DENTAL CARIES AND METHODS OF USING THE SAME

RELATED APPLICATION INFORMATION

This application claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Patent

Application No. 61/925,474, filed January 9, 2014, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support in part under Grant No. DE017737 awarded by the National Institutes of Health and the National Institute of Dental and Craniofacial Research. The government has certain rights in the invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled

5656-58WO_ST25.txt, 2,496 bytes in size, generated on January 9, 2015 and filed via EFS- Web, is provided in lieu of a paper copy. The Sequence Listing is incorporated herein by reference into the specification for its disclosures. FIELD OF THE INVENTION

The present invention relates to compositions that interfere with the adherence of oral streptococci to the tooth surface, and consequently can prevent the colonization and infection that lead to tooth decay. Gal( 1 -3-GalNac has been identified to be an inhibitor of adherence of oral streptococci to the tooth surface. The compositions of the present invention, methods of using the same and methods of identifying compositions that interfere with the adherence of oral streptococci to the tooth surface are related to the prevention and inhibition of the formation of dental caries.

BACKGROUND OF THE INVENTION

The attachment of bacteria to human tissue and other surfaces within the oral cavity is thought to be an essential first step in pathogenesis, and microbes utilize surface proteins (pili, fimbrae) to effectively adhere to a variety of molecules and surfaces. While the oral cavity is home to a number of microbes this study focuses on oral streptococci, where the mutans streptococci (S. mutatis, S. sobrinus), are the known etiological agents in dental caries, whereas the viridians streptococci (S. gordonii, S. sanguis) are considered to be commensal flora. Among the surface proteins on oral streptococci, Antigen I/II (Agl/II) homologs (also known as P 1 , PAc, SpaP, SR in S. mutatis. SspA and SspB in S. gordonii, Pas in S. intermedins, etc. are the most extensively studied. These Agl/II homologs adhere to tooth immobilized salivary agglutinin (SAG) secreted by salivary glands. Typically, Agl/II homologs carry a signal sequence at the N-terminus, followed by the alanine-rich (A), variable (V) and proline-rich (P) regions, succeeded by the C-terminal region and the membrane spanning domain that anchors to the bacterial cell wall (FIG. 1 A). In earlier studies, two SAG adherence regions have been identified on Agl/II: one, the V -region, at the apex of the molecule (A3VP ₁ ); and the other, the CiC ₂ region, at the C-terminus (C ₁₂₃ ), specifically the C i and C ₂ domains that adopt the DEv-IgG fold, a variant of the classical IgG-fold, near to where Agl/II is attached to the streptococcal cell surface (FIG. I B). More importantly we determined that these two regions adhere to SAG in a non-competitive manner, indicating the presence of two different surfaces on SAG, pointing towards bacterial heterogeneity (multivalency) in adherence. Thus far all these interactions have been studied with purified SAG (some groups have now begun to address SAG as Gp340) extracted from single or multiple donors, and in some cases with saliva itself.

SAG is a large glycoprotein complex that contains glycoprotein 340 (Gp340), slgA and an unknown 80 kDa protein. Among these the major component Gp340 is known to aggregate several species of bacteria, including mutans, viridan streptococci and H. pylori and is thereby considered an innate immune response factor. Gp340 orthologs are observed in various mammalian species including mouse, rabbit, rat, pig, cow and rhesus monkey. Gp340 is a 340 kDa protein that contains 14 SRCR (scavenger receptor cysteine rich) domains, 2 CUB (C l r/Cl s Uegf Bmpl ) and one ZP (zona pellucida) domain (FIG. 2). The 13 SRCR domains are present in tandem at the N-terminus, followed by an intriguingly nested 14 ^th SRCR domain between two CUB domains, with a ZP domain at the C-terminus. The SRCR domains are interspersed with regions termed SID, an acronym for the SRCR interspersed domains. Except between the 4 ^th and the 5 ^th SRCR domain, all other tandem repeats contain the SID domain. These SRCR domains belong to an ancient class of proteins and are present in protozoan parasites like Cryptosporidium, Toxoplasma, Plasmodium and in the algae Chi am ydomonas . They also appear in the entire animal kingdom beginning with sponges, and are highly conserved, where a single SRCR domain usually contains 100-1 10 amino acids. The SRCR domains of Gp340 were recently shown to aid in transcytosis of HIV into vaginal epithelial cells. This highlights the role of the Gp340 SRCR domains in infection, where it serves as a portal of entry into the host for both bacteria and viruses that result in various human diseases. Therefore, Gp340 and its major constituent the SRCR domain has now become the focus of a number of recent reviews that highlight the importance of Gp34() in bacterial and viral pathogenesis.

In a systematic study conducted with various oral streptococci, Loimaranata et al. classified the bacterial recognition properties of Gp340 into three different groups, where group I strains both aggregated by and adhered to gp340, group II preferentially adhered, and group III preferentially aggregated. Using a peptide based approach, Bikker et al. identified a consensus peptide SRCRP2 (QGRVEVLYRGSWGTVC, SEQ ID NO.l) derived from the 14 SRCR domains of Gp340, which aggregated several species of bacteria, and also inhibited the adherence of Agl/II to SAG. In a subsequent study using alanine scanning, the most important residues involved in aggregation were deduced to reside within the 'VEVLXXXXW (SEQ ID NO:2) motif. In these studies, the SID domains that are predicted to host the glycosylation sites were classified into two different groups namely, S1D20 and SID22 based on sequence homology, and neither one displayed aggregation nor adherence to bacteria. However, it is not believed to date that the interaction between the oral streptococci and the SRCR domains has been characterized.

The ability to adhere strongly to human receptors within the oral cavity is a necessity for bacterial survival, or else they will be washed into the acidic gut. Bacteria that colonize the oral cavity have multiple proteins on its surface for specific adherence to human receptors. The present invention is related to the S. mutans surface receptor Agl/II and its homologs, for example, S. gordonii SspB, and the development of inhibitors of the interaction of Agl/II and its homologs with Gp340, which is considered to be the first step in adherence of oral streptococci to the tooth surface. The adherence of oral streptococci subsequently leads to colonization and infection, and among these the mutans streptococci are known etiological agents in dental caries. Thus, compositions that can inhibit the adherence of oral streptococci to the tooth surface can provide an avenue for the development of compositions suitable for preventing the development of dental carries. SUMMARY OF THE INVENTION

The present invention utilizes the inhibition of the interaction between Agl/II, and/or its homologs, and SAG, more particularly with the SRCR domains found on Gp340 within the SAG complex which provides the basis for the compositions and methods of the present invention. Thus, in an aspect of the invention, provided is a composition for the prevention and/or inhibition of the formation of dental caries in a subject. In another aspect of the invention, provided is a composition for the prevention and/or inhibition of the formation of denture plaques in a subject.

In yet another aspect of the invention, the compositions of the present invention comprise inhibitors of the interaction of Agl/II, and/or its homologs, with SAG. In a particular aspect of the invention, the inhibitor of the composition is a Gal(ll-3-GalNac glycan. In another particular aspect of the invention, the inhibitor is a peptide. In yet another aspect of the invention, inhibitor binds to Agl/II and/or its homologs.

In yet another aspect of the invention, provided are formulations that comprise the composition for the prevention and/or inhibition of the formation of dental caries in a subject. In a further aspect of the invention, provided are formulations that comprise the composition for the prevention and/or inhibition of the formation of denture plaques in a subject.

In yet another aspect of the invention, provided is a method for inhibiting the interaction of Agl/II, and/or its homologs, with SAG, the method comprising the administration of the composition, compositions or formulations of the present invention.

In yet another aspect of the invention, provided is a method for preventing, inhibiting and/or treating the fonnation of dental caries in a subject, the method comprising the administration of the composition, compositions or formulations of the present invention.

In yet another aspect of the invention, provided is a method of identifying compounds that inhibit the interaction of Agl/II, and/or its homologs, with SAG.

In yet another aspect of the invention, provided is a method of identifying compounds for preventing, inhibiting and/or treating the formation of dental caries in a subject.

The foregoing and other objects and aspects of the present invention are explained in detail in the drawings and specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 A depicts the primary sequence layout of S. mutatis UA159 Agl/II and S. gordonii DL l SspB including the extents of the recombinant fragments used herein.

FIG. I B depicts the structure for Agl/H as derived from crystal structures of AjVP _t and C ₁₂₃ , and from electron microscopy.

FIG. 2 shows a schematic representation of the primary sequence layout of Gp340 from human saliva depicting the overall architecture. FIG. 3 depcits recombinantly expressed and purified fragments of S. gordonii DL1 FL ^SspB , A ₃ VP, ^SspB , and C _{l 23} ^SspB on a 12.5% SDS-PAGE gel stained with coomassie blue.

