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1.  Staphylococcus aureus SasA Is Responsible for Binding to the Salivary Agglutinin gp340, Derived from Human Saliva 
Infection and Immunity  2013;81(6):1870-1879.
Staphylococcus aureus is a major human pathogen that can colonize the nasal cavity, skin, intestine, and oral cavity as a commensal bacterium. gp340, also known as DMBT1 (deleted in malignant brain tumors 1), is associated with epithelial differentiation and innate immunity. In the oral cavity, gp340 induces salivary aggregation with several oral bacteria and promotes bacterial adhesion to tissues such as the teeth and mucosa. S. aureus is often isolated from the oral cavity, but the mechanism underlying its persistence in the oral cavity remains unclear. In this study, we investigated the interaction between S. aureus and gp340 and found that S. aureus interacts with saliva- and gp340-coated resin. We then identified the S. aureus factor(s) responsible for binding to gp340. The cell surface protein SasA, which is rich in basic amino acids (BR domain) at the N terminus, was responsible for binding to gp340. Inactivation of the sasA gene resulted in a significant decrease in S. aureus binding to gp340-coated resin. Also, recombinant SasA protein (rSasA) showed binding affinity to gp340, which was inhibited by the addition of N-acetylneuraminic acid. Surface plasmon resonance analysis showed that rSasA significantly bound to the NeuAcα(2-3)Galβ(1-4)GlcNAc structure. These results indicate that SasA is responsible for binding to gp340 via the N-acetylneuraminic acid moiety.
doi:10.1128/IAI.00011-13
PMCID: PMC3676022  PMID: 23439307
2.  Human Common Salivary Protein 1 (CSP-1) Promotes Binding of Streptococcus mutans to Experimental Salivary Pellicle and Glucans Formed on Hydroxyapatite Surface 
Journal of proteome research  2010;9(12):6605-6614.
Summary
The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins which interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/ml). However, S. mutans cells exposed to rCSP-1 (10 μg/ml) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto tooth surface.
doi:10.1021/pr100786y
PMCID: PMC2997150  PMID: 20858015
Common Salivary Protein-1; Human Saliva; Saliva-Microbial Interaction; Affinity and Lectin Chromatography; Mass Spectrometry
3.  Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus 
Infection and Immunity  2013;81(4):1364-1373.
Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1gp-340, mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.
doi:10.1128/IAI.00825-12
PMCID: PMC3639616  PMID: 23403559
4.  Multiple components contribute to ability of saliva to inhibit influenza viruses 
Introduction
Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied.
Methods
We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro.
Results
Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study.
Conclusions
These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.
doi:10.1111/j.1399-302X.2008.00468.x
PMCID: PMC2848456  PMID: 19121065
histatins; innate immunity; MUC5B
5.  Fluid- or Surface-Phase Human Salivary Scavenger Protein gp340 Exposes Different Bacterial Recognition Properties  
Infection and Immunity  2005;73(4):2245-2252.
Salivary scavenger receptor cysteine-rich protein gp340 aggregates streptococci and other bacteria as part of the host innate defense system at mucosal surfaces. In this article, we have investigated the properties of fluid-phase gp340 and hydroxylapatite surface-adsorbed gp340 in aggregation and adherence, respectively, of viridans group streptococci (e.g., Streptococcus gordonii and Streptococcus mutans), non-viridans group streptococci (e.g., Streptococcus pyogenes and Streptococcus suis), and oral Actinomyces. Fluid-phase gp340 and surface-phase gp340 bioforms were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction with gp340. Group I streptococci were aggregated by and adhered to gp340, and group II streptococci preferentially adhered to surface-bound gp340, while group III streptococci were preferentially aggregated by gp340. Each species of Streptococcus tested was found to contain strains representative of at least two of these gp340 interaction groupings. The gp340 interaction modes I to III and sugar specificities of gp340 binding strains coincided for several species. Many gp340 interactions were sialidase sensitive, and each of the interaction modes (I to III) for S. gordonii was correlated with a variant of sialic acid specificity. Adherence of S. gordonii DL1 (Challis) to surface-bound gp340 was dependent upon expression of the sialic acid binding adhesin Hsa. However, aggregation of cells by fluid-phase gp340 was independent of Hsa and involved SspA and SspB (antigen I/II family) polypeptides. Conversely, both gp340-mediated aggregation and adherence of S. mutans NG8 involved antigen I/II polypeptide. Deletion of the mga virulence regulator gene in S. pyogenes resulted in increased cell aggregation by gp340. These results suggest that salivary gp340 recognizes different bacterial receptors according to whether gp340 is present in the fluid phase or surface bound. This phase-associated differential recognition by gp340 of streptococcal species of different levels of virulence and diverse origins may mediate alternative host responses to commensal or pathogenic bacterial phenotypes.
