceutical compositions according to the invention can be
administered by any suitable route known in the art. In cases where the
infection is
localized, the pharmaceutical composition can be administered topically to
infected
area. In cases where the infection is systemic, the pharmaceutical composition
may be
administered orally, intravenously, or parenterally.
[00158] For injection, the agents of the invention may be formulated in
aqueous
solutions, preferably in physiologically compatible buffers such as Hanks'
solution,
Ringer's solution, or physiological saline buffer.
[00159] For oral administration, the compounds can be formulated readily by
combining the active compounds with pharmaceutically acceptable carriers well
known in the art. Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries,
suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical
preparations for oral use can be obtained by solid excipient, optionally
grinding a
resulting mixture, and processing the mixture of granules, after adding
suitable
auxiliaries, if desired, to obtain tablets or dragee cores. Suitable
excipients are, in
particular, fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol, or
cellulose preparations such as, maize starch, wheat starch, rice starch,
potato starch,
gelatin, gum .tragacanth, methyl cellulose, hydroxypropyhnethyl-cellulose,
sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone. If desired,
disintegrating agents
may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic
acid or a
salt thereof such as sodium alginate.
[00160] 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 conlprises 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, W002/44230.
[00161] A further aspect of the present invention provides the use in the
preparation of a medicament for administration to a mammalian patient to
alleviate
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37
dental caries, of viable, transfected S. mutans genetically modified to
produce a CSP
derivative which inhibits transformation efficiency.
[00162] 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 biofihn
formation.
[00163] 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.
[00164] The phannaceutical compositions according to the invention can be
administered by any suitable route known in the art. In cases where the
infection is
localized, the pharmaceutical composition can be administered topically to
infected area.
In cases where the infection is systemic, the pharmaceutical composition may
be
administered orally, intravenously, or parenterally.
[00165] In some instances, it may be desirable to administer one or more
pharmaceutical compositions according to the invention to treat an infection
caused by
more than one type of bacteria. For example, it may be desirable to administer
a
phaimaceutical composition comprising an antisense oligonucleotide with a
pharmaceutical composition comprising a CSP peptide analog. In another
instance, it
may be desirable to administer a pharmaceutical composition comprising an
antibody
with a pharmaceutical composition comprising a CSP peptide analog.
Alternatively, the
pharmaceutical compositions may be prepared with one or more types of CSP
inhibitors
to yield a unitary dosage form.
[00166] In some instances, it may be desirable to administer the
pharmaceutical
compositions according to the invention with a known antibacterial agent such
as an
antibiotic. In some instances, the pharmaceutical compositions which repress
biofilm
formation are also useful for rendering the bacterial cells more susceptible
to antibiotics.
[00167] The term "antibiotic" as used herein refers to any compound known to
one of ordinary skill in the art that will inhibit the growth of, or kill,
bacteria. The term
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38
"antibiotic" includes, but is not limited to, beta-lactams (penicillins and
cephalosporins),
vancomycins, bacitracins, macrolides (erythromycins), lincosamides
(clindomycin),
chloramphenicols, 'tetracyclines, aniinoglycosides (gentamicins),
amphotericins,
cefazolins, clindamycins, mupirocins, sulfonamides and trimethoprim,
rifampicins,
metronidazoles, quinolones, novobiocins, polymixins, gramicidins or any salts
or
variants thereof. The antibiotic used will depend on the type of bacterial
infection.
[00168] The therapeutically effective dosage of the pharmaceutical
compositions according to the invention will depend on the CSP inhibitor, the
type
and severity of the infection and whether the pharmaceutical composition
comprises a
further active ingredient such as an antibiotic. Generally, the
therapeutically effective
dose is the minimal amount sufficient for controlling biofilm formation and
which is
not toxic to the human or animal treated. Methods for determining effective
dosages
and toxicity are known in the art.
Anti-biofilm Compositions and Uses Thereof
[00169] In a further aspect, the present invention provides compositions
useful
for inhibiting and disrupting biofilms in Streptococcus spp. The compositions
comprise at least one peptide analogue of S. inutans CSP which inhibits or
disrupts
biofihn formation in a Streptococcus spp. The peptide analogues useful for
practicing
the invention include: Fl [SEQ ID NO. 37], Hl [SEQ ID NO. 39], B2 [SEQ ID NO.
41], C2 [SEQ ID NO. 42], E2 [SEQ ID NO. 44], and B3 [SEQ ID NO. 47]. In a
prefeiTed embodiment of the invention, the compositions are prepared using the
E2
[SEQ ID NO. 44] peptide.
