It is widely accepted that saliva is the primary source of carbon and nitrogen for bacterial growth in dental plaque biofilm communities. Little is known however about which salivary components support this growth and whether all or only certain oral species utilize these components as growth substrates. To address these questions, 32 strains representing 16 genera commonly found in early plaque biofilms were sub-cultured in whole saliva that was first heat-treated to destroy endogenous enzymes and dialyzed to remove low molecular weight growth substrates. In comparable cultures set up with such saliva, densities of Streptococcus gordonii, S. oralis and S. mitis strains, together with the type strain of S. sanguinis, reached from one million to ten million cells/ml as measured by qPCR, while strains of S. mutans and Gemella haemolysans only grew to about half a million cells/ml. In contrast, little or no growth was seen for certain strains of S. sanguinis, as well as for strains of S. salivarius, S. vestibularis, S. sobrinus, Actinomyces spp., Abiotrophia defectiva, and Rothia dentocariosa. Examination of saliva from growth cultures by SDS PAGE and lectin blotting revealed species-dependent differences in degradation and deglycosylation of highly basic proline-rich glycoproteins for strains of S. gordonii or S. oralis, minimal changes in the salivary proteome for strains of S. mitis and no changes for species that that did not grow. Differences in growth on saliva of pioneer species have important implications for establishment of biofilm communities and for cross-feeding among their members. Growth of Streptococcus gordonii DL1 in saliva may depend on a number of glycoside hydrolases (GHs) produced by this oral commensal including three cell wall-anchored proteins that are homologues of pneumococcal β-galactosidase (BgaA), β-N-acetylglucosaminidase (StrH) and endohexosaminidase D (EndoD). To assess the role of these enzymes, we deleted the corresponding genes from S. gordonii DL1 in different combinations for comparative studies of growth and salivary glycan foraging. Overnight growth of wild type DL1 in saliva was reduced 3- to 10-fold by deletion of any one or two genes and approximately 20-fold by deletion of all three genes. The only notable change in salivary proteome associated with growth of S. gordonii DL1 involved a shift in the apparent molecular weight of basic proline-rich proteins (PRB) from approximately 70 to 55 kDa, which was accompanied by the loss of terminal galactose- and mannose-containing receptors for specific lectin probes (i.e. ECA and GNA, respectively). ECA binding to Gal receptors of PRB was also abolished in saliva cultures of mutants that expressed cell surface BgaA alone or together with either StrH or EndoD. The subsequent loss of GNA-reactive Man receptors was however only seen in saliva co-cultures of different mutants that together expressed all three cell surface GHs. The findings indicate that growth of S. gordonii DL1 in saliva depends to a significant extent on the sequential action of BgaA followed by StrH and EndoD on N-linked glycans of PRB. Formation of the early plaque biofilm community is thought to involve specific inter- and intra-species bacterial cell-cell recognition (i.e. coaggregations). Little is known however about how frequently such interactions occur between different bacteria within the biofilm community of a single patient or about the coaggregation phenotypes of several species prominent in early plaque. In the present study, HOMIM (molecular) analysis of early plaque biofilm communities from two healthy individuals verified the expected species composition with few obvious inter-individual differences. Culture collections from each individual were used to assess the extent of coaggregation within each community. Extensive specific coaggregations were noted between isolates within each collection. Importantly, these interactions were not limited to well-studied Streptococcus and Actinomyces spp. but were also were prominent among the poorly characterized genera Rothia, Haemophilus, and Neisseria. Antibodies against strains of the latter genera were used to document the presence of these bacteria within the community in situ. Furthermore, two previously uncultivated streptococcal phylotypes were isolated that showed extensive coaggregation and reacted with antibodies against well-characterized coaggregation-mediating receptor polysaccharides. The widespread occurrence of specific coaggregations between bacteria within individual biofilm communities is consistent with an essential role for these interactions in early plaque development. Structures of Streptococcus pneumoniae capsular polysaccharides (CPS) are essential for defining antigenic as well as genetic relationships between CPS serotypes. The four serotypes that comprise CPS serogroup 35 (i.e. types 35F, 35A, 35B and 35C) are known to cross-react with genetically related types 20, 29, 34, 42 or 47F. While the structures of CPS35A and 35B are known, those of CPS35F and 35C are not. Repeat unit structures of the latter two CPS serotypes have now been determined by high-resolution hetero-nuclear magnetic resonance (NMR) spectroscopy and glycosyl composition analyses. Importantly, CPS35F, the immunizing serotype for production of group 35 serum, more closely resembles CPS34 and CPS47F than other members of serogroup 35. Moreover, CPS35C is distinct from either CPS35F or CPS35B but closely related to CPS35A and identical to de-O-acetylated CPS42. The findings provide the first overall view of structural and genetic relations that exist between the members of CPS serogroup 35 and other cross reactive serotypes.
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