FIG 4 shows confocal microscopic images displayed the interaction between S. mutatis UA159 (stained with blue DAP I) and z ^' SRCRs (stained with green Anti-His tag Alexa fluor 488 antibody). The observed green fluorescence indicated the adherence of both z ^' SRCRi (panel A) and z ^' SRCRs _? (panel B) with S. mutans, where z ^' SRCRs adhered more profoundly. S. mutans displayed counterstaining only with DAPI in the absence f z ^' SRCRs (panel C).

FIG. 5 shows confocal microscopic images of S. gordonii DLl interaction with z ^' SRCRs similar to FIG. 4, panel A. Even in these images, z ^' SRCRi ₂₃ displayed more profound interaction compared to z ^' SRCRi.

FIG. 6 depicts histograms constructed from FACS analysis describe the interaction of z ^' SRCRi and z ^' SRCRi 23 with S. mutans in Panel A and with S. gordonii in Panel B.

FIG. 7 shows aggregation of (panel A) S. mutans UA 159 and (panel B) S. gordonii DLl cells in the presence of z ^' SRCRs or SAG. Bacterial cells in buffer alone were used as control. The results are plotted as percentage of aggregation measured at OD ₇₀ o at 5 minute intervals for 1 hour. Difference in aggregation detected between groups were analyzed using One-way ANOVA, where *P<().05 was considered significant, and error bars represent the standard deviation.

FIG. 8 depicts aggregation of S. mutans UA159 and S. gordonii DLl cells in the presence of SRCRP2 peptide at different concentration. Bacterial cells in buffer alone were used as control. The results are plotted as percentage of aggregation measured at OD ₇₀ o at 5 minute intervals for 1 hour. Difference in aggregation detected between groups were analyzed using One-way ANOVA, where *P<0.05 was considered significant, and error bars represent the standard deviation.

FIG. 9 shows binding of SRCRs with different concentrations (lng-^g) of (A) A g I/I I of S. mutans and (B) SspB of S. gordonii analyzed using EL1SA. The dotted line (— ) represent z ^' SRCRi and the bold line (— ) represent z ^' SRCRi ₂₃ . The data shows FL ^{A I} " and FL ^SspB binds to z ^' SRCRi and z ^' SRCR ₁₂₃ with higher affinity compared to its subfragments.

FIG. 10 depicts an ELISA assay illustrating the binding of Fluorescent tagged

SRCRP2 peptide at different concentration with (— ) Agl/II of S. mutans and (— ) SspB of S. gordonii. FIG. 11 shows sensorgrams from surface plasmon resonance studies showing the interaction of FL ^AgI " and FL ^{Ss B} at various concentrations with immobilized /SRCRi, /SRCRm and SAG.

FIG. 12 shows sensorgrams from surface plasmon resonance studies showing the interaction of A ₃ VPi ^AgI/n and A ₃ VP, ^SspB at various concentrations with immobilized /SRCRi, I ^' SRCRm and SAG.

FIG. 13 shows sensorgrams from surface plasmon resonance studies showing the interaction of Cm ^AgVn and Ci ₂₃ ^SspB at various concentrations with immobilized /SRCRi, /SRCR ₁₂₃ and SAG.

FIG. 14 depicts surface plasmon resonance studies illustrating binding of (2 μΜ)

Lysozyme but not (2 μΜ) thaumatin with /SRCRi or /SRCRi 23 immobilized on CMS sensor chip.

FIG. 15 depicts concentration of FL ^{Agl n} and FL ^SspB bound to immobilized

/SRCRi and /SRCRi ₂₃ on CM5 sensor chip.

FIG. 16 shows competition experiments with FL ^{AgI I} , A ₃ VP, ^{A l} ", and C ₁₂₃ ^{A , u} conducted with immobilized /SRCRi (panel A) immobilized /SRCRi ₂₃ (panel B) on Biaeore CM5 chip. The direct binding of the fragment prior to competition is shown in bold, followed by the fragments that were tested for their inhibitory activity. Similarly, competition of

FL ^SspB , A ₃ VPi ^SspB , Ci ₂₃ ^SspB with immobilized /SRCRi, /SRCR, ₂₃ and SAG are shown in panels C, D and E. All experiments were carried out in triplicates and the error bars represent standard deviations.

FIG. 17 depicts the binding of 2 μΜ FL and subfragments of Agl/II of S. mutans in the presence and absence of 2.5mM CaCl ₂ with immobilized (panel A) /SRCRi and (panel B) /SRCRi ₂₃ on CM5 sensor chip.

FIG. 18 depicts the binding of 2 μΜ FL and subfragments of SspB of S.gordonii in the presence and absence of 2.5 mM CaCl ₂ with immobilized (panel A) /SRCRi and (panel B) iSRCRi ₂₃ on CM5 sensor chip

FIG. 19A shows CD studies demonstrating spectral changes of /SRCRi on addition of various concentration of calcium ions (1 mM-100 mM).

FIG. 19B shows CD studies demonstrating spectral changes of /SRCR) ₂₃ on addition of various concentration of calcium ions (1 mM-100 mM).

FIG. 20 depicts DSC showing the stability of /SRCR, at various temperature with dose dependent increase of calcium ions. FIG. 21 depicts Glycoprotein stained SDS-PAGE gel containing /SRCRi, /SRCR 12 , horse radish peroxidase (HRP. positive control) and soybean trypsin inhibitor (SBTI, negative control).

FIG. 22 A shows SPR studies of Gaipi-3-GalNac carbohydrates at different concentrations (0.010 mM-1 raM) with FL ^{A , u} and FL ^SspB and its subfragments of Agl/II of S. mutans and SspB of S. gordonii over ZSRCRi immobilized CMS sensor chip.

FIG. 22B shows SPR studies of Gaipi-3-GalNac carbohydrates at different concentrations (0.010 mM-1 mM) with FL ^{A l} " and FL ^SspB and its subfragments of Agl/II of S. mutans and SspB of S. gordonii over /SRCR ₁₂₃ immobilized CM5 sensor chip.

FIG. 23 A depicts inhibition studies SRCRP2 peptide at different concentrations (0.005 mM-0.200 mM) with 2 μΜ FL ^{AgI 11} and FL ^SspB and sub-fragments on /SRCR, immobilized CM5 sensor chip using surface plasmon resonance analysis.

FIG. 23B depicts inhibition studies SRCRP2 peptide at different concentrations (0.005 mM-0.200 mM) with 2 μΜ FL ^Agl " and FL ^SspB and sub-fragments on /SRCR ₎₂₃ immobilized CM5 sensor chip using surface plasmon resonance analysis.

FIG. 24 shows the binding of different concentrations (0.250 μΜ-2 μΜ) of SRCRs with immobilized /SRCR) and /SRCRi ₂₃ on CM5 sensor chip.

FIG. 25 shows models for the adherence of Agl 'II to Gp34(): (A) Adherence to Gp340 may occur through a single site of Agl/II, (B) AglTI uses both sites to adhere to an elongated Gp340, (C) Each site on Agl/H adheres to different Gp340 (D) Or both sites on Agl/H adhere to a very large SAG high molecular weight (HMW) complex. SRCRs are shown as medium grey circles, CUB is shown as medium grey squares and ZP is shown as in light grey circles.

FIG. 26 depicts the effect of Galpl-3GalNac (core-1 ) carbohydrate on bacterial surface proteins interaction with immobilized Salivary Agglutinin on a CM5 sensor chip.

FIG. 27A depicts a sensorgram from surface plasmon resonance studies showing the interaction of SRCR peptide ETNDANVVARQL (SEQ ID NO: 1 0) with immobilized Agl/II Vhel.

FIG. 27B depicts a sensorgram from surface plasmon resonance studies showing the interaction of SRCR peptide ETNDANVVARQL (SEQ ID NO: 10) with immobilized SspB Vhel.

FIG. 27C depicts a sensorgram from surface plasmon resonance studies showing the interaction of SRCR peptide ETNDANVVARQL (SEQ ID NO: 10) with immobilized GbpC. DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, embodiments of the present invention are described in detail to enable practice of the invention. Although the invention is described with reference to these specific embodiments, it should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. All publications cited herein are incorporated by reference in their entireties for their teachings.

The invention includes numerous alternatives, modifications, and equivalents as will become apparent from consideration of the following detailed description.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Also as used herein, the terms "treat," "treating ^" or ""treatment" may refer to any type of action that imparts a modulating effect, which, for example, can be a beneficial and/or therapeutic effect, to a subject afflicted with a condition, disorder, disease or illness, including, for example, improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression f the disorder, disease or illness, delay of the onset of the disease, disorder, or illness, and/or change in clinical parameters of the condition, disorder, disease or illness, etc., as would be well known in the art.