[00170] The compositions according to the invention are effective for
inhibiting and disrupting biofilm formation in Streptococci which employ a
quorum
sensing system. The compositions are effective for both oral and non-oral
Streptococcus spp. Examples of oral streptococci infections which can be
modulated
using the compositions of the invention include, but are not limited to:
Streptococcus
sobrinus, Streptococcus sanguis, Streptococcus gordonii, Streptococcus oralis
and
Streptococcus mitis. Examples of non-oral streptococci infections which can be
modulated using the compositions of the invention, include, but are not
limited to:
Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus
agalactiae.
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39
[00171] In an embodiment of the invention, the composition may be formulated
as a pharmaceutical composition suitable for administration to a human or
animal
patient suffering an infection with one or more Streptococcus spp. The
pharmaceutical composition may comprise one or more of Fl [SEQ ID NO. 37], H1
[SEQ ID NO. 39], B2 [SEQ ID NO. 41], C2 [SEQ ID NO. 42], E2 [SEQ ID NO. 44],
and B3 [SEQ ID NO. 47] peptide analogues or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier. In a preferred embodiment
of the
invention, the pharmaceutical composition comprises the E2 [SEQ ID NO. 44]
peptide analogue.
[00172] The peptide analogues may be coupled to lipids or carbohydrates. This
increases their ability to adhere to the infected surface such as teeth,
either by
prolonging the duration of the adhesion or by increasing its affinity, or
both.
[00173] In some instances, it may be desirable to administer the
pharmaceutical
compositions according to the invention with a known antibacterial agent such
as an
antibiotic, examples of which are listed above. The antibiotic used will
depend on the
type of bacterial infection. For example, in some instances the biofilm
inhibiting
pharmaceutical compositions according to the invention also useful for
rendering the
bacterial cells more susceptible to antibiotics. Alternatively, the
pharmaceutical
compositions may be prepared with one or more active ingredients such as an
antibiotic,
in addition to the' CSP peptide analogue, to yield a unitary dosage form.
[00174] In addition to conventional oral pharmaceutical formulations such
tablets, capsules and syrups, the compositions according to the invention can
be
administered to humans or animals in the form of food, food additives,
dentrifice gels,
toothpaste, mouthwash, dental floss, denture wash, denture adhesive, chewing
gum,
candy, biscuits, soft drinks, or sports drinks.
[00175] 1 The antimicrobial composition may further comprise additional
ingredients including but not limited to: a surfactant, a chelating agent, an
antibody,
an antiseptic, and an antibiotic (see examples listed above).
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[00176] In a further aspect, the present invention provides a method of
treating
or preventing a Streptococcus spp infection in a patient in need thereof,
compri sing
administering a therapeutically effective amount of at least one peptide
having an
ainino acid sequence selected from a group consisting of: Fl [SEQ ID NO. 37],
H1
[SEQ ID NO. 39], B2 [SEQ ID NO. 41], C2 [SEQ ID NO. 42], E2 [SEQ ID NO. 44],
and B3 [SEQ ID NO. 47]. In a preferred embodiment, the method comprises
administering a therapeutically effective amount of E2 [SEQ ID NO. 44]
peptide.
The peptide analogues may be administered in the form of any of the
pharmaceutical
compositions encompassed by the present invention. In some instances, the
method
of treatment or prevention of a Streptococcus spp infection further comprises
the
administration of a therapeutically effective amount of an additional active
agent such
as an antibiotic (see examples above). The antibiotic may be administered
concurrently with the peptide analogue. In circumstances where it is desirable
to
enhance penetration of the antibiotic into the biofilm, it is preferably to
administer the
peptide analogue first to disrupt the biofilm.
[00177] The "therapeutically effective amount" or "therapeutically effective
dosage" of the pharmaceutical compositions according to the invention will
depend
on the peptide analogue, the type and severity of the infection and whether
the
pharmaceutical composition comprises a further active ingredient such as an
antibiotic. Generally, the therapeutically effective dose is the minimal
amount
sufficient for controlling biofilm formation and/or eliminating the infection,
which is
not toxic to the human or animal treated. Methods for detemlining effective
dosages
and toxicity are known in the art.
[00178] The method encompasses the treatment of infections caused by both
oral and non-oral Streptococcus spp. In the case of oral Streptococcus spp,
such as
Streptococcus spp. including: Streptococcus sobrinus, Streptococcus sanguis,
Streptococcus gondonii, Stj-eptococcus oralis, and Streptococcus rnitis, the
pharmaceutical compositions are useful for reducing and controlling dental
plaque.
