It has been suggested that host-derived salivary proteins such as lysozyme, lactoferrin and lactoperoxidase modulate the composition of the oral microflora by binding to the microbial cells and causing aggregation and/or direct killing, while other salivary proteins including amylase enhance bacterial binding to tooth surfaces 5
. In parallel, salivary and microbial proteins selectively bind onto tooth enamel forming the acquired enamel pellicle 36–38
. The glucosyltransferases (Gtfs) secreted by S. mutans
, for example, bind avidly to the pellicle formed on the tooth surface where they are highly active; i.e. when exposed to sucrose, the adsorbed Gtfs form a layer of glucans on the surface within minutes 39, 40
. The polysaccharides on the pellicle provide specific binding sites for bacterial colonization, particularly mutans streptococci 30, 41
. Thus, specific host receptors (e.g. agglutinins, proline-rich proteins, amylases) and glucans synthesized in situ
by bacterial Gtfs bound to the pellicle act as bacterial anchor sites, and along with cell-surface proteins dictate the composition of initial microbial tooth colonizers 9, 30, 39, 42
cells attach initially to saliva coated surfaces through sucrose-independent mechanisms mediated primarily by lectin-like interactions between specific pellicle proteins (e.g. agglutinins) and adhesins (e.g. P1) present on the bacterial cell surface 9, 42
. Furthermore, S. mutans
cells also bind to the glucan-coated surfaces, and more importantly, in larger numbers and with higher adhesion strength than to saliva-coated surfaces 30, 43
through expression of several glucan-binding proteins 44
. Binding of S. mutans
to the tooth surface is critical for its establishment and initiation of pathogenic biofilm formation. Thus, any molecule that modulates adherence of S. mutans
, especially glucan-mediated binding, may influence colonization and further accumulation on the tooth surface. The majority of the studies conducted thus far have been focused on identification of streptococcal surface proteins responsible for mediating bacterial binding to the salivary pellicle 42
. Considering that S. mutans
cells (and, in fact, all other oral pathogens) are present in and coated with whole saliva in the mouth, it is critical to identify which salivary proteins bind to this pathogenic organism and whether the surface-bound host protein mediates their initial attachment to the tooth surface, affect bacterial viability and/or cause aggregation.
The present study identified by multi-dimensional protein identification technology 90 non-redundant salivary proteins bound to S. mutans
, suggesting their potential significance in oral defense and colonization in the oral cavity. Different conditions (low to high salt and urea) were used to differentiate between proteins with weak, moderate and strong binding affinities to S. mutans
surface. Of the proteins identified, gp340 and CSP-1 were identified as the most abundant, tightly bound proteins on the microbial surface. Salivary agglutinin is a well known bioactive (defense) salivary molecule associated with aggregation capacity of saliva, which may modulate implantation and colonization of cariogenic bacteria (such as S. mutans
) on tooth surfaces 14
. It is noteworthy that calgranulin B, a component of the acquired pellicle 38
, was also detected in high abundance on S. mutans
surface. Although there is no evidence showing this protein mediates S. mutans
binding, calgranulin family contains a calcium-binding domain possibly involved in enamel deposition 38
which may have a potential significance for caries disease.
CSP-1 appears to be expressed mainly in human salivary tissue and to a lesser extent in trachea and prostate gland, based on large-scale analysis of the transcriptome of 79 human tissues 34
. CSP-1 was identified as hypothetical protein (IPI00060800.3) in this study, and is the human ortholog of rat CSP-1 and mouse Dcpp (Demilune cell and parotid proteins). Human CSP-1 was also reported as hypothetical protein (accession number IPI00060800) by Denny et al. 2
, while it was reported as “similar” to common salivary protein (accession number gi3 21687060) by Wilmarth et al 45
. This difference in terminology and accession numbers was due to the use of different protein databases; Denny et al. used the EBI protein databases, while the NCBI non-redundant database was used by Wilmarth et al. for protein identifications. As compared to other known salivary proteins such as lysozyme (~21 μg/ml) 46
, IgA (109 μg/ml) 47
, CSP-1 is a moderately abundant proteins with its concentration estimated as 10 μg/ml in parotid saliva. CSP-1 shares a similarity of 38.1% with rat CSP-1 and 37.4% with mouse sublingual demilune protein (also called as SPT2) 32
. The similarities between human CSP-1 and these related proteins include a classical NH2
-terminal signal sequence, a putative jacalin-related lectin (JRL) domain, and potential N-linked glycosylation sites 48
. Members of the JRL protein family bind to glycoproteins, are ubiquitously expressed throughout the plant and animal kingdom 49
, and perform functions such as cell agglutination and antimicrobial activity 34
. Future studies are required to determine if the strong binding of CSP-1 to S. mutans
cells is mediated by the JRL domain in CSP-1, or if other functional domains in CSP-1 bind to S. mutans
either directly or as part of a protein complex.
Considering that CSP-1 binds tightly to S. mutans, we examined whether bacterial cells exposed to CSP-1 display (i) changes in their binding activity to experimental pellicle (sHA) and glucans (gsHA) formed on apatitic surface, (ii) altered viability/growth rate and/or (iii) enhanced cell aggregation. This was the first step toward identifying additional novel salivary proteins that potentially modulate the functional and biological activities of S. mutans.