As used herein, the terms "prevent," '"preventing ^*" or "prevention of (and grammatical variations thereof) may refer to prevention and/or delay of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset and/or progression of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. In representative embodiments, the term "prevent, ^" ""preventing." or '"prevention of ^" (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of a metabolic disease in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom (s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the present invention.

An "effective amount" or "therapeutically effective amount" may refer to an amount of a compound or composition of this invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, during the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount or therapeutically effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example. Remington, The Science and Practice of Pharmacy (latest edition)). Compositions

The present invention is based on the inhibition of the interaction between Agl/ l, and/or its homologs, with SAG, more particularly the interaction between Agl/ll and the SRCR domains found on Gp340 within the SAG complex, compositions that inhibit this interaction for the prevention and/or inhibition of the formation of dental caries, or the prevention and/or inhibition of the formation of denture plaques, in a subject. In some embodiments of the invention, the inhibitor of the interaction between SAG and Agl/II, and/or its homologs, is a glyean. In one embodiment, the glycan is Gaip i -3-GalNac glycan. In other embodiments of the invention, the inhibitor of the interaction between SAG and Agl/II, and/or its homologs, is a peptide. In some embodiments, the peptide inhibitor of the interaction between Agl/II, and/or its homologs, and SAG, binds to Agl/II, and/or its homologs. In other embodiments, the peptide has sequences identical to or homologous to a scavenger receptor cysteine rich (SRCR) domain from SAG. In one embodiment, the peptide is ETNDANVVARQL (SEQ ID NO: 10).

In an embodiment of the invention, provided is a pharmaceutical composition, comprising a therapeutically effective amount of the inhibitor of the interaction between Agl/II, and/or its homologs, with SAG. In other embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier ^' as used herein refers to any substance, not itself a therapeutic agent, used as at least in part a vehicle for delivery of a therapeutic agent to a subject. Non-limiting examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions or water/oil emulsions, microemulsions, and various types of wetting agents. Further, in preparing such pharmaceutical compositions comprising the active ingredient or ingredients in admixture with components necessary for the formulation of the compositions, other conventional pharmacologically acceptable additives may be incorporated, for example, excipients, stabilizers, wetting agents, emulsifying agents, lubricants, sweetening agents, coloring agents, flavoring agents, isotonicity agents, buffering agents, antioxidants and the like. Additives may include, for example, starch, mannitol, sorbitol, precipitated calcium carbonate, crystalline cellulose, carboxym ethyl cellul se, dextrin, gelatin, acacia. EDTA, magnesium stearate, talc, hydroxypropylmethylcellulose, sodium metabisulfite, and the like.

Formulations suitable for administering the composition of the present invention may be suitable for oral or buccal (sublingual) administration. The formulation may either be in the form of a solid or a liquid. In some embodiments, forms of formulations suitable for oral administration of the compositions of the present invention include, but are not limited to, a tooth paste or dentifrice composition, an oral hygiene product, for example, an oral hygiene tablet, an oral care composition, for example, an oral rinse, a gel or an additive to a digestible product. Formulations suitable for buccal (sub-lingual) administration include lozenges, tablets, capsules, chewing gum and the like, comprising the active compound, with suitable carriers and additives that would be appreciated by one of skill in the art, for example, binders, diluents, lubricants, disintegrating agents and the like.

Formulations for the prevention of denture plaques may include liquid solutions and/or rinses, either when worn by a subject, or when removed and not being worn by the subject, for example, a solution or rinse for soaking the dentures for a period of time therein.

Liquid formulations include, but are not limited to, solutions, emulsions, dispersions, suspensions and the like with suitable carriers. Additives may include water, alcohols, oils, glycols, preservatives and the like.

In some embodiments, formulations suitable for administering the composition of the present invention may also include additives that may provide greater patient compliance, for example, coloring agents, flavoring agents and the like.

In some other embodiments, the formulations for administering the composition of the present invention may further comprise an additional agent or agents. Such agents may include, but are not limited to, agents for removing plaque, whitening and/or remineralizing teeth, and the like. In still other embodiments, the formulation may further comprise a delivery system, for example, a film or a strip of material, which can be placed against the surface of the teeth of the subject in order to deliver the formulation, for example, as set forth in U.S. Patent Nos. 5,989,569 and 6,045,811. Methods of Administration and Use

Another embodiment of the present invention provides a method for administering to a subject in need thereof a compound or pharmaceutical composition as described herein. For administration, either the compound or pharmaceutical composition is understood as being the active ingredient and capable of administration to a subject, and thus, in some instances, the terms are interchangeable.

Subjects suitable to be treated with the composition, compositions and formulations of the present invention include, but are not limited to mammalian subjects. Mammals according to the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, humans and the like, and mammals in utero. Any mammalian subject in need of being treated or desiring treatment according to the present invention is suitable. Human subjects of any gender (for example, male, female or transgender) and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult, elderly) may be treated according to the present invention.

The method of administration of the compound or pharmaceutical composition as described herein is not particularly limited, and any method that would be appreciated by one of skill in the art for the compound or pharmaceutical composition in a particular formulation as described herein. Methods of Identification and Screening

In yet other embodiments of the invention, provided are methods of screening for and identifying inhibitors of the interaction between SAG and Agl/II, and/or homologs thereof, which may be utilized alone or in combination with information on the inhibitors described above to generate still additional inhibitors.

For example, active agents may also be developed by generating a library of molecules, selecting for those molecules which act as ligands for a specified target, and identifying and amplifying the selected ligands. Techniques for constructing and screening combinatorial libraries of oligomeric biomolecules to identify those that specifically bind to a given receptor protein are known. Suitable oligomers include peptides, oligonucleotides, carbohydrates, nonoligonucleotides and nonpeptide polymers. Peptide libraries may be synthesized on solid supports, or expressed on the surface of bacteriophage viruses (phage display libraries). Known screening methods may be used by those skilled in the art to screen combinatorial libraries to identify compounds that antagonize the interaction between SAG and Agl/II, and/or homologs thereof. Techniques are known in the art for screening synthesized molecules to select those with the desired activity, and for labeling the members of the library so that selected active molecules may be identified.

As used herein, "combinatorial library" refers to collections of diverse oligomeric biomolecules of differing sequence, which can be screened simultaneously for activity as a ligand for a particular target. Combinatorial libraries may also be referred to as "shape libraries ^' ", i.e., a population of randomized polymers which are potential ligands. The shape of a molecule refers to those features of a molecule that govern its interactions with other molecules, including Van der Waals, hydrophobic, electrostatic and dynamic.

As noted above, potential active agents or candidate compounds as described can be readily screened for activity in inhibiting the interaction between SAG and Agl/ll, and/or a homolog thereof. The method may comprise the steps of: (a) adding or contacting a test compound to an in vitro system comprising SAG and Agl/II, and/or a homolog thereof (this term including binding fragments thereof sufficient to bind to the other); then (b) determining whether the test compound is an inhibitor of the interaction between SAG and Agl/II, and/or homologs thereof; and then (c) identifying the test compound as active or potentially active in inhibiting the formation of dental caries when the test compound is an inhibits the interaction between SAG and Agl/II, and/or homologs thereof. The in vitro system may be in any suitable format that would be appreciated by one o skill in the art. In some embodiments, the in vitro system may be a cell-free system, such as an aqueous preparation of SAG and Agl/II, and/or homologs thereof, or the binding fragments thereof. The contacting, determining and identifying steps may be are carried out in any suitable manner, such as manually, semi- automated, or by a high throughput screening apparatus. The determining step may be carried out by any suitable technique, such as by precipitation, by labeling one of the fragments with a detectable group, all of which may be carried out in accordance with procedures well known to those skilled in the art.

The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1: Inhibition of SAG adherence on Agl/II and SSpB

A: MATERIALS AND METHODS

Expression and Purification of Proteins used in this study. z ^' SRCRi and SRCR _i23 were expressed and purified as described recently by Sangeetha et.al [36], and similarly Agl/II fragments used in this study were prepared as described previously [15]. SspB constructs (FL ^SspB , A ₃ VP, ^SspB and Ci ₂₃ ^SspB ), were cloned into pET23d vector (Novagen, Inc) using primers listed in Table 1, restriction enzymes Ncol, Not], BamHl, and the template plasmid containing the SspB gene FIG. 1. Similar to methods described above for S. mutatis Agl/II, the SspB fragments were purified over three columns, HisPrep Nickel affinity, MonoQ and Superdex 200 10/300 GL gel filtration. The purified fragments were analyzed by SDS-PAGE (FIG. 3).