doi:10.1128/IAI.73.4.2245-2252.2005
PMCID: PMC1087402  PMID: 15784568
6.  Salivary-agglutinin-mediated adherence of Streptococcus mutans to early plaque bacteria. 
Infection and Immunity  1991;59(10):3446-3450.
Interspecies binding is important in the colonization of the oral cavity by bacteria. Streptococcus mutans can adhere to other plaque bacteria, such as Streptococcus sanguis and Actinomyces viscosus, and this adherence is enhanced by saliva. The salivary and bacterial molecules that mediate this interaction were investigated. Salivary agglutinin, a mucinlike glycoprotein known to mediate the aggregation of many oral streptococci in vitro, was found to mediate the adherence of S. mutans to S. sanguis or A. viscosus. Adherence of S. mutans to saliva- or agglutinin-coated S. sanguis and A. viscosus was inhibited by antibodies to the bacterial agglutinin receptor. Expression of the S. sanguis receptor (SSP-5) gene in Enterococcus faecalis increased adhesion of this organism to saliva- or agglutinin-coated S. sanguis and A. viscosus. This interaction could be inhibited by antibodies to the agglutinin receptor. The results suggest that salivary agglutinin can promote adherence of S. mutans to S. sanguis and A. viscosus through interactions with the agglutinin receptor on S. mutans.
PMCID: PMC258905  PMID: 1894355
7.  Mapping of B-cell epitopes on the polypeptide chain of the Epstein-Barr virus major envelope glycoprotein and candidate vaccine molecule gp340. 
Journal of Virology  1992;66(2):1246-1251.
The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.
Images
PMCID: PMC240837  PMID: 1370550
8.  Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries 
Background
Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of Streptococcus mutans (implicated in caries), harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of S. mutans.
Methods
A case-referent study was performed in 12-year-old Swedish children with high (n = 19) or low (n = 19) caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of S. mutans using the partial least squares (PLS) multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms.
Results
All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively). The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37) and saliva adhesion of S. mutans Ingbritt (VIP = 1.47). The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of S. mutans strains Ingbritt and NG8 and Lactococcus lactis expressing AgI/II adhesins (SpaP or PAc) compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins.
Conclusion
Gp-340 I behaves as a caries susceptibility protein.
doi:10.1186/1471-2334-7-57
PMCID: PMC1894970  PMID: 17562017
9.  Functions of Cell Surface-Anchored Antigen I/II Family and Hsa Polypeptides in Interactions of Streptococcus gordonii with Host Receptors 
Infection and Immunity  2005;73(10):6629-6638.
Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surface receptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements into biologically productive phenomena.
doi:10.1128/IAI.73.10.6629-6638.2005
PMCID: PMC1230909  PMID: 16177339
10.  Structural and Functional Variation within the Alanine-Rich Repetitive Domain of Streptococcal Antigen I/II  
Infection and Immunity  2002;70(11):6389-6398.