The compositions are also use for the treatment and prophylaxis of a condition
caused
by dental plaque, including for example, dental caries, periodontal disease,
such as
gingivitis, and endocarditis. In such cases, it is generally desirable to
topically
administer the pharmaceutical compositions to the infected areas. For example,
the
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pharmaceutical compositions can be administered in the form of food, food
additives,
dentrifice gels, toothpaste, mouthwash, dental floss, denture wash, denture
adhesive,
chewing gum, candy, biscuits, soft drinks, or sports drinks.
[00179] In the case of infections caused by non-oral Streptococcus spp. such
as,
but not limited to: Streptococcus pn.eumoniae, Streptococcus pyogenes, and
Streptococcus agalactiae, the pharmaceutical compositions can be administered
either
locally or systemically depending on the nature, and severity of the
infection.
[00180] The precise dose for any of the pharmaceutical compositions of the
present invention will depend on a number of factors which will be apparent to
those
skilled in the art and in light of the disclosure herein. In particular these
factors
include: the identity of the corimpounds to be administered, the formulation,
the route
of administration employed, the patient's gender, age, and weight, and the
severity of
the condition being treated. Methods for determining dosage and toxicity are
well
known in the art with studies generally beginning in animals and then in
hunians if no
significant animal toxicity is observed. For example, in instances where the
pharmaceutical compositions are used to treat an infection, the
appropriateness of the
dosage can be assessed by monitoring the severity of the infection using
conventional
methods known in the art. Where the dose provided does not cause bacterial
levels to
decline to normal or tolerable levels, following at least three to fourteen
days of
treatment, the dose can be increased. The patient should be monitored for
signs of
adverse drug reactions and toxicity.
[00181] In a preferred embodiment of the invention, the therapeutically
effective amount of the peptide analogue will be between 1 g/ml and 1 mg/ml
daily.
In a further preferred embodiment, the therapeutically effective amount of the
peptide
analogue will be between 1 g/ml and 100 g/ml. In a still further preferred
embodiment, the peptide analogue administered is the E2 [SEQ ID NO. 44]
peptide in
an amount of between 1 g/ml and 100 gg/ml. In instances which include the
concurrent administration of an additional active ingredient such as an
antibiotic, the
amount of peptide analogue may be reduced. Alternatively, it may be
appropriate to
reduce the amount of the conventional dose of the antibiotic.
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Antimicrobial compositions
[00182] The CSP inhibitors described above for use in the preparation of
pharmaceutical compositions can also be used to prepare antimicrobial
compositions
such as disinfectants useful for inhibiting biofilm formation on various
surfaces.
[00183] Antimicrobial compositions for inhibiting biofilm formation may
comprise any of the peptide, antisense and antibody CSP inhibitors described
above.
In a preferred embodiment of the invention, the CSP inhibitor is used to
prepare the
antimicrobial composition is a peptide analogue of S. mutans CSP which
inhibits
biofilm formation. More preferably, the CSP inhibitor is one or more of the
following
S. mutans CSP peptide analogues: B3 [SEQ ID No:47], C2 [SEQ ID No:42], E2 [SEQ
ID No:44], and Fl [SEQ ID No:37]. In further preferred embodiment, the
antimicrobial
compositions are prepared using the E2 [SEQ ID No:44] peptide.
[00184] The antimicrobial composition may further comprise additional
ingredients including but not limited to: a surfactant, an antispetic and an
antibiotic
(see examples listed above).
[00185] Where the CSP inhibitor is one or more of S. mutans CSP peptide
analogues B3 [SEQ ID No:47], C2 [SEQ ID No:42], E2 [SEQ ID No:44], and Fl [SEQ
ID No:37], and the amount of the CSP inhibitor is preferrably between 1 g/ml
to 1
mg/ml. In preferred embodiments, wherein the CSP inhibitor is the E2 [SEQ ID
No:44]
peptide, the amount of E2 [SEQ ID NO:44] peptide is preferably is preferrably
between
1 g/ml to 100 g/ml.
Vaccines
[00186] Antibodies directed against the CSP [SEQ ID NO:2 or SEQ ID NO:30]
would provide protection against caries. Antibodies may be manufactured as
described below. Alternatively, a disclosed peptide [SEQ ID NO:2 or SEQ ID
NO:30] 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 rnutans" (in press). In order to
enhance the
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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. Patent Nos. 5,679,352, 5,194,254 and
4,950,480.
Techniques for preparing vaccines involving site-directed mutagenesis are
described
in U.S. Patent 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 c6ntrast 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, tootlipastes, mouthwashes or chewing gum.
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[00187] 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
[00188] hihibitors are preferably directed towards CSP [SEQ ID NO:2 or SEQ
ID NO:30] to block S. fnutans competence, low pH tolerance and biofilm
formation.