Confocal microscopy. S. mutans UA159 and S. gordonii DL1 were grown overnight in TSY (30 g/L of Trypticase soy broth and 0.5 g/L yeast extract, pH 7.2) media on an eight- well Lab Tek Chamber slide system (Sigma). The cells were fixed with 3% paraformaldehyde, washed with binding buffer (20 mM HEPES pH 7.4, 150 mM NaCl and 2.5 mM CaCl ₂ ), and thereafter iSRCR, or iSRCR ₁₂₃ (10 μΜ) were added to the cells and incubated for 60 min. The unbound SRCRs were removed by repeated washing using the binding buffer. Subsequently, Alexa fluor 488 conjugated Anti-His-tag antibody (EMD Millipore, Inc) (1 :50 dilution) that can bind to the His Tag on SRCRs was added. After 60 min incubation the unbound antibody was washed away thoroughly using binding buffer, and the chamber walls were gently removed. Cover slips were then mounted with 15 μΐ of fluoromount-G with DAPI (Southern Biotech Inc) to stain bacterial nuclei and were sealed until ready to be imaged. The experiment without SRCRs served as control. All slides were imaged using Leica SPl UV Confocal Laser Scanning Microscope and Zeiss LSM 710 Confocal Laser Scanning Microscope at the UAB-High resolution imaging facility (UAB- HRIF).

Flow cytometric analysis. S. mutans UA159 and S. gordonii DL1 cells were grown overnight in TSY broth media at 37 °C and washed thoroughly with FACS buffer (20 mM HEPES pH 7.4, 150 mM NaCl and 5% non-fat dry milk) to reduce non-specific binding. Subsequently, 10 μΜ of SRCR, or /SRCR _]23 were added to 100 μΐ of cells (1 x 10 ⁷ cells/ml) in binding buffer (FACS buffer + 2.5mM CaCl ₂ ) and incubated for 60 min at 37°C. The cells were later washed thoroughly with binding buffer to remove unbound SRCRs. Subsequently, Anti-His tag Alexafluor 488 antibody ( 1 :50 dilution) (EMD Millipore, Inc) that adheres to the His tag present on SRCRs was added to the cells and incubated for 30 min, washed three times and resuspended in 200 μΐ of binding buffer. Samples without /SRCRs served as control. Samples were then assayed using the FACScan machine (BD Biosciences) at the Analytical and Preparative Cytometry Facility (APCF) at UAB, and data obtained was analyzed using Flow Jo 7.2.4 software.

Aggregation assays. Aggregation assays were performed as described earlier [37] with slight modifications. Briefly, S. mutatis DAI 59 and S. gordonii DLl cells were grown in TSY broth media overnight at 37 ^° C in the presence of 5% C0 ₂ . The bacteria were centrifuged at 5000 x g and washed with a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl and re-suspended to an approximate OD700 of 1. The bacterial suspension (900 μΐ) was mixed with 100 μΐ of SAG or zSRCRj ( 10 μΜ) or z ^' SRCR ₁₂₃ ( 10 μΜ) or commercially synthesized (Think Peptides. Inc) SRCRP2 peptide with fluorescein amidite (FAM) at the carboxyl end (QGRVEVLYRGSWGTVCK-[FAM]) (SEQ ID NO:9, at both 400 μ^τηΐ and 1 mg/ml) in the presence of 6 mM CaCl ₂ and aggregation was measured by recording OD ₇₀ o over 60 min at 5 min intervals, where the buffer alone was used as control. All experiments were carried out at least five times, and the results were analyzed with One-way ANOVA. Post-hoc testing where *, P 0.05 was considered statistically significant and results were presented as the percentage of cells aggregated.

ELISA. The binding between SRCRs and oral streptococci fragments were analyzed using ELISA. Both /SRCRi and z ^' SRCR ₁₂₃ (10 μ^\νε11) in carbonate-bicarbonate buffer (pH 9.6) were coated on a black ELISA plate individually, washed with binding buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 2.5 mM CaCl ₂ (pH 7.2) and blocked with 3% non-fat dry skim milk. Various concentrations of Alexafluor 488 conjugated Anti-His-tag antibody (Millipore, Inc) ranging from 10 μg/ml to 0.1 ng/ml was added to 10 μg of each of the FL or A3VP 1 or C _t23 fragments of S. mutatis or S. gordonii for 3 hours at room temperature and were dialyzed into the binding buffer. Two hundred microliters of the fluorescently labeled fragments of Agl/II and SspB were added to the wells containing immobilized / ^' SRCRs and incubated for 3 hours. SRCR immobilized wells without fluorescently labeled analytes were used as controls. Later these wells were washed three times with binding buffer and data were recorded at an excitation wavelength of 495 nm with the emission at 519 nm using Synergy 2- multimode mi crop late reader ( Synergy, Inc). The assays were performed in triplicates and thereafter results were analyzed.

The binding between synthesized SRCRP2 peptide and SRCR were analyzed utilizing the FAM label on the SRCRP2 peptide. Briefly, both /SRCR, and /SRCR ₁₂₃ ( 10 μ^ννεΐΐ) in carbonate-bicarbonate buffer (pH 9.6) were coated on a black EL1SA plate individually, washed with binding buffer containing 20 mM HEPES, pH 7.5, 15 mM NaCl and 2.5 mM CaCl ₂ (pH 7.2) and blocked with 3% non-fat dry skim milk. Serial dilution of the SRCRP2 peptide (200 μΐ) ranging from 3ng/ml to O.OOlmg/ml in binding buffer were incubated with SRCRs for 3 hours at room temperature. SRCR immobilized wells without fiuorescently labeled SRCRP2 peptides were used as controls. Later the wells were washed with binding buffer and the data was recorded at an excitation wavelength of 495 nm with the emission at 519 nm using Synergy 2- multimode microplate reader (Synergy, Inc).

Surface Plasnion Resonance. Real time binding analyses of the SRCR domains with Agl/II fragments were carried out using the BIAcore 2000 system. The CM5 chip was labeled with ligands / ^' SRCRi or /SRCR ₁₂₃ SAG (a gift from Dr. Jeannine Brady, University of Florida, Gainesville, prepared as previously described [37,38], using the amine coupling kit (GE healthcare, Inc). The control and experimental surfaces were blocked using 1 M ethanolamine. Various concentrations of analytes (0.125 μΜ to 2 μΜ) of S. mutans Agl/II or S. gordonii SspB fragments and (Table 2) 2 μΜ of Lysozyme (Positive control) and Thaumatin (Negative control) were injected over the prepared chip surfaces and dissociation were measured for 8-10 minutes at a flow rate of 20 μΐ/min of binding buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 2.5 mM CaCI ₂ )at 25°C. Self association of iSRCR l or /SRCR123 (at 2 μΜ) were also determined in a similar manner as described above. Between these experiments the regeneration of the surface was accomplished using solutions as indicated in Table 2. Finally to determine the the effect of calcium SPR analysis was carried out by dialyzing the analytes and ligands in binding buffer devoid ofCaCl ₂ .

On-chip Deglycosylation of the SRCRi and /SRCR 12? was carried out after immobilizing them on CM5 sensor chip. Enzymatic deglycosylation was done to remove N- and O- 1 inked carbohydrates from ZSRCRi and /SRCR ₁₂₃ . Briefly, after immobilization of /SRCR) and /SRCR ₁₂₃ , deglycosylation was carried out at native conditions by incubating the entire chip surface externally with a cocktail containing a total reaction volume of 40 μΐ made up of 4 μΐ of 10X G7reaction buffer, 4 μΐ of 10% NP40, 4 μΐ of Neuraminidase (Sigma), 18 μΐ water and 10 μΐ of O-glycosidase (New England Biolabs, Inc.,) and similarly following manufacturers protocols for EndoH (New England Biolabs, Inc). Later, the chip was sealed and incubated overnight at 37°C. Subsequently the chip was thoroughly washed with binding buffer (20 mM HEPES, 150mM NaCl, 2.5mM CaCl ₂ ) to remove the deglycosylating enzymes and other remnants. Binding studies with FL ^AgVn and FL ^SspB and subfragments were then carried out as described above and regenerated as described in Table 2.

The utilization of a bivalent adherence model to elucidate the kinetics had inherent difficulties in clearly distinguishing affinities for each region, particularly for FL ^{AgI 11} and FL ^SspB . In addition, the SRCR holding two distinct surfaces compounded the elucidation of individual kinetics, and presently there are no modeling protocols available to determine the individual affinities for such a system, therefore for simplicity we have utilized a single site 1 : 1 Langmuir model. All experiments were carried out in triplicates and the kinetics of the association (K _A ) and dissociation (K _D ) rate constants were deduced using the 1 : 1 Langmuir Kinetic model on the BIA-Evaluation software [39]. The concentration (C in μΜ) of analyte (FL ^{A i n} or FL ^SSpB at 2 μΜ) that adhered to the immobilized ligand (i SRCRi, SRCR ₁₂₃ ) within the flow cell was calculated using the formula C = (RU/MW) x (1/V), where RU is resonance unit (1 RU = 1 pg of bound protein), MW is molecular weight of analyte and V is volume of flow cell ( 1 .2 10 ^"10 L).

