Salivary proteins and glycoproteins are the primary source of carbon and nitrogen for growth of oral bacteria in dental plaque biofilm communities. Little is known, however, about how different bacteria degrade salivary macromolecules for use as growth substrates and if cross-feeding between species influences the growth and composition of biofilm communities. We previously found that commensal Streptococcus gordonii DL1 grows in saliva and that cariogenic S. mutans UA159 does not. These studies have now been extended to additional strains of these species and to other members of the plaque biofilm community. Overnight growth of S. gordonii, S. oralis and S. mitis strains, as measured by qPCR, was transferable from primary to secondary salivary cultures and was associated with salivary glycoprotein degradation as shown by lectin-blotting of saliva from growth cultures. In contrast, growth of S. mutans, S. sobrinus, S. sanguinis, S. salivarius, S. vestibularis, S. parasanguinis, Granulicatella elegans, Gr. adiacens, Abiotrophia defective and Rothia dentocariosa was not transferable and did not result in detectable degradation of salivary glycoproteins. We are currently establishing biofilm co-cultures of different species to determine whether bacteria that can grow alone in saliva promote growth of other oral species, including cariogenic S. mutans. Growth of S. gordonii DL1 in saliva may depend on three putative cell-wall anchored glycoside hydrolases (GHs). To test this hypothesis, S. gordonii deletion mutants lacking genes for one, two or all three enzymes were compared with wild type DL1 for growth in saliva. Growth of mutants lacking genes for one or two cell surface GHs was reduced 3- to 10-fold while growth of the mutant that lacked all three GHs was reduced approximately 20-fold. Lectin blotting of saliva from cultures of wild type DL1 revealed partial deglycosylation of a major salivary glycoprotein identified as basic proline-rich protein 3 (PRB3). This glycoprotein was detected in control saliva by lectins specific for galactose (Gal), mannose (Man), L-fucose (Fuc) or N-acetyl-glucosamine (GlcNac) in N-linked oligosaccharides. In contrast, PRB3 in saliva cultures of wild type S. gordonii DL1 was reactive with GlcNAc- and Fuc-binding but not with Gal- and Man-binding lectin probes. Lectin blotting of saliva from mono- or co-cultures of different mutant strains established that the removal of Gal from PRB3 was associated with expression of a specific cell surface GH and that the subsequent removal of Man was dependent on the sequential action of all three cell-surface GH activities of wild type S. gordonii DL1. GH-specific antibodies have also been prepared and are being used in conjunction with available anti-RPS antibodies to assess possible GH-dependent cross-feeding between different RPS-producing bacteria in plaque biofilm communities. Further insights into dental plaque biofilm development were gained from ongoing studies to characterize and compare biofilm communities that form during intra-oral incubation of retrievable enamel chips in different subjects. Spatial arrangements of different bacteria in biofilm communities are being assessed by labeling with antibodies against streptococcal RPS and Actinomyces spp. fimbriae. Bacteria that were not labeled with available antibodies are being identified by Human Oral Microbiome Database (HOMD) microarray of total biofilm DNA. Targeted isolation of all major taxa detected by microarray is also underway to obtain representative culture collections from different subjects for comprehensive studies of interbacterial adhesion between different isolates from the same biofilm. The extent and diversity of coaggregations noted between members of the same biofilm community is striking. For example, isolates of Rothia spp. and Haemophilus parainfluenzae, species not previously noted for their interactions with other bacteria, have been found to participate in extensive coaggregations with a broad array of other biofilm isolates. The underlying mechanism of these coaggregations appears to be protein-carbohydrate binding between complementary bacterial cell surfaces. Current findings clearly suggest that Rothia spp. and Haemophilus spp. have important, but as yet unappreciated, roles in the ecology of initial dental biofilms. In addition to characterizing the coaggregation properties of these bacteria in vitro, they (and their coaggregation partners) are being localized in chip biofilms by labeling with specific antibodies. We anticipate that findings from these studies will provide important new insight into the role of interbacterial adhesion in early biofilm development. Structural studies of S. oralis RPS and closely related S. pneumoniae capsular polysaccharide (CPS) serotypes were extended to S. pneumoniae CPS34, CPS39 and CPS47F. Common as well as unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the alpha 1-2 transfer of Galp to ribitol-5-phosphate in synthesis of CPS47F and CPS34 but alpha 1-1 transfer of Gal to ribitol-5-phosphate in CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled wefM of S. oralis, which was previously associated with alpha 1-1 transfer of Galp to ribitol-5-phosphate in synthesis of RPS4Gn. These findings identify linkages from Galp to ribitol-5-phosphate and from this residue to adjacent Galf in these polysaccharides as important sites of CPS genetic, structural and antigenic diversity. We anticipate these findings will contribute to development of rapid molecular approaches for serotyping not only pneumococci, but also RPS-bearing oral streptococci.
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