[00189] A method of identifying a compound which reduces the interaction of
CSP [SEQ ID NO:2 or SEQ ID NO:30] with HK [SEQ ID NO:4], can include:
contacting (i) CSP [SEQ ID NO:2 or SEQ ID NO:30] with (ii) HK [SEQ ID NO:4], a
CSP-binding fragment of HK [SEQ ID NO:4] 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:2 or SEQ ID NO:30] and HK [SEQ ID
NO:4]. 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.
[00190] In one embodiment, the invention includes an assay for evaluating
whether test compounds are capable of acting as agonists or antagonists for
CSP
[SEQ ID NO:2], or a peptide having CSP functional activity, including
culturing cells
containing DNA which expresses CSP [SEQ ID NO:1], 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:2 or SEQ ID NO:30] 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. Patent No. 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
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[00191] The CSP [SEQ ID NO:2 or SEQ ID NO:30] peptide 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:2] binding to HK
[SEQ ID NO:4]. Antibodies are preferably targeted to the entire CSP [SEQ ID
NO:2]
sequence. The CSP peptide [SEQ ID NO:2 or SEQ ID NO:30] may be conjugated to
other compounds, in order to increase immunogenicity.
[00192] We generate polyclonal antibodies against the CSP [SEQ ID NO:2 or
SEQ ID NO:30], 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. Patent 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. Patent 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 [SEQ ID NO:2 or SEQ ID NO:30] can be employed to
screen organisms or tissues containing CSP peptide [SEQ ID NO:2] or CSP-like
peptides. The antibodies are also valuable for immuno-purification of CSP or
CSP-
like peptides from crude extracts.
[00193] An antibody (preferably the antibody described above) may be used to
detect CSP [SEQ ID NO:2] 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:2] 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 inununolgical complex to ascertain the presence of
CSP
[SEQ ID NO:2] or a similar peptide. Certain embodiments of the invention also
include a kit for the ifa vitro detection of the presence or absence of CSP
[SEQ ID
NO:2] or a similar peptide in a biological sample, wherein the kit preferably
includes
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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:2] or a similar peptide in a biological sample. Further background on the
use of
antibodies is provided, for example in U.S. Patent Nos. 5,695,931 and
5,837,472,
which are incorporated by reference in their entirety.
Assay of genetic competence
[00194] The ability of the peptide to activate the HK [SEQ ID NO:4] and RR
[SEQ ID NO:6] 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 Irnmun, 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 [SEQ ID NO:1] (preferably plasmid
DNA) to a S. mutans culture (or culture of a bacteria expressing CSP [SEQ ID
NO:1]
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 g/ml plasmid DNA or 10 g/ml of chromosomal DNA is added to the culture. To
assay induction of competence, synthetic competence stimulating peptide,
(SCSP)
[SEQ ID NO:14] 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 g/ml plasmid
or
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
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lower ratio of transformants/recipients, indicating that the inhibitor is
effective at
blocking activity of the CSP [SEQ ID NO:2]. CSP deficient cells [SEQ ID NO: 1
or
SEQ ID NO:2] may also be used in a variation of these assays. One can identify
compounds that inhibit CSP [SEQ ID NO:2] 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.
[00195] 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:2]/HK [SEQ ID NO:4] /RR [SEQ ID
NO:6]. One such promoter occurs immediately 5' proximal to the S. mutans conx
gene. S. nautans cells grown in microtiter wells are exposed to the CSP [SEQ
ID
NO:2] 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:2] stimulation (and
conversely inhibitor activity). One can identify compounds that inhibit CSP
[SEQ ID
NO:2] or variants thereof by adding a test compound to the mixture to
determine if
the quantitative measure of CSP [SEQ ID NO:2] stimulation is decreased by the
addition of the test compound.
Assay of acid resistance tolerance
[00196] The ability of CSP [SEQ ID NO:2] 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 C with 5% CO2 for 2 hours. These cells are then collected by centrifugation
at
8,000 x g for 10 min and resuspended in 2 ml of fresh TYG (pH 5.5) at various
cell
densities as determined by O.D600. The cells are induced for acid adaptation
by
incubation at pH 5.5 for 2 h at 37 C with 5% CO2. 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 2Ø
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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 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 C
with 5%
CO2 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 [SEQ ID
NO:2]. CSP [SEQ ID NO:1 or SEQ ID NO:2] 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 [SEQ ID NO:1 or SEQ ID NO:2]
deficient cell. One can identify compounds that inhibit CSP [SEQ ID NO:1 or
SEQ
ID NO:2] 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
[00197] Cells transformed with a nucleic acid molecule disclosed herein
(histidine kinase [SEQ IlD NO:3], CSP [SEQ ID NO:1] or response regulator [SEQ
ID
NO:5]) are useful as research tools. For example, one may obtain a cell (or a
cell line,
such as an immortalized cell oulture or a primary cell culture) that does not
express
histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO: 1] or response regulator [SEQ
ID
NO:5], insert a histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or response
regulator [SEQ ID NO:5] nucleic acid molecule in the cell, and assess the
level of
expression and activity. Alternatively, histidine kinase [SEQ ID NO:3], CSP
[SEQ
ID NO:1] or response regulator [SEQ ID NO:5] nucleic acid molecules may be
over-
expressed in a cell that expresses a histidine kinase [SEQ ID NO:3], CSP [SEQ
ID
NO:l] or response regulator [SEQ ID NO:5] nucleic acid molecule. In another
example, experimental groups of cells may be transformed with vectors
containing
different types of histidine kinase [SEQ ID NO:3], CSP [SEQ ID NO:1] or
response
regulator [SEQ ID NO:5] nucleic acid molecules to assess the levels of
polypeptides
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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 [SEQ
ID NO:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] activity or
structure. For example, the polypeptides and peptides produced can be used for
microscopy or X-ray crystallography studies.