Our data indicate that S. mutans cells exposed to rCSP-1 (10 μg/ml) in saliva significantly increased bacterial binding to sHA and gsHA compared to bacterial cells exposed to saliva alone. In contrast, sHA or gsHA exposed to rCSP-1 prior to incubation with S. mutans cells (which had not been pre-treated with rCSP-1) displayed negligible effects on bacterial adherence on these surfaces. These results demonstrate that the presence of rCSP-1 in saliva may contribute to the initial binding of S. mutans to both salivary pellicle and glucans formed in the pellicle by interacting directly with bacterial cell surface rather than affecting binding sites on the apatitic surface (i.e. receptor). It is noteworthy that our CHWS preparation is free of significant levels of native CSP-1 and also glucosyltransferases-Gtfs which could interfere with the interpretation of the data. The concentration of native CSP-1 in our CHWS preparation (which is diluted in adsorption buffer and clarified by centrifugation) is negligible in causing effects on bacterial binding in vitro; which may indicate CSP-1 association with high-molecular weight salivary proteins (during clarification process); S. mutans cells exposed to CHWS (no added rCSP-1) showed no difference on bacterial binding compared to those exposed to PBS only (no added rCSP1).
The adhesion of this bacterium to dental surfaces involves multiple potential mechanisms, including those mediated by hydrophobic or electrostatic forces (low affinity and non-specific) and highly-specific adhesion-receptor interactions between bacterial cell and acquired enamel pellicle formed on tooth enamel surfaces 42
. S. mutans
expresses multiple highly specific surface adhesins that are able to mediate attachment of the bacteria to host-derived and bacterium-derived binding sites on tooth surfaces. For example, the bacterial surface-protein P1 recognizes and attaches to saccharide receptors in the salivary glycoproteins constituents of the pellicle (e.g. agglutinins) 42
, whereas glucan-binding proteins on S. mutans
membrane bind glucans synthesized by surface adsorbed Gtfs, such as those from GtfB and GtfC activity 44
. Our data suggest that the enhanced binding of S. mutans
to sHA and gsHA are mediated by distinct but complementary mechanisms where a host-derived protein bound to microbial membrane could (1) act as an additional adhesin-like component recognizing and attaching to salivary proteins and glucans present within salivary pellicle and/or (2) modify the properties of the microbial surface influencing the non-specific adherence forces between bacterial and pellicle surfaces 50
It is noteworthy, however, that the enhancement of bacterial adherence was most robust when S. mutans
cells were exposed to rCSP-1 in saliva indicating that the presence of other salivary proteins (as occurs in the mouth) may be mediating the attachment of rCSP-1 to the cell-surface and/or between the bacterium and pellicle/glucan surfaces, possibly by forming protein complexes, thereby influencing adhesion. For example, it is well-known that mucins are highly glycosylated and form heterotypic complexes with specific salivary proteins, i.e. immunoglobulin A, lactoferrin agglutinin, cystatin, PRPs, histatins 51–54
. These complexes may influence the biological property of individual molecules 55
or may serve as a bridge between S. mutans
and other salivary proteins, including those in the pellicle 56
. Similarly α-enolase interacts with mucin either for the microbial attachment to oral tissues or successful removal from the oral cavity 51
. Further studies are needed to elucidate whether CSP-1 form complexes with specific proteins in saliva in the fluid phase or adsorbed state, and how these interactions enhance its ability to promote adherence of S. mutans
cells to saliva and glucan-coated apatitic surfaces. In addition, it is possible that glycosylation in insect cell line may be different from native form of the protein found in saliva which may affect formation of protein complexes.
Although CSP-1 shares a lectin-like domain, which suggests a possible effect on cell agglutination and microbial growth 34, 57
, rCSP-1 at concentrations tested in this study did not show any detectable effects on these parameters. Thus, it appears that CSP-1 may not participate directly in host defense in the oral cavity but rather on modulation of bacterial binding to apatitic surfaces. Consequently, the biological importance of such differences highlights that saliva-induced aggregation and saliva-mediated adhesion of bacterial cells may be independent processes, and presumably mediated by different salivary proteins 58
Collectively, CSP-1 and gp340 were identified as major proteins in parotid: SM/SL saliva that strongly binds to S. mutans cells, each with distinctive biological functions that could influence the survival and colonization of this ubiquitous pathogen in the oral cavity. Furthermore, we demonstrated that host-derived CSP-1 may play a role in modulating S. mutans adherence to tooth surface, which is critical for initial microbial colonization and further development into pathogenic biofilms. Additional studies are warranted to determine how CSP-1 bridges the specific interaction(s) between the bacterial and pellicle/glucan surfaces by (i) identifying the specific proteins and/or sugars on the surface of S. mutans that recognize and bind CSP-1, (ii) identifying the pellicle and glucan component/structure that bind cell surface-adsorbed CSP-1, and (iii) examining whether CSP-1 form complexes with other salivary proteins. The overall results of this study should encourage future research to consider the importance and to elucidate the exact mechanisms involved in the complex interplay between specific proteins in saliva and microbial surfaces which will advance our current understanding of the pathogenesis of dental caries and other oral infectious diseases.