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Human saliva contains a high-molecular-weight glycoprotein (agglutinin) which binds to specific streptococci in a calcium-dependent reaction leading to the formation of bacterial aggregates. We report the cloning of a gene encoding a surface antigen from Streptococcus sanguis M5 and show that the expressed protein inhibits agglutinin-mediated aggregation and specifically binds the salivary agglutinin in a calcium-dependent fashion. Clones isolated from the immunological screening of S. sanguis M5 genomic libraries with polyclonal antibodies against whole cells were assayed for the ability to compete with S. sanguis for agglutinin. One clone, pSSP-5, expressed antigens of 165 and 130 kilodaltons (kDa) possessing this activity. A 3-kilobase-pair (kbp) insert fragment from this clone was used to screen a genomic library in lambda EMBL3 which resulted in the isolation of clone SSP-5A. This clone contained an insert of 17 kb and expressed proteins of 170 to 205 kDa that reacted with the anti-S. sanguis antibodies. Subcloning of a 5.3-kbp EcoRI-BamHI fragment from SSP-5A produced pEB-5, which expressed streptococcal components that were indistinguishable from SSP-5A. The streptococcal antigen was purified by gel permeation and ion exchange chromatography and shown to potently compete with S. sanguis M5 cells for agglutinin. The antigen also bound purified salivary agglutinin in the presence of 1 mM CaCl2. This binding was inhibited by EDTA. Both the SSP-5 antigen and a 205-kDa protein in surface protein extracts from S. sanguis M5 cross-reacted with antibodies directed against antigen B from S. mutans and SpaA from S. sobrinus 6715. These results indicate that a 205-kDa surface protein that is antigenically related to SpaA and antigen B is involved in the binding of salivary agglutinin to S. sanguis M5.

Using a quantitative assay to measure saliva-mediated bacterial aggregating activity, we have surveyed 20 streptococcal strains with saliva samples obtained from a large population study. Individual saliva samples demonstrated characteristic levels of aggregating activity for Streptococcus sanguis M5. In general, high activity for this strain was associated with high activity for other strains of S. sanguis. Streptococcus mitis, and Streptococcus salivarius. The population distribution of aggregating activity for Streptococcus mutans, however, was different.

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.

A study of saliva-mediated aggregation and adhesion has been carried out in a group of caries-resistant (CR) and caries-susceptible (CS) individuals. The submandibular saliva of the CS group had a much greater potency, as determined by dilution, in promoting adherence to hydroxyapatite beads than did the saliva of CR group. In contrast, the CR group demonstrated a twofold enhancement of saliva-mediated aggregation compared with the CS group. These observations support the hypothesis that saliva-mediated aggregation and adherence are important factors in caries resistance.

Two new assays for saliva-mediated aggregation of oral bacteria have been developed, based on the use of [3H]thymidine-labeled cells. One assay separates free cells from aggregated cells by centrifugation through sucrose, whereas the other utilizes membrane filters (8 micrometers, Nuclepore) to effect the separation. Comparison of these assays with the turbidity method reveals that they are faster (X20 to 40) and require 10 times less saliva and bacteria. The aggregation of Streptococcus sanguis M5, as determined with these assays, is complete in 5 min and is dose dependent on added cells and saliva. The reaction exhibits a temperature optimum of 42 degrees C with no reaction at 0 degrees C. If the pH is reduced to below 5, saliva-dependent aggregation is inhibited. The salivary factor(s) are heat labile, losing 100% of their activity after 100 degrees C, 10 min or 70 degrees C, 30 min.

Aggregation of bacteria by zinc and lysozyme was studied and compared with aggregation induced by a high-molecular-weight salivary agglutinin. Each ligand was found to exhibit a unique profile of properties when examined by both a microradiochemical centrifugation assay and a turbidimetric assay. Significant differences in rate of aggregation and bacterial species specificity were noted. Zinc- and lysozyme-mediated aggregations were shown to be calcium independent and to proceed rapidly at 0 degree C, in contrast to the salivary agglutinin. Zinc produced large, asymmetric aggregates, saliva produced intermediate-sized aggregates, and lysozyme produced the smallest aggregates. These size differences are consistent with many of the observed reaction properties.

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.

A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polymorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease.

Human and animal sera contain potent inhibitors of saliva-mediated aggregation of oral streptococci. The inhibitors consist of a high-molecular-weight heat-labile factor and a lower-molecular-weight heat-activated factor. The latter appears to be serum albumin. Analyses of purified blood-derived proteins indicated that several high-molecular-weight proteins (fibrinogen, fibronectin, and ferritin) were able to inhibit aggregation at low concentrations. These data suggest that high-molecular-weight proteins may modulate the aggregation process.