[00198] The histidine kinase [SEQ ID NO:3 and SEQ ID NO:4], CSP [SEQ ID
NO:1 and SEQ ID NO:2] or response regulator [SEQ ID NO:5 and SEQ ID NO:6]
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.
funetion 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:4],
CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6] by: (a) culturing cells
containing a nucleic acid molecule which expresses histidine kinase [SEQ ID
NO:4],
CSP [SEQ ID NO:1] or response regulator peptides [SEQ ID NO:5] (or fragments
or
variants thereof having histidine kinase , 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:4], CSP
[SEQ ID
NO:2] or response regulator [SEQ ID NO:6] 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:4], CSP [SEQ ID NO:2] or response regulator [SEQ ID NO:6]
activity.
[00199] Suitable assays may be adapted from, for example, US patent no.
5,851,788.
EXAMPLES
Materials and Methods
Growtli conditions of cells
[00200] Cells are grown in Todd Hewitt yeast extract medium at various
dilutions with and without 5% horse serum and 0.01% hog gastric mucin.
Pcotocol for Trafasforfization of Biofilna grown Cells
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[00201] 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:2]/receptor [SEQ ID NO:6]/kinase [SEQ ID NO:4] system.
Formation of biofilms is initiated by inoculating 20 ul of cell suspension
into each
well containing 2 ml of biofilm medium (4X diluted Todd-Hewitt Yeast Extract
supplemented with final concentration of 0.01% hog gastric mucin) for
overnight
incubation at 37 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 x g for 5 min. Both biofilm and
planktonic
cells are resuspended into 200 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
[00202] Homologues of the Streptococcus pneumoniae conaD [SEQ ID
NO:3]/E [SEQ ID NO:5] genes encoding a histidine kinase [SEQ ID NO:4]/
response
regulator [SEQ ID NO:6] system were identified. This sequence was used to
design
primers to amplify the region from a number of S. nzutans 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:2] in length, the precursor of
the 21-
amino acid CSP [SEQ ID NO:30].
PCR affaplification and fzucleotide sequefacing
[00203] The comCDE genes [SEQ ID NO:21] were amplified from the
genomes of several S. fnutans isolates by PCR using primers designed based on
the
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51
genome database sequence and their nucleotide sequences determined. The
deduced
amino acid sequences are compared among the isolates by sequence alignment to
confium identity.
Gene inactivations
[00204] Genes are inactivated by integration of internal homologous fragments
into the suicide vector pVA8912. Mutants defective in each of the individual
genes
(comC [SEQ ID NO:l], comD [SEQ ID NO:3], comE [SEQ ID NO:5] ) are
inactivated and their phenotypes are compared to the parent strain NG8 for
their
abilities to.form biofihns, tolerate acidic pH (pH 2-4), and transport and
incorporate
DNA. The knockout mutants of com D [SEQ ID NO:3] and E [SEQ ID NO:5] were
constructed by insertion-duplication mutagenesis, whereas the knockout comC
[SEQ
ID NO:1] mutant was created by allelic exchange via insertion of an
erythromycin
resistance determinant into the comC [SEQ ID NO:1] 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 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 K2HP04, 15 mM KH2PO4, 10 mM (NH4)2SO4,
35 m1V! NaC1, 2 mM MgSO2-7H2O, 0.2% (wt/vol) Casamino Acids and was
supplemented with filter-sterilized vitamins, (0.04 mM nicotinic acid, 0.1 mM
pyridoxine HC1, 0.01 mM pantothenic acid, 1 M riboflavin, 0.3 M thiamin HC1,
and 0.05 M D-biotin), amino acids (4 mM L-glutamic acid, 1 mM L-arginine HC1,
1.3 mM L-cysteine HC1, and 0.1 mM L-tryptophan) and 20 niM glucose.