Thaumatin /SRCRi /SRCRiza SAG 20 mM HEPES, pH 1 M NaCI, 10 mM HCI

(negative 8.0, 150 mm NaCI, 20m M EDTA

control) 2.5 mM CaCI ₂ pH 7.2

Lysozyme /SRCRi /S CRi23 SAG 20 mM HEPES, pH 1 M NaCI, 10 mM HCI

(positive 8.0, 150 mm NaCI, 20m M EDTA

control) 2.5 mM CaCI ₂ pH 7.2

SRCRi /SRCRi /SRCR ₁₂₃ 20 mM HEPES, pH 10 mM HCI 10 mM HCI

8.0, 150 mm NaCI,

2.5 mM CaCI ₂

/SRCR ₁₂₃ /SRCRi /SRCR ₁₂₃ 20 mM HEPES, pH 10 mM HCI 10 mM HCI

8.0, 150 mm NaCI,

2.5 mM CaCI ₂

Competition adherence assays. To determine whether Agl/II domains bound to the same site on SRCR domains, competitive binding SPR experiments were conducted in triplicates as previously described [16] where each fragment of Agl/II (FL, A3VP1, or C 123) or SspB (FL, A3 VP] or C] ₂₃ ) was initially passed over the chip surface immobilized with either /SRCRi, /SRCR 12 or SAG for 60 seconds to saturate available binding sites. The response curve of Agl/11 or SspB first fragment was recorded, where the maximal RU (RUi) was considered as the base line for the second injection, and thereafter the competing fragment was injected and its response was recorded as RU ₂ . The adherence of the second fragment was then calculated (RU ₂ -RUi) for all SPR competing assay as reported earlier [16].

Circular Dichroism. Spectroscopic studies were carried out on an Olis DSM 100 circular dichroism speetrophotom eter with 0.2 mm path length quartz cell. Recombinant /SRCRi or /SRCR 12 at concentration of 1 mg/ml in a buffer containing 20 mM HEPES pH 7.4, 150 mM NaCI and 2.5mM CaCl ₂ at 22°C were scanned between 200-260 nm and the spectra was recorded ( 10 times). Similarly, the conformational changes of SRCRs on addition of different concentrations of calcium (2,4,6,8 and 10 mM) in binding buffer as well as SRCR samples devoid of calcium (control) were analyzed by scanning the spectra between 200-260 nm for nearly 10 times. Using CONTIN/LL algorithm implemented in CDPRO [40] the protein secondary structure were assigned.

Differential scanning calorimetry. The thermostability of SRCRs in the presence of calcium ions was analyzed using Microcal MC-II differential scanning calorimeter (GE HealthCare, USA) as described earlier [41 ]. Briefly, /SRCR] or /SRCR 12 at concentration of 1 mg/ml was mixed and incubated with different concentration of CaCl ₂ ranging from (0- 100 mM) to final volume of 400 μΐ of buffer containing 20 mM HEPES, 150 mM NaCI), and buffer without SRCRs served as control. Data were recorded with cal ori metric scanning rates that ranged from 3()°C/h to 90°C/hat 30 psi pressure. The data collected was analyzed for the unfolding temperature (T _t ), and the calorimetric (AH _cal ) and van't Hoff (Δ// _ν ) unfolding enthalpies using the Origin software package (MicroCal).

Glycoprotein staining and GC-MS analysis of SRCRs carbohydrates. The SRCRi and SRCR ₁₂₃ proteins were electrophoretically separated on a 12.5% SDS-PAGE gel and stained by glycoprotein staining kit (Pierce. Inc), where Horse radish peroxidase (HRP) and Soybean trypsin Inhibitor (SBTI) were used as positive and negative control respectively. The glycosyl composition analysis of purified /SRCRi and 1SRCR ₁₂₃ were done by the preparation and gas chromatograph-mass spectrometry (GC-MS) of trimethylsilyl (TMS) methyl glycosides as previously described [42].

Adherence/Inhibition studies. Carbohydrates Gaipi -3-GalNac and Man nose

(identified from glycan profile analysis) at different concentrations (0.010, 0.050, 0.1 , 0.5 and 1 mM) were incubated with 2 μΜ of each FL, A3VP1 and Q2 ₃ of Agl/II and SspB and the interaction with immobilized /SRCRi and /SRCRi _2? with running buffer containing 20 mM HEPES, 150 m NaCl and 2.5 mM CaCl ₂ , pH 7.4 at 25°C and with flow rate of 20 μΐ/min was analyzed. Direct adherence of these carbohydrates alone served as the control and all calculations were carried out using the BIAevaluation software. Using the same protocol above at varying concentrations the effect of SRCRP2 peptide on the adherence inhibition was assessed.

Analytical Ultracentrifugation. /SRCRi or /SRCR1 ₂₃ (0.5 mg/ml) in a buffer containing 20 mM Tris pH 8.0, 150 mM NaCl, and 1 mM EDTA were subjected to sedimentation velocity experiments on a Beckman Optima XL- A as previously described [15]. Briefly, the samples were centrifuged to 45,000 rpm with the temperature maintained at 20°C, and where absorbance at 280 nm across the cell recorded every 5 min. Using Sednterp, buffer density values of 1.0052 g/ml, protein partial specific volumes of 0.720 and 0.714g/ml, and hydration values of 0.365 and 0.370 g/g for /SRCRi and /SRCR123, respectively were calculated [43,44].

B: RESULTS

Agl/II and SspB constructs. Constructs /SRCRi (15kDa) and SRCR ₁₂₃ (43 kDa) were prepared as described earlier [36]. S. mutatis Agl/II constructs FL ^{A I 11} ( 167.5 kDa, earlier referred to as CG I 4). A ₃ VP, ^AgL " (54.4 kDa) and C ₁₂₃ ^AgI/I1 (57.3 kDa) were expressed and purified as described earlier [ 15, 16]. The equivalent constructs for S. gordonii SspB, _FL Ss _P B _{( 1 5 1 9 kDa)i} A ₃ VPi ^SspB (46.3 kDa) and Ci ₂₃ ^SspB (56.2 kDa) were cloned, expressed and purified for this study as described in the materials and methods section. The purity of proteins was qualitatively assessed to be >95% from SDS-PAGE gels (FIG. 3).

Confocal microscopy. Adherence of / ^' SRCRs to S. mutans and S. gordonii cells were qualitatively analyzed using confocal microscopy. In FIGs 1 and 2 bacterial nuclei stained with DAPI are displayed in blue, and in green the Anti-His tag Fluor 488 antibody that recognizes the His Tag on the SRCRs. The controls in the absence of SRCRs were only stained by DAPI (FIG. 4, panel A), whereas green fluorescence observed (FIG. 4, panel B and C) confirms the binding of /SRCR] and SRCR ₁₂₃ domains to S. mutans cells. Similar adherence was observed with S. gordonii cells (FIG. 5, panels A-C). These findings indicate that z ^' SRCRs interact with both S. mutans and S. gordonii, and that the z ^' SRCRi adheres poorly compared to z ^' SRCRi ₂₃ , thus indicating that multiple SRCR domains have better adherence capability compared to that f a single SRCR domain. Z view of the confocal microscopy picture clearly shows that the SRCRs adhere only to the top surface of immobilized S. mutans and S. gordonii cells and not to the plate.

Flow cytometry. From representative histograms, / ^' SRCR 123 shows nearly a 100 fold increase in fluorescence intensity compared to the control, whereas z ^' SRCRi displays only a 10 fold increase (FIG. 6, panel A). In the case of S. gordonii, z ^' SRCRi and / ^' SRCR 123 display lower adherence (FIG. 6, panel B), not as profoundly as evidenced with S. mutans. Taken together. / ^' SRCR123 definitively adheres better than z ^' SRCRi again indicating that multiple domains play a co-operative role in this interaction. Furthermore, these results also demonstrate that S. mutans has more pronounced adherence to z ^' SRCRi 23 compared to S. gordonii.