Members of the antigen I/II family of cell surface proteins are highly conserved, multifunctional adhesins that mediate interactions of oral streptococci with other oral bacteria, with cell matrix proteins (e.g., type I collagen), and with salivary glycoproteins, e.g., gp340. The interaction of gp340 (formerly designated salivary agglutinin) with Streptococcus mutans requires an alanine-rich repetitive domain (A region) of antigen I/II that is highly conserved in all members of this family of proteins. In this report, we show that the A regions from the two Streptococcus gordonii M5 antigen I/II proteins (SspA and SspB) interact differently with the salivary gp340 glycoprotein and appear to be structurally distinct. Recombinant polypeptides encompassing the A region of SspA or from a highly related S. mutans antigen I/II protein (SpaP) competitively inhibited the interaction of gp340 with intact S. gordonii and S. mutans cells, respectively. In contrast, an A region polypeptide from SspB was inactive, and furthermore, it did not bind to purified gp340 in vitro. Circular dichroism spectra suggested that all three polypeptides were highly α-helical and may form coiled-coil structures. However, the A region of SspB underwent a conformational change and exhibited reduced α-helical structure at pH 8.5, whereas the A region polypeptides from SspA and SpaP were relatively stable under these conditions. Melt curves also indicated that at physiological pH, the A region of SspB lost α-helical structure more rapidly than that of SspA or SpaP when the temperature was increased from 10 to 40°C. Furthermore, the SspB A region polypeptide denatured completely at a temperature that was 7 to 9°C lower than that required for the A region polypeptide of SspA or SpaP. The full-length SspB protein and the three A region peptides migrated in native gel electrophoresis and column chromatography with apparent molecular masses that were approximately 2- to 2.5-fold greater than their predicted molecular masses. However, sedimentation equilibrium ultracentrifugation data showed that the A region peptides sedimented as monomers, suggesting that the peptides may form nonglobular intramolecular coiled-coil structures under the experimental conditions used. Taken together, our results suggest that the A region of SspB is less stable than the corresponding A regions of SspA and SpaP and that this structural difference may explain, at least in part, the functional variation observed in their interactions with salivary gp340.
doi:10.1128/IAI.70.11.6389-6398.2002
PMCID: PMC130335  PMID: 12379719
11.  The salivary scavenger and agglutinin binds MBL and regulates the lectin pathway of complement in solution and on surfaces 
The salivary scavenger and agglutinin (SALSA), also known as gp340, salivary agglutinin and deleted in malignant brain tumor 1, is a 340-kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A, and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan-binding lectin (MBL) as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit Candida albicans-induced complement activation. Thus, SALSA has a dual complement activation modifying function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid phase. These activities are mediated via a direct interaction with MBL. This suggests that SALSA could target the innate immune responses to certain microorganisms and simultaneously limit complement activation in the fluid phase.
doi:10.3389/fimmu.2012.00205
PMCID: PMC3397308  PMID: 22811680
complement regulation; gp340; lectin pathway; mannan-binding lectin; mucosal immunity; salivary agglutinin; scavenger receptor cysteine-rich
12.  Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system. 
Infection and Immunity  1981;34(1):208-214.
Resting human whole saliva inhibited acid production by glucose-stimulated, homologous plaque. The degree of inhibition of plaque acid production correlated positively with the concentration of hypothiocyanite (OSCN-) ions in saliva. Supplementation of saliva with an appropriate combination of thiocyanate and hydrogen peroxide resulted in a significant increase in the concentration of OSCN- ions and in more effective inhibition of plaque acid production. In most cases, the inhibition was complete when the supplements were added directly to saliva-plaque mixtures. Acid production resumed when the inhibitory effect of OSCN- was reversed by addition of thiols. Among the oral defense factors, the salivary lactoperoxidase system seems to play an important role by producing highly reactive antibacterial products (including OSCN-) which can regulate bacterial metabolism in the human mouth. The concentration of OSCN- in normal human whole saliva seems to be just below the threshold level required for plaque inhibition. Therefore, enhancement of this system in vivo may be effective in the regulation of plaque acid production.