Creation of comD deletion mutant.
[00205] An S. mutans UA159 cornD 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 conaD deletion mutant are P1-HK13 (5'-
CACAACAACTTATTGACGCTATCCC-3'), P2-HK13 (5'-
GGCGCGCCAACTGGCAACAGGCAGCAGACC-3'), P3-HK13 (5'-GGCC
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52
GGCCTCAAAACGATGCTGTCAAGGG-3'), P4-HK13 (5'-AGATTATCATTGGC
GGAAGCG-3'), Erm-19 (5'-GGCGCGCCCCGGGCCCAAAATTTGTTTGAT-3'),
and Erm-20 (5'- GGCCGGCCAGTCGGCAGCGACTCATAGAAT-3').
Synthesis of syntlzetic peptide
[00206] 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 ofpeptide analogs
[00207] The sequences of the peptide analogs used in this study are listed in
Table 4. The peptides were synthesized by methods known in the art.
TABLE 4: S nthetic CSP Analogues
Peptide Amino acid sequence
Fl SGSLSTFFRLFNRSFTQALK (SEQ ID NO. 37)
H1 SGSLSTFFRLFNRSFTQLGK (SEQ ID NO. 39)
B2 SGSLSTFFVLFNRSFTQALGK (SEQ ID NO. 41)
C2 SGSLSTFFALFNRSFTQALGK (SEQ ID NO. 42)
E2 SGSLSTFFRLFNASFTQALGK (SEQ ID NO. 44)
B3 SGTLSTFFRLFNRSFTQA (SEQ ID NO. 47)
[00208] Competence stimulating peptide (CSP) analogues were synthesized
based on the sequence . of the mature 21 amino acids CSP
(SGSLSTFFRLFNRSFTQALGK). The CSP peptide analogues (Fl [SEQ ID No:37],
H1 [SEQ ID No:39], B2 [SEQ ID No:41], C2 [SEQ ID No:42], E2 [SEQ ID No:44],
and B3 [SEQ ID No:47]) were synthesized by the Advanced Protein Technology
Centre, Peptide Synthesis Facility of Hospital for Sick Children (Toronto, ON)
and
Mimotopes (San Diago, CA). While the Fl and H1 analogues were generated by
deleting the 2nd and 4th residues from the C' termini, separately, the B2 and
C2
analogues in which the charged residues were substituted with neutral
(alanine) or
hydrophobic (valine) residues. In E2 analogue, second arginine (from the C'
terminus) was substituted with neutral alanine. The B3 analogue was generated
by
substituting 3rd residue from the N' terminus with threonine and by deleting
lst, 2nd
and 3rd residues from the C' terminus.
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53
[00209] The peptides were dissolved to 1 mg per ml in sterile distilled
deionized water. To any insoluble peptides, 10% (vol/vol) acetic acid, 2Q%
(vol/vol)
acetonitrile or 100% (vol/vol) dimethylformamide (DMF) was subsequently added.
Peptides were stored at -20 C until used.
Restoration of phenotypic defects by addition of CSP
[00210] To determine if the synthetic peptide [SEQ ID NO:14] could restore
defective phenotypes of the comC [SEQ ID NO:2] mutants, a chemically
synthesized
21-amino acid competence-stimulating peptide (CSP) [SEQ ID NO:14] (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 g/ml 2 h after inoculation of
bacterial
cells.
Growth rates
[00211] 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 l of cell suspension into each
well
containing 200 l of fresh THYE medium. The cell suspensions were pre-adjusted
to
the same optical density at O.D600 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 growtla conditions
[00212] 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,
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54
the medium was supplemented with either erythromycin (Em) (10 g/ml) or
kanamycin (Km) (500 g/ml).
[00213] 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 C in air with 5% C02. 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 conunercial plasmid preparation kit (Qiagen). When
needed,
antibiotics were added as follows: 10 g erythromycin per ml or 500 g
kanamycin
per ml for S. mutans, and 50 g kanamycin per ml for E. coli.
[00214] Streptococcus spp. including S. sobrinus, S. sanguis, S. gordonii, S.
oralis, S. naitis and Streptococcus pneumoniae were also used to study the
inhibitory
effects of the synthetic peptide analogues. They were grown in Todd-Hewitt
broth
containing 0.3% yeast extract (THYE) at pH 5.5 or 7.5. They were subcultured
routinely on THYE agar plates and incubated at 37 C in an anaerobic chamber
(5%
C02). In liquid media, cultures were incubated in closed screw-cap tubes
without
agitation at 37 C in an anaerobic chamber (5% C02).