Aggregation assays. SAG has been well documented to have aggregation properties particularly with S. mutans and S. gordonii [35,45,46]. In this regard, we tested whether individual SRCR domains possess aggregation property as that of SAG. In the presence of z ^' SRCRi23, 69% of S. mutans and 48% of S. gordonii aggregated while z ^' SRCRi aggregated 17% of S. mutans and 13% of S. gordonii (FIG. 7, panels A and B). The positive control SAG aggregated S. mutans by 74% and S. gordonii by 72%. Earlier studies with consensus peptide, SRCRP2 derived from SRCR domains, aggregated a variety of bacteria [20,34] but however in our current study compared to z ^' SRCRi and i ^' SRCRi ₂₃ , the SRCRP2 peptide displayed limited aggregation with S. mutans (12%) and S. gordonii (11%). The SRCRP2 peptide even at higher concentrations like 1 mg/'ml was able to aggregate S. mutans (18%) and S. gordonii (12%) only minimally (FIG. 8). ELISA. Both FL ^Agl/l1 and FL ^SSPB displayed better adherence to / ^' SRCR, and /SRCR 123 compared to their sub fragments A ₃ VP ₁ and Cj ₂₃ (FIG. 9, panels A and B). Similarly, the SRCRP2 peptide bound to FL ^{A l 11} and FL ^SSPB with higher affinity compared to their sub fragments A ₃ VP, and C _i23 of Agl/II or SspB (FIG. 10).

Adherence assays and quantitation. SPR was used to quantify the affinities between immobilized z ^' SRCRs and the analytes FL, A ₃ VPj and Ci ₂₃ of Agl/II and SspB (Table 3; FIGS. 11-13). The adherence to lysozyme (positive control) and not to thaumatin (negative control) confirmed the specificity of the SRCR domains (FIG. 14). The interaction of the _FL Agi ^/ n _with SAG is one order of magnitude lower (3.33 x 10 ^"8 M [15]) than that of the FL ^SSPB (6.15 x 10 ^"9 M), and indicates that SspB adheres with higher affinity to SAG. While FL ^SSPB interacts with higher affinity to SAG, the reverse is observed with the individual SRCR domains, where FL ^Ag,/ " displays a higher affinity with both z ^' SRCRi (7.69 x 10 ^" M vs 2.56 x l O ^"7 M) and SRCR ₁₂₃ (7.46 x 10 ^~9 M vs 4.21 x 10 ^"8 M). In all other cases, the FL ^{A l} " and FL ^SSPB had similar or higher affinities compared to the individual fragments except for A ₃ VPi ^SspB which displays one order higher affinity (3.41 x l O ^"8 M) with /SRCR,. Interestingly. Ci ₂₃ of Agl/II and SspB, which is located near the streptococcal cell wall (FIG. I) displayed similar affinities to the z ^' SRCRs (1.51 x 10 ^"7 M and 4.64 x 10 ^~7 M with /SRCR, and 8.70 x 10 ^"8 M and 6.21 x 10 ^"7 M with /SRCR 123) . These affinities indicate that the binding mechanism adopted by the A ₃ VP ₁ region at the apex of the molecule may vary between species. Overall, the similarity in the affinities observed between all Agl/II and SspB fragments with both z ^' SRCRi, z ^' SRCRi ₂₃ and SAG, strongly proves that a single SRCR domain contains the adherence sites for Agl/II and SspB. Although FL ^{A l} " and FL ^SspB displayed similar affinities, the quantity of protein that adhered to z ^' SRCR ₁₂₃ was 16% and 43% higher than z ^' SRCR] (FIG. 15), and this result is in conjunction with our earlier whole cell assays, where z ^' SRCR] ₂₃ displayed better adherence and aggregation.

Competitive binding experiments. We previously demonstrated that the interaction of Agl/II with SAG was multivalent, where A ₃ VPi ^{A l} " as well as C ₁₂₃ ^AgI711 interacted with two distinct surfaces on SAG [16]. To determine if the individual SRCR domains contain these distinct surfaces, competitive binding experiments with FL ^{Agl 11} and FL ^SspB and their fragments were analyzed by SPR using immobilized z ^' SRCRs on CM5 sensor chip and the results of which are summarized in (FIG. 16, panels A-E). While FL ^{A l} " was able to inhibit the binding of A ₃ VPi ^AgI/n and C _m ^AgI/I1 by 46%, and 36% respectively, FL ^SspB inhibited A ₃ VPi ^SspB and Ci ₂₃ ^SspB by 54% and 23% with immobilized /SRCR, . Similar inhibition was observed with immobilized iSRCRi ₂₃ domains where FL ^Ag, " adherence inhibited the binding of A ₃ VP, ^AgVU and C ₁₂₃ ^{AgI n} by 44% and 25%. However, FL ^SspB had limited inhibitory effects with immobilized /SRCR] ₂₃ , where A ₃ VPi and C] ₂₃ displayed 68% and 76% inhibition respectively. This points out that the surface proteins of S. mutans and S. gordonii may display different characteristics in their adherence, although they are highly homologous. In all other cases, A VP| or C ₁₂₃ of Agl/II and SspB did not significantly inhibit the adherence of each other indicating that there are indeed two distinct surfaces within the SRCR domains that specifically bind Agl/II and SspBs A ₃ VP] as well as Ci ₂₃ fragments.

Role of calcium (calcium mediated adherence/stability). We tested to see if these SRCRs require calcium for adherence similar to that of SAG with FL and subtragments of Agl/II and SspB, and in the absence of calcium there was no adherence (FIGS. 8 and 9), and therefore we set out to determine the role of calcium ions in this adherence. In CD studies, the SRCRs clearly demonstrated a notable change in secondary structural content, particularly a reduction in alpha helices and an increase in beta sheet content in the presence of calcium (Table 4, FIG. 19, panels A and B), whereas no such changes were observed with Agl/II or SspB (data not shown). In addition, the stability (thermal unfolding) of z ^' SRCRi increased in a dose dependent manner (Table 5, FIG. 20), and highly stable unfolding only at 90°C ( 100 mM CaCl ₂ ). While the thermal unfolding curves of /SRCR] were simple and easy to interpret, those of the /SRCR] was complex, with many peaks due to unfolding of multiple domains (data not shown).

Table 5: DSC Studies

Samples ΔΗ ΔΗ _ν Tm C°C)

/SRCRi 5.703E ⁴ ± 216 6.016E ⁴ ± 281 56.5

/SRCRi + 2.5 mM CaCI ₂ 7.828E ⁴ ± 280 8.455E ⁴ ± 374 78.1

/ ^' SRCRi + 10 mM CaCI ₂ 6.708E ⁴ ± 412 1.117E ⁵ ± 854 86.4 SRCRi + 100 mM CaCI ₂ 9.736E ⁴ ± 1.55E ³ 1.111E ⁵ ± 2.24E ³ 92.8

While homologous structures of SRCR domains from both group A and group B have been determined [47,48], to date there are no crystal structures of the SRCR domains or Gp340. CD results summarized in Table 4 and FIG. 19, panels A and B indicate that both the /SRCRi and /SRCR ₁₂₃ domains possess similar secondary structures compared to the solved crystal structures (PDB2JA4, PDB1BY2, PDB1P57 [47,49,50]. This indicates that the SRCR domains of Gp340 could adopt a similar SRCR fold compared to the solved crystal structures.

Effect of carbohydrates on the binding of Agl II. The presence of glycosylation on

/SRCRi and SRCRi ₂₃ was initially confirmed using glycoprotein staining (FIG. 21). While EndoH (N-glycosidase) did not have any measurable effect (data not shown), O-glycosidases had profound effects on the adherence kinetics. Deglycosylation of /SRCRi and /SRCRi ₂₃ by O-glycosidases did not affect the adherence characteristics of A3VP1 of Agl/II, but decreased the adherence of the Cm ^{A m} by two orders of magnitude (Table 3), indicating that carbohydrate binding could arise from the domains close to the cell surface for S. mutatis Agl/II. In the case of SspB, both the apical A? VP, as well as Ci ₂₃ displayed reduced kinetics, and thus indicating that both these regions could possess adherence sites to carbohydrates.

Following these above observations, glycan profile analysis of both /SRCRi and /SRCR123 indicated that they are predominantly O-glycosylated with Gaipi-3-GalNac and Mannose type of carbohydrates (Table 6). Enumerating the role of Galpl-3-GalNac and Mannose in adherence/inhibition experiments, we observed that the carbohydrate controls by themselves did not have any interactions with SRCRs, however, at lower concentrations (0.010 niM-0.500 mM) of Galpl-3-GalNac enhanced the adhesion of all Agl/II and SspB fragments with /SRCRi and /SRCR ₁₂₃ , thus indicating an important role they play in the adhesion process. However, at 1 .0 mM concentration Gai i -3-GalNac significantly inhibited the adhesion of FL ^AgI ", A ₃ VP, ^{A l} " and C _X2 ^ ^m to /SRCRi by 85%, 79% and 73% respectively. Similarly, Galpl-3-GalNac significantly inhibited the adhesion of FL ^SspB , A ₃ VPi ^SspB and C ₁₂₃ ^SspB to /SRCRi by 64%, 61% and 32% respectively. In the case of zSRCR ₁₂₃ adhesion to FL ^{A l} ", A ₃ VP, ^AgI/11 , C ₁₂₃ ^AgI/I1 was also significantly inhibited by 73%, 75% and 47%. whereas Galpl-3-GalNac did not greatly inhibit the adhesion FL ^SspB , A ₃ VP ₁ ^SspB and C _{1 2} 3 ^SspB to SRCR, ₂ 3 (37%, 60% and 40%) (FIG. 22. panels A and B). Although, it is difficult to directly correlate these results, the apical A3 VP] f both Agl/II and SspB appear be the most inhibited fragment by Gal| l -3-GalNac. When testing for the role of Mannose (data now shown), it was clear that it neither inhibited nor enhanced the adhesion of FL, A ₃ VP, and C ₁₂₃ of Agl/II and SspB to both /SRCR, and iSRCR _]23 .