PMCID: PMC350844  PMID: 7298182
13.  Interactions of Streptococcus mutans Fimbria-Associated Surface Proteins with Salivary Components 
Streptococcus mutans has been implicated as the major causative agent of human dental caries. S. mutans binds to saliva-coated tooth surfaces, and previous studies suggested that fimbriae may play a role in the initial bacterial adherence to salivary components. The objectives of this study were to establish the ability of an S. mutans fimbria preparation to bind to saliva-coated surfaces and determine the specific salivary components that facilitate binding with fimbriae. Enzyme-linked immunosorbent assay (ELISA) established that the S. mutans fimbria preparation bound to components of whole saliva. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot techniques were used to separate components of whole saliva and determine fimbria binding. SDS-PAGE separated 15 major protein bands from saliva samples, and Western blot analysis indicated significant binding of the S. mutans fimbria preparation to a 52-kDa salivary protein. The major fimbria-binding salivary protein was isolated by preparative electrophoresis. The ability of the S. mutans fimbria preparation to bind to the purified salivary protein was confirmed by Western blot analysis and ELISA. Incubation of the purified salivary protein with the S. mutans fimbria preparation significantly neutralized binding of the salivary protein-fimbria complex to saliva-coated surfaces. The salivary protein, whole saliva, and commercial amylase reacted similarly with antiamylase antibody in immunoblots. A purified 65-kDa fimbrial protein was demonstrated to bind to both saliva and amylase. These data indicated that the S. mutans fimbria preparation and a purified fimbrial protein bound to whole-saliva-coated surfaces and that amylase is the major salivary component involved in the binding.
PMCID: PMC103730  PMID: 10225843
14.  HIV Envelope Binding by Macrophage-Expressed Gp340 Promotes HIV-1 Infection.1 
The scavenger receptor cysteine-rich protein gp340 functions as part of the host innate immune defense system at mucosal surfaces. In the genital tract, its expression by cervical and vaginal epithelial cells promotes HIV trans-infection and may play a role in sexual transmission. Gp340 is an alternatively spliced product of the deleted in malignant brain tumors 1 (DMBT1) gene. In addition to its innate immune system activity, DMBT1 demonstrates instability in multiple types of cancer and plays a role in epithelial cell differentiation. We demonstrate that monocyte derived macrophages express gp340 and HIV-1 infection is decreased when envelope cannot bind it. Inhibition of infection occurred at the level of fusion of M-, T-, and dual-tropic envelopes. Additional HIV-1 envelope binding molecules, such as dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN), mannose binding lectin, and heparan sulfate, enhance the efficiency of infection of the cells that express them by increasing the local concentration of infectious virus. Our data suggest that gp340, which is expressed by macrophages in vivo, may function to enhance infection in much the same manner. Its expression on tissue macrophages and epithelial cells suggests important new opportunities for HIV-1 pathogenesis investigation and therapy.
PMCID: PMC2561920  PMID: 18641344
Monocytes/Macrophages; AIDS; Cell Surface Receptors; Human
15.  Deleted in Malignant Brain Tumors-1 Protein (DMBT1): A Pattern Recognition Receptor with Multiple Binding Sites 
Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.
doi:10.3390/ijms1112521
PMCID: PMC3100851  PMID: 21614203
dental caries; innate immunity; mucosal protection; SRCR domains
16.  Characteristic differences between saliva-dependent aggregation and adhesion of streptococci. 
Infection and Immunity  1982;35(1):86-90.
Comparison of saliva-mediated aggregation of Streptococcus sanguis, Streptococcus mitis, and Streptococcus mutans and adhesion of these organisms to saliva-coated hydroxyapatite showed that there was no relationship between these two activities. Adsorption of salivary aggregating activity to bacteria appears to have little effect on the ability of the residual saliva to support adherence; conversely, adsorption of salivary adherence factors to hydroxyapatite does not affect aggregation. Although heating saliva significantly reduces bacterial aggregation, it has little or no effect on adherence. A comparison of aggregation and adhesion with serial dilutions of saliva demonstrated that adhesion could still be detected at 100 to 500-fold-lower concentrations of salivary protein that bacterial aggregation. These findings support the concept that aggregation and adherence involve two distinct mechanisms of microbial clearance in the oral cavity.