Assay for S. rnutans biofilnas formed on polystyrene microtiter plates (a)
[00215] Biofilms were developed on polystyrene microtiter plates to provide a
rapid and simple method for assaying genetic transformation. A 4X 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
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 C for 20 h under an anaerobic condition, fluid medium was
removed
for viable cell counts. The wells were rinsed once with 10mM 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, OH) and incubated at 37
C under
CA 02630088 2008-05-16
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an anaerobic condition. Formation of biofilms was quantified by viable cell
counts
after 48 h of incubation.
Assay for S. anutans biofilms formed on polystyrene microtiter plates (b)
[00216] Biofilms were developed in 96-well polystyrene microtiter plates. The
growth of the biofilm was initiated by inoculating 10 l of an overnight S
mutans
UA159 culture into 300 l of SDM-glucose containing different concentrations
(0,
0.1, 0.5, 2, and 5 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 C in air with 5% COa 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 biofihns at 490 nm with a microplate reader (model
3550;
Bio-Rad Laboratories, Richmond, CA).
Assay for S. mutans, S. sobrinus, S. sanguis, S. gordozzii, S. oralis, S.
mitis and S.
pneumonaie biofilms formed on polystyrene nzicrotiter plates
[00217] To determine the anti-biofilm activity of synthetic E2 peptide against
Streptococcus spp. including S. sobrinus, S. sanguis, S. gordonii, S. oralis
and S.
mitis, the growth of biofilms on 96-well polystyrene microtiter plate was
initiated by
inoculating 10 l of an overnight Streptococcus spp. culture into 300 l of
semi-
defined minimal medium (58 mM K2HPO4, 15 mM KH2PO4, 10 mM (NH4)2 SO4, 35
mM NaCI, and 2mM MgSO4.7H20) supplemented with filter-sterilized vitamins
(0.04
mM nicotinic acid, 0.1 mM pyridoxine HC1, 0.1 mM pantothenic acid, 1 M
riboflavin, 0.3 M thiamine HC1, 0.05 M D-biotin), amino acids (4 mM L-
glutamic
acid, 1 mM L-arginine HCI, 1.3 mM L-cysteine HCI, 0.1 mM L-tryptophan), 0.2%
casamino acids, and 20 mM glucose containing E2 peptide (0 and 5 g/ ml) in
the
individual wells of a 96-well microtiter plate. Wells witliout cells were used
as blank
controls. The microtiter plates were then incubated at 37 C in an anaerobic
chamber
(5% C02) for 24 hours without agitation. After the incubation, the growth was
measured at 600 nm with a microplate reader. The planktonic cells were
carefully
removed and plates were air dried overnight. The plates were then stained with
0.4%
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WO 2006/060903 PCT/CA2005/001845
56
crystal violet for 10 minutes, rinsed with sterile distilled water and air
dried for 15
minutes. Biofilm was quantified by measuring the absorbance of stained biofilm
at
630 mu with a microplate reader.
S. mutans eompetence assay
[00218] 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. snutans UA159 were
diluted
(1:20) with prewarmed THYE broth and incubated at 37 C in air with 5% COZ
until
an optical density (OD) of approximately 0.1 at 600 nni was reached. The
culture was
then divided into six aliquots containing 1 g/ml of plasmid pDL289 and
different
concentrations (0, 0.1, 0.5, 2, and 5 g per inl) of peptide analogs. The
cultures were
incubated at 37 C in air with 5% COa for 2.5 h, gently sonicated for lOs to
disperse
the streptococcal chains, and spread on THYE plates containing kanamycin.
Plates
were incubated at 37 C in air with 5% CO2 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 nuniber of recipient cells.
S. mutans acid resistance assay
[00219] 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 C in air
with
5% COa until an OD600 of approximately 0.4 was reached. A 20-fold dilution was
made into 400 l of either THYE pH 7.5 or THYE pH 5.5 broth containing
different
concentrations (0, 0.1, 0.5, 2, and 5 jig 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 C. Measurements were taken every 20 min with shaking to prevent cell
aggregation.
Assay for "steady-state" biofilnis
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57
[00220] Biofilms were also grown in a chemostat-based biofihn fermentor to
defme and optimize the conditions for genetic competence of biofilm-grown
cells.
The biofihn 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 cma area immersed in fluid medium), providing abiotic
surfaces for
accumulation of biofilms. Temperature in the chemostat vessel was maintained
at
37 C 0.1 by a temperature controller (Model R-600F, Cole Parmer Instrument
Cop.,
Vernon Hill, IL). The culture pH was controlled by a pH control unit (Digital
pH
Meter/Controller, Model 501-3400, Barnant Corp. Barrington, IL) through the
addition of 1M KOH or 1M HC1. 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 punlping
fresh 4X
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
mean generation times), glass rods were aseptically inserted into the
chemostat for
the initiation of biofihn formation. Then, biofilms of different ages were
removed
from the cultures for both genetic transformation and quantification of
biofilms using
viable cell counts.