SRCRP2 (Bikker Peptide). Initial ELISA assays demonstrated that the SRCRP2 peptide adheres well with Agl/II and SspB and their subtragments. When incubated at low concentration (0.005 mM) with FL ^AgI " and A ₃ VP] ^AgI/11 , the SRCRP2 peptide improved the adherence by 8% and 13% respectively to SRCRi, and 8% and 16% respectively to SRCRi ₂₃ , whereas C, ₂₃ ^AgI/n had no notable changes in adherence (2.3% with /SRCR, and 7% with SRCR ₁₂₃ ). Only FL ^SspB improved the adherence by 97% with /SRCR, and 91 % with z ^' SRCRi ₂₃ at lower concentration (0.005 mM), whereas A ₃ VP, ^SspB and Ci ₂₃ ^SspB did not alter the adherence characteristics to either / ^' SRCR, (3% and 4%) or SRCR123 (3% and 5%) respectively FIG. 23. panels A and B). Also, the SRCRP2 alone (control) did not show any binding with SRCRs. These results clearly indicate that SRCRP2 peptide does not bind and inhibit the adherence of Agl/II and SspB to z ^' SRCRi and z ^' SRCRi, indicating that the adherence site may be different from that of the aggregation sites present on Agl/II and SspB.

Self adhesion. Interaction of SRCRs with each other was tested using SPR. The z ^' SRCRi and z ^' SRCRi strongly interacted with each other. Analytes z ^' SRCRi (1.13 X 1(T ¹⁰ ) and z ^' SRCR]23 (5.68 XI 0 ^"9 ) demonstrated high affinity with immobilized z ^' SRCRi. Similarly, analyte with z ^' SRCRi (1.2 X 1 ( ^)" '') and z ^' SRCRi ₂₃ (6.72 X 10 ^"9 ) interacted with immobilized z ^' SRCRi 23 with nanomolar affinities (FIG. 24, panels A-D). These results clearly showed that the SRCRs have self adhesion property as well.

Analytical Ultracentrifugation. We sought to answer the question of the spatial organization of the SRCR domains, particularly whether they might be elongated similar to Agl/II through ultracentrifugation experiments. From their observed frictional ratios (z ^' SRCRi = 1.59, z ^' SRCR ₁₂₃ = 1.80), resultant prolate ellipsoid ratios (z ^' SRCRi = 7.18, z ^' SRCR _]23 = 10.36) and calculated dimensions (z ^' SRCRi = 12.60 x 1.75 nm, SRCR ₁₂₃ = 22.08 x 2.13 nm), it is clear that both z ^' SRCRi and z ^' SRCR ₁₂₃ will have extended structures (Table 7). However, these may not be extended as linear rigid structures and instead may exist in a flexible nonlinear conformation forming curvy tertiary structures. Models for the interaction of Agl/II and SspB with SRCR domains of Gp340 are discussed further below.

C: DISCUSSION

The ability to adhere strongly to human receptors within the oral cavity is a necessity for bacterial survival, or else they will be washed into the acidic gut [51]. Therefore bacteria that colonize the oral cavity have multiple proteins on its surface for specific adherence to human receptors [52]. As set forth herein, the oral streptococcal surface receptor Agl/II and its homologs have been examiner to develop both small molecule/peptide inhibitors as well as passive immunization [35,53,54]. The interaction Agl/II with Gp340 is considered to be the first step in adherence to tooth surface, which subsequently leads to colonization and infection, and among these the mutans streptococci are known etiological agents in dental caries [22,55]. For the past three decades this interaction has been studied using Gp340 extracted from saliva of either single or multiple donors [33,56,57].

Using recombinantly expressed SRCR domains of Gp340 by means of the Drosophila expression system, this interaction has been examined to elucidate the intricate components involved in this bacterial adhesion. The minimal Agl/H adherence region on Gp340 has been identified, and the species-specific differences in adherence among the Agl/II homologs and the role of glycolysations and metal ions have been examined.

Expression and purification of SRCRs has been reported previously [36], and herein, the specific adherence of the SRCRs using lysozyme (positive control) and thaumatin (negative control) has been shown (FIG. 14). Our results from confocal microscopic images (FIGS. 1 and 2), FACS analyses (FIG. 6) and aggregation assays (FIG. 7, panels A and B) clearly revealed that SRCRs bind to S. mutans and S. gordonii cells, where /SRCR 12 attaching more profusely compared to /SRCRi. Through calculations based on protein adherence to chip surface (see methods) it has been discovered that higher amounts of FL ^{A I 11} and FL ^SspB adhered to immobilized /SRCR 123 than /SRCRi (FIG. 15) and is in conjunction with the whole cell assays described here above. It appears from these results that more than one SRCR domain is required for efficient aggregation of bacteria. As such, longer tandem SRCR domains may more efficiently agglutinate various bacteria, and warrants further validation in future studies. Knowing that Gp34() is an innate immunity molecule, the number of tandem repeats it takes to efficiently agglutinate bacteria could have been evolutionarily determined, and it is interesting to note that in humans, Gp340 contains 14 SRCR domains, in which thirteen of them are tandem repeats, whereas in other vertebrates the number of tandem repeats are comparatively lower [24,30]. The interaction of /SRCRi and SRC nj with Agl/ll-homologs was further characterized to determine their kinetic coefficients. Initial ELISA (FIG. 9, panels A and B) assays provided clues of efficient binding of recombinant iSRCRi and i ^' SRCR ₁₂₃ to FL ^{A I} " and FL ^SspB and their individual SAG binding regions A ₃ VP _t and C ₁₂₃ . Further characterization with SPR established the existence of nanomolar affinity interactions between the SRCRs and Agl/II-homologs (Table 3 and FIGS. 11-13. It is here that significant differences in the adherence kinetics of A ₃ VP , (apex region) of both Agl/II and SspB were discovered, whereas the adherence kinetics of the Ci ₂₃ domain (present near cell the wall) to the SRCRs were not notably different. Inspite of indistinguishable kinetics, it needs to be noted here that the Ci ₂ ^SspB also displayed characteristic sensorgrams with a tendency not to remain bound to the immobilized SAG or SRCR domains (FIG. 13). The observed differences in adherence between Agl/II and the apex adherence site A ₃ VP) of SspB could be attributed to species-specific recognition and perhaps attributable to the apical V- regions of Agl/II and SspB as they are structurally distinct (37 kDa Vs 31 kDa) [58]. Overall, these results imply that the only known SAG binding protein Agl/II of pathogenic S. mutans could possibly contain a locking mechanism to maintain adherence, whereas the commensal S. gordonii, with two tandem gene repeats containing SspA and SspB, could adopt a different approach in its mode of adherence to SAG.

While it is known that A ₃ VP] and C _{) 2} 3 adheres to two distinct surfaces on SAG,[16], this study demonstrates that the SRCRs contains these two binding surfaces that are recognized by A ₃ VP] and C ₁₂₃ . These points to the fact that a single SRCR domain contains both the surfaces and is therefore the minimal adherence region for Agl/II and its homologs (FIG. 7, panels A-E). This result is highly significant as it would now render focus on the SRCR domains of Gp340 to further elucidate the multivalent adherence mechanisms of Agl/II homologs.

It is known that calcium plays a crucial role in the interaction between Gp340 and Agl/II homologs [59,60]. The analysis herein confirms that calcium is essential for mediating the interaction between SRCRs and Agl/H homologs (FIGS. 17 and 18). Furthermore, CD studies clearly indicate that calcium influences secondary structural changes in both z ^' SRCRi and z ^' SRCR ₁₂₃ (Table 4, FIG. 19, panels A and B), and DSC analysis indicates that calcium increases the thermostability of SRCRs (Table 5, FIG. 20). These data confirm that not only the SRCRs undergo conformational change, but also get stabilized in the presence of calcium. The oral cavity is subject to environmental changes including pH and temperature. Perhaps the thermal stability that was observed may be a direct consequence of evolution, wherein these molecules have developed ability to withstand temperature changes (hot and cold food and beverages) that they face within the oral cavity. It would be interesting to see whether the SRCR domains from sea urchin possess these thermal properties which would directly link it to evolution of the SRCR domains within the human oral cavity.