PMCID: PMC350999  PMID: 6274804
17.  Salivary pellicles on titanium and their effect on metabolic activity in Streptococcus oralis 
BMC Oral Health  2013;13:32.
Background
Titanium implants in the oral cavity are covered with a saliva-derived pellicle to which early colonizing microorganisms such as Streptococcus oralis can bind. The protein profiles of salivary pellicles on titanium have not been well characterized and the proteins of importance for binding are thus unknown. Biofilm bacteria exhibit different phenotypes from their planktonic counterparts and contact with salivary proteins may be one factor contributing to the induction of changes in physiology. We have characterized salivary pellicles from titanium surfaces and investigated how contact with uncoated and saliva-coated titanium surfaces affects metabolic activity in adherent cells of S. oralis.
Methods
Salivary pellicles on smooth titanium surfaces were desorbed and these, as well as purified human saliva, were subjected to two-dimensional gel electrophoresis and mass spectroscopy. A parallel plate flow-cell model was used to study binding of a fresh isolate of S. oralis to uncoated and saliva-coated titanium surfaces. Metabolic activity was assessed using the BacLight CTC Vitality Kit and confocal scanning laser microscopy. Experiments were carried out in triplicate and the results analyzed using Student’s t-test or ANOVA.
Results
Secretory IgA, α-amylase and cystatins were identified as dominant proteins in the salivary pellicles. Selective adsorption of proteins was demonstrated by the enrichment of prolactin-inducible protein and absence of zinc-α2-glycoprotein relative to saliva. Adherence of S. oralis to titanium led to an up-regulation of metabolic activity in the population after 2 hours. In the presence of a salivary pellicle, this effect was enhanced and sustained over the following 22 hour period.
Conclusions
We have shown that adherence to smooth titanium surfaces under flow causes an up-regulation of metabolic activity in the early oral colonizer S. oralis, most likely as part of an adaptation to the biofilm mode of life. The effect was enhanced by a salivary pellicle containing sIgA, α-amylase, cystatins and prolactin-inducible protein which was, for the first time, identified as an abundant component of salivary pellicles on titanium. Further studies are needed to clarify the mechanisms underlying the effect of surface contact on metabolic activity as well as to identify the salivary proteins responsible for enhancing the effect.
doi:10.1186/1472-6831-13-32
PMCID: PMC3726426  PMID: 23866104
Bacteria; Microbial biofilm; Dental implant; Streptococci
18.  Analysis of Candida albicans adhesion to salivary mucin. 
Infection and Immunity  1993;61(5):1940-1949.
Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.
Images
PMCID: PMC280787  PMID: 8478083
19.  Taking the Starch out of Oral Biofilm Formation: Molecular Basis and Functional Significance of Salivary α-Amylase Binding to Oral Streptococci 
α-Amylase-binding streptococci (ABS) are a heterogeneous group of commensal oral bacterial species that comprise a significant proportion of dental plaque microfloras. Salivary α-amylase, one of the most abundant proteins in human saliva, binds to the surface of these bacteria via specific surface-exposed α-amylase-binding proteins. The functional significance of α-amylase-binding proteins in oral colonization by streptococci is important for understanding how salivary components influence oral biofilm formation by these important dental plaque species. This review summarizes the results of an extensive series of studies that have sought to define the molecular basis for α-amylase binding to the surface of the bacterium as well as the biological significance of this phenomenon in dental plaque biofilm formation.
doi:10.1128/AEM.02581-12
PMCID: PMC3553756  PMID: 23144140
20.  Estimation and Comparison of Salivary Secretory Leukocyte Protease Inhibitor in Human Immunodeficiency Virus Patients and Healthy Individuals 
Aim:
Transmission of human immunodeficiency virus (HIV) in the oral cavity is a rare event, despite detectable virus in saliva and oropharyngeal tissues of infected persons, unlike other mucosal sites. Secretory leukocyte protease inhibitor (SLPI) has been suggested as the main soluble factor responsible for the HIV inhibitory effect of saliva. The study was designed to estimate and compare the salivary SLPI levels in HIV patients and healthy controls. Furthermore, the relationship between salivary SLPI levels and disease severity was also investigated.