Scanfzifzg electrotz microscopy (SEM)
[00221] To examine spatial distribution and biofihn 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 KPO4 and
fixed with 2 ml of 3.7% formaldehyde in 10 mM KPO4 buffer overnight. The
samples
were then dehydrated with a series of alcohol baths (30%, 50%, 70%, 95% and
100%), critical point dried with liquid C02, mounted and sputter coated with
gold.
The samples were then examined using a scanning electron microscope (Model S-
2500, Hitachi Instruments, San Jose, CA).
CA 02630088 2008-05-16
WO 2006/060903 PCT/CA2005/001845
58
Transfornaation protocol
[00222] 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. Biofihns 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 C. After 2h incubation, a final concentration of 1
g/ml
plasmid DNA or 10 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:14], the cultures were incubated for 30 min and a final
concentration of 500 ng/ml of SCSP [SEQ ID NO:14] was added to each well.
After a
30 min incubation, equal amounts of DNA was added to each well (1 g/rnl
plasmid
or 10 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 X g for 5 min. Both biofilm and planktonic cells were resuspended into
200 l
of fresh medium and were immediately spread on THYE agar plates plus
appropriate
antibiotics. For the biofilms developed in the chemostat, rods with biofihn
cells were
removed and placed into 2 ml of pre-watmed, 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 biofilni
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 g DNA per viable
recipient at the time of DNA added.
Donor DNA
[00223] Both plasmid and chromosomal DNA were used as donor DNA to
assay genetic transformation in this study. Plasmid DNA included an
integrative
CA 02630088 2008-05-16
WO 2006/060903 PCT/CA2005/001845
59
plasmid, pVAGTFA carrying an erythromycin resistance (Emr) determinant and a
fragment of the S. mutans gtfA gene. The replicative plasmid, pDL289 carrying
a
kanamycin resistance gene (Kmo was also used. Chromosomal DNA harboring an
Emr gene was prepared from a recombinant S. mutans strain harboring a
chromosomally integrated copy of pVAGTFA.
[00224] 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.
[00225] 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.
[00226] Example 1- CSP Peptide Analo$!ues Inhibit Biofilm Formation in
Streptococcus nzutans
[00227] An in vitro assay was performed as described in Materials and
Methods to determine the effects of six synthetic analogues of CSP (Fl, H1,
B2, C2,
E2 and B3; TABLE 4) on biofilm formation in Streptococcus mutans. The
analogues
were synthesized as described in Materials and Methods. The anti-biofilm
activity of
analogues against S. mutans in terms of percentage inhibition varied from 0 to
80%
(FIG 18). The E2 peptide was selected for further in vitro studies as it
showed the
highest anti-biofilm activity (80% inhibition) among six synthetic CSP
analogues
tested.
[00228] Example 2- E2 CSP Peptide Analogue Inhibits Biofilm Formation
in S. sobrinus, S. sauguis, S. gordonii, S. oralis and S. mitis
CA 02630088 2008-05-16
WO 2006/060903 PCT/CA2005/001845
,
[00229] An in vitro assay was performed as described in Materials and
Methods to determine whether E2 analogue of CSP could inhibit biofihn
formation in
Streptococcus spp. such as S. sobrinus, S. sanguis, S. gordonii, S. oralis and
S. mitis.
E2 peptide at a concentration as low as 5 g/mi showed inhibitory effects on
both
growth and biofihn formation in all the five organisms tested. The percent
inhibition
of biofihn formation in these organisms varied from 40 to 75% (FIGS. 19, 20,
21, 22,
and 23). Furthermore, the anti-biofilm activity of E2 peptide was tested
against mixed
culture of the above Streptococcus spp. It also showed a significant
inhibitory effect
on the mixed culture biofihn formation (data not shown).
[00230] Example 3 - E2 CSP Peutide Analoeue Inhibits Biof.ilm Formation
in Streptococcus pneuinoniae
[00231] This example illustrates the effect of S. mutans CSP analogue on the
CSP-mediated quorum-sensing signaling system in a non-oral streptococcal
pathogen
was studied. An in vitro assay was performed as described in Materials and
Methods
to determine whether E2 analogue of CSP could inhibit biofilm formation in
StYeptococcus pneumoniae. E2 peptide at a concentration as low as 5 g/mi
showed a
considerable antibiofilm activity against S. pneumoniae (25% inhibition, FIG.
24).
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61
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