Gp340 is decorated with glycosylations [24,61], and were previously shown to play an important role in the adherence of Agl/II and its homologs [62]. While glycostaining of recombinant SRCRs indicated the presence of glycans (FIG. 21), we further expounded their composition using glycan profile analysis (Table 6), which showed predominant O- glycosylation. Deglycosylation with O-glycanase reduced the adherence of Agl/II and SspB by 10 fold (Table 3), whereas the deglycosylation with EndoH (N-glycanase) had no significant effect (data not shown) thus indicating a role for the glycosylations. While these results implicate O-glycosylations to be the major player, it is not possible to currently rule out the effect of N-glycosylations in adhesion. In a series of further experiments, where Agl/II and SspB and their sub-fragments were incubated with various concentrations of Gaipi-3-GalNac, it appears that there seems to be a significant additive effect (more of Agl/II adhered) at lower concentrations of the glycosylations, indicating a role in adhesion, as well as inhibition at higher concentrations with SRCRs, possibly saturating the adherence sites (FIG. 22, panels A and B). These above exemplify that glycosylations play a role, albeit peripheral compared to the calcium ions, in adherence.

The SRCRP2 peptide has been shown to adhere and aggregate bacteria [20,34].

However, the aggregation by the SRCRP2 peptide (FIG. 6, panels A and B) was very limited, even at high concentration (FIG. 3), compared to that of z ^' SRCRj and SRCR ₁₂₃ , indicating that the folded protein may possess additional sites that result in efficient aggregation. While fluorescence based assays indicated adherence of the SRCRP2 peptide to Agl/II and SspB (FIG. 9), incubation of SRCRP2 peptides with the apex (A ₃ VPi) and proximal cell surface (C ₁₂₃ ) SAG adherence domains did not significantly inhibit the adherence, but rather surprisingly increased the adhesiveness f only FL ^SspB , indicating the presence of a nonspecific aggregation property (FIG. 22, panels A and B). These results now demonstrate that the peptide while possessing an aggregating property, is very limited in its ability compared to the full length folded protein domains, and more importantly it neither adheres to nor blocks the GP340 binding motif/site on either Agl/II or SspB.

It is known that Gp340 exists as a higher order complex, and these aggregates could be as large as 5000 kDa [22,63]. The aggregation property of Gp340 has been attributed to the Zona Pellucida (ZP) domain, as in other mammalian proteins, the ZP domain was shown to be involved in self-aggregation [64]. In this context, we tested for the ability of the SRCR domains for self-interaction, and surprisingly found that they associate with nano-molar affinities, and thus indicating that this association as highly specific, as non-specific interactions traditionally appears to fall within the micro-molar range (FIG. 24, panels A-D). These results now for the first time implicate the SRCR domains in self-aggregation, and opens up several possible models for bacterial aggregation, wherein one potential model could simulate the bacterial proteins to be sandwiched between two SRCRs (Gp340s) (FIG. 25). It is here that the tertiary architecture of tandem SRCR domains were examined, and identified through ultraeentrifugation studies that the SRCR domains may not strictly form a linear elongated structure (Table 7) but could form a curvy centipede-like extended structure, similar to that observed in electron microscopy images of Gp340 [31].

While that which is exemplified herein is generally focused on Agl/II and its homologs, it has been shown that the SRCR domains of Gp340 play a pivotal role in mediating HIV adhesion/clearance through Gpl20 within the oral cavity [65,66]. While Gp340 acted as a clearance mechanism in the oral cavity, the case was very different on the vaginally derived Gp340, which is immobilized on the cell surface, where this was shown to mediate trancytosis from apical to basolateral surface in both genital tract epithelial cells in culture and with endocervical tissue [67]. Similarly, in our SPR experiments, immobilized SRCRs adhere tightly to Agl/II homologs, while in fluid phase SRCRs aggregate S. mutans and S. gordonii, a double faceted property, where on the one hand acts as a portal of entry for microbes while immobilized, on the other as a clearance mechanism within the oral cavity in fluid state. This property indicates that SRCRs may adopt different secondary structural conformations in fluid and immobilized states and this conformation could be induced by calcium ions.

That which is exemplified herein indicates that the minimal adherence region is restricted to a single SRCR domain, which carries the two distinct surfaces that adhere to A ₃ VP ₁ as well as Ci ₂₃ of both Agl/II and SspB with increasing number of SRCR domains for better adherence and aggregation of bacteria. Calcium mediated structural changes are essential for the adherence of Agl/II and SspB, and the SRCR domains become more stable at higher concentrations of calcium. Biophysical characterization indicates that the SRCR domains may adopt a curvy centipede like structure. That which is exemplified herein also establish that glycosylations do play a role in the adherence to Agl/II and SspB. While there are similarities in the binding of Agl/II and SspB, there are certainly distinct differences pointing toward species specificity in their adherence. Overall, that which is exemplified herein indicate that focusing on the SRCR domains and the interactions at a molecular level between Agl/Il homologs and SRCR can assist in identifying interventional therapeutics in the form of small molecule inhibitors, or development of passive immunization therapies that can impede oral streptococcal adherence to tooth surfaces and alleviate the global burden of dental caries.

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other bacterial surface proteins by Gaipi-3GalNac (core-1)

Inhibition of SAG adherence to Agl/II, SspB, Pas, along with glucan binding protein C (GbpC) and collagen binding protein (rcnM) by Gaip i -3GalNac (core-1) was determined as is described in Example 1 and as described below.

Method: Recombinant full length (FL) constructs of surface proteins (homologs of Agl/II) Agl/II, SspB, Pas, along with GbpC (Glucan binding protein C) and rcnM (collagen binding protein) were incubated with various concentrations (0.010 mM to 2 mM) of Gaipi - 3GalNac at room temperature and their binding interaction with immobilized salivary agglutinin on a CM5 sensor chip was determined. In control experiments, 2m Gaipi- 3GalNac did not show direct adherence to SAG.

Results: Results of SAG inhibition on other bacterial surface proteins are depicted in FIG. 1. At a concentration of 2 mM, Gal(31 -3GalNac inhibited SAG adherence of Agl/II by 94%, SspB by 65%, GBPC by 72%, rcnM by 69%, another Agl/II homolog, Pas, exhibited 28% inhibition. These results in the inhibition SAG adherence by Gaipi-3GalNac shown in FIG. 26 clearly point towards the use of Gal l -3GalNac as a broad range inhibitor of SAG adherence that can target more than one surface protein. In addition, these results are: indicative of how each surface protein interacts with SAG through carbohydrates; indicative that Gaipi-3GalNac can effectively inhibit attachment of pathogenic oral streptococci to SAG; and indicative that Gai i-3GalNac can serve the worldwide populace with dental caries.

EXAMPLE 3: Binding of SRCR

peptide to Agl/II, SspB and GbpC Binding of the SRCR peptide ETNDANVVARQL (SEQ ID NO: 10) to immobilized bacterial surface proteins was determined using procedures as described in Example 1.

The interaction of the SRCR peptide ETNDANVVARQL (SEQ ID NO: 10) with immobilized bacterial surface proteins Agl/II, SspB and GbpC as examined by surface plasmon resonance studies are shown in FIG 27A, 27B and 27C respectively, and binding affinities determined from these studies are listed in Table 8. Table 8. Surface plasmon resonance studies with SRCR peptide k _a (1/ms) kd(l/s) Rmax (Ru) l/M) MM) Chi2

Agl/ll Vhel (S. mutans) 1.08 x 10 ⁷ 0.696 43.3 1.56 x 10 ⁸ 6.42 x 10 ^"9 8.72

SspB Vhel (S. gordonii) 4.44 x 10 ⁵ 0.0312 110 1.42 x 10 ^s 7.02 x.lO ⁹ 6.99

GbpC (S. mutans) 1.47 x 10 ⁷ 0.0573 232 2.57 x 10 ⁸ 3.89 x 10 ^"9 25.1

As shown by the dissociation constants _D listed in Table 8, the SRCR peptide ETNDANVVARQL (SEQ ID NO: 10) binds with nanomolar affinity (3.89-7.02 x 10 ^" '' M) to bacterial surface proteins Agl/II, SspB and GbpC.

These results indicate that the peptide ETNDANVVARQL (SEQ ID NO: 10) may be a peptide inhibitor of the interaction between Agl/ll, and its homologs, at SAG.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.