Materials and Methods:
Unstimulated whole saliva specimens were collected from 60 HIV-infected and 20 healthy subjects. Disease severity was determined by CD4 count in HIV subjects, who were divided into two groups: ≥200 cells/μL (n = 30) and < 200 cells/μL n = 30. Salivary SLPI levels were determined by enzyme-linked immunosorbent assay.
Results:
Numerically higher SLPI levels were observed in HIV subjects 193.342 ng/mL vs. 190.587 ng/mL; P = 0.517. A nonsignificant negative correlation was noted between CD4 counts and SLPI levels r = −0.037, P = 0.781.
Conclusion:
The salivary anti-HIV factor, SLPI, is not only preserved in HIV infection but its concentration may even get enhanced in the infection. However, the clinical significance of SLPI levels and disease severity should be investigated further with a larger sample of patients.
doi:10.4103/0973-1075.125551
PMCID: PMC3931238  PMID: 24600179
CD4 count; Human immunodeficiency virus; Saliva; Secretory leukocyte protease inhibitor
21.  Impaired Histatin-5 Levels and Salivary Antimicrobial Activity against C. albicans in HIV Infected Individuals 
Journal of AIDS & clinical research  2013;4(193):1000193.
HIV-infected individuals constitute a population highly susceptible to opportunistic infections, particularly oral candidiasis caused by the most pathogenic human fungal species Candida albicans. Host-produced salivary antimicrobial peptides are considered to be an important part of the host innate immune system involved in protection of the oral cavity against colonization and infection by microbial species. Histatin-5 (Hst-5) specifically has exhibited potent anti-candidal properties in vitro. However, its importance in protecting the oral mucosa against candidal colonization and importantly, its contribution to the observed enhanced susceptibility of HIV-infected individuals to candidiasis has not been previously investigated. To that end, a novel immunoassay was used to demonstrate significant decrease in salivary Hst-5 levels in HIV+ individuals concomitant with enhanced candidal prevalence. Further, saliva’s anti-candidal potency was found to be proportional to Hst-5 concentration and significantly compromised in HIV+ subjects compared to controls. The key role for Hst-5 was further confirmed upon exposure to the Hst-5-specific antibody where saliva’s initial killing activity was substantially compromised. Combined, these findings identify Hst-5 as a key anti-candidal salivary component and demonstrate its decreased levels in HIV infection providing new insights into oral Innate immune defense mechanisms and the enhanced susceptibility of HIV+ individuals to oral candidiasis.
doi:10.4172/2155-6113.1000193
PMCID: PMC3666036  PMID: 23730535
HIV/AIDS; Opportunistic infections; Oral candidiasis; Innate immunity; Salivary antimicrobial peptide; Histatin-5; C. albicans
22.  Identification of Saliva-Regulated Genes of Streptococcus gordonii DL1 by Differential Display Using Random Arbitrarily Primed PCR 
Infection and Immunity  2000;68(8):4834-4837.
Attachment of Streptococcus gordonii to the acquired pellicle of the tooth surface involves specific interactions between bacterial adhesins and adsorbed salivary components. To study saliva-regulated gene expression in S. gordonii, we used random arbitrarily primed PCR (RAP-PCR). Bacteria were incubated in either brain heart infusion medium or saliva. Total RNA from both conditions was purified and RAP fingerprinted and then PCR amplified with an arbitrary primer. The differentially displayed DNA fragments were cloned, sequenced, and analyzed using the BLAST search network service. Three DNA products were up-regulated. One was identified as that of the sspA and -B genes, which encode the salivary agglutinin glycoprotein-binding proteins SspA and SspB of S. gordonii; another had 79% identity with the Lactococcus lactis clpE gene, encoding a member of the Clp protease family; and the third product showed no significant homology to known genes. Five down-regulated genes were identified which encode proteins involved in bacterial metabolism. We have shown, for the first time, direct induction of sspA and -B in S. gordonii by human saliva.
PMCID: PMC98450  PMID: 10899901
23.  Characterization of SIV in the Oral Cavity and in Vitro Inhibition of SIV by Rhesus Macaque Saliva 
Abstract
Human immunodeficiency virus (HIV) infections are rarely acquired via an oral route in adults. Previous studies have shown that human whole saliva inhibits HIV infection in vitro, and multiple factors present in human saliva have been shown to contribute to this antiviral activity. Despite the widespread use of simian immunodeficiency virus (SIV)-infected rhesus macaques as models for HIV pathogenesis and transmission, few studies have monitored SIV in the oral cavity of infected rhesus macaques and evaluated the viral inhibitory capacity of macaque saliva. Utilizing a cohort of rhesus macaques infected with SIVMac251, we monitored virus levels and genotypic diversity in the saliva throughout the course of the disease; findings were similar to previous observations in HIV-infected humans. An in vitro infectivity assay was utilized to measure inhibition of HIV/SIV infection by normal human and rhesus macaque whole saliva. Both human and macaque saliva were capable of inhibiting HIV and SIV infection. The inhibitory capacity of saliva samples collected from a cohort of animals postinfection with SIV increased over the course of disease, coincident with the development of SIV-specific antibodies in the saliva. These findings suggest that both innate and adaptive factors contribute to inhibition of SIV by whole macaque saliva. This work also demonstrates that SIV-infected rhesus macaques provide a relevant model to examine the innate and adaptive immune responses that inhibit HIV/SIV in the oral cavity.
doi:10.1089/aid.2009.0235
PMCID: PMC2957632  PMID: 20672998
24.  Inactivation of Streptococcus gordonii SspAB Alters Expression of Multiple Adhesin Genes  
Infection and Immunity  2005;73(6):3351-3357.
SspA and SspB (antigen I/II family proteins) can bind Streptococcus gordonii to other oral bacteria and also to salivary agglutinin glycoprotein, a constituent of the salivary film or pellicle that coats the tooth. To learn if SspA and SspB are essential for adhesion and initial biofilm formation on teeth, S. gordonii DL1 was incubated with saliva-coated hydroxyapatite (sHA) for 2 h in Todd-Hewitt broth with 20% saliva to develop initial biofilms. Sessile cells attached to sHA, surrounding planktonic cells, and free-growing cells were recovered separately. Free-growing cells expressed more sspA-specific mRNA and sspB-specific mRNA than sessile cells. Free-growing cells expressed the same levels of sspA and sspB as planktonic cells. Surprisingly, an SspA− SspB− mutant strain showed 2.2-fold greater biofilm formation on sHA than wild-type S. gordonii DL1. To explain this observation, we tested the hypothesis that inactivation of sspA and sspB genes altered the expression of other adhesin genes during initial biofilm formation in vitro. When compared to wild-type cells, expression of scaA and abpB was significantly up-regulated in the SspA− SspB− strain in sessile, planktonic, and free-growing cells. Consistent with this finding, ScaA antigen was also overexpressed in planktonic and free-growing SspA− SspB− cells compared to the wild type. SspA/B adhesins, therefore, were strongly suggested to be involved in the regulation of multiple adhesin genes.
doi:10.1128/IAI.73.6.3351-3357.2005
PMCID: PMC1111841  PMID: 15908361
25.  Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis. 
Infection and Immunity  1994;62(8):3372-3380.
Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.
Images
PMCID: PMC302968  PMID: 8039907

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