Molecular Biology of Oral Microbial Adhesins and Receptors

Dental & Craniofacial Research

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Abstract

Seven structural types of RPS have been identified, each composed of a somewhat different hexa- or heptasaccharide repeating unit. Each repeating unit contains a host-like motif, either beta-GalNAc1-3Gal (Gn) or beta-Gal1-3GalNAc (G) that functions as a recognition site for adhesin binding and a non-host-like antigenic region. The adhesins present on certain oral species recognize both Gn and G types of RPS whereas those present on other species are specific for one receptor type or the other. The gene clusters for these polysaccharides encode six or seven putative glycosyltransferases, the number required for synthesis of the corresponding lipid-linked hexa- or heptasaccharide repeating unit, a flippase (Wzx), a polysaccharide polymerase (Wzy) and other genes that do not directly affect polysaccharide structure.[unreadable] Although genes for glycosyltransferases can frequently be identified by sequence homologies with the existing database, the donor and acceptor specificity or the linkage and anomeric specificity of encoded enzymes are generally not predictable, and thus, must be determined experimentally. Our studies illustrate a novel approach to this important problem, which involves the structural characterization of recombinant polysaccharides. We replace genes for specific transferases in the rps cluster of one streptococcal strain with a cassette for antibiotic resistance and then precisely replace this cassette with genes for different but complementary transferases from another strain. The first step abolishes cell surface RPS production as detected by the reaction of RPS-specific antibody while the second step restores production of an immunoreactive polysaccharide, which is isolated for complete structural characterization by high-resolution nuclear magnetic resonance. The results of our studies indicate that a relatively small number of encoded glycosyl or glycosyl-1-phosphotransferases act in concert to determine the diverse structural and biological properties of a major RPS group. Considered together, our findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and function. They also provide a framework for molecular typing of RPS-producing streptococci.[unreadable] Studies of A. naeslundii provided early evidence for the presence of adhesive fimbriae or pili on gram positive bacteria. Although similar structures have now been identified on a wide range of other gram positive species, A. naeslundii remains the only organism for which the corresponding adhesion receptors have been structurally identified. Peptide motifs in adsorbed salivary proline-rich proteins are the receptors of A. naeslundii type 1 fimbriae while the Gn- and G-motifs displayed on RPS-bearing streptococci and similar motifs in host glycoconjugates are the receptors of type 2 fimbriae. The genes for the structural subunits of type 1 and type 2 fimbriae, respectively, are known. However, Fab fragments of antibodies against the corresponding encoded proteins (i.e. FimP and FimA, respectively) fail to block fimbriae-mediated adhesion, thereby raising the possibility that these proteins are not adhesins.[unreadable] Results of collaborative studies between the Microbial Receptors Unit and other laboratories have revealed two comparable fimbrial gene clusters in different A. naeslundii strains, fimQ-fimP-srtC1 for type 1 fimbriae and fimB-fimA-srtC2 for type 2 fimbriae production. Like the major fimbrial subunits FimP and FimA, the predicted features of FimQ and FimB include an N-terminal signal sequence and a C-terminal cell wall anchoring domain. The identification of FimQ and FimB as fimbriae-associated proteins was established from results from immunogold electron microscopy, and for FimQ, by the identification of FimQ-derived peptides in digests of purified type 1 fimbriae by liquid chromatography-tandem mass spectrometry. Importantly, double immunogold labeling experiments revealed a clear difference in the distributed of FimQ and FimP in type 1 fimbriae and FimB and FimA in type 2 fimbriae. Antibody against FimQ or FimB appeared to react primarily at the tips of fimbriae, whereas antibody against FimP or FimA reacted along the length of these structures, consistent with the earlier identification of these proteins as major subunits. The identification of FimQ and FimB at the tips of type 1 and type 2 fimbriae, respectively, make these proteins highly attractive candidates for the long sought fimbrial adhesins of A. naeslundii. The genes srtC1 and srtC2 encode different fimbriae-specific, class C sortases. Deletion of srtC1 or srtC2 abolished type 1 or type 2 fimbriae production, respectively, resulting in the synthesis of the corresponding unassembled, monomeric fimbrial subunits. These findings are consistent with the hypothesis that the role of sortase in fimbriae biogenesis involves the covalent linkage of different fimbrial subunits. Further studies are underway to elucidate the molecular and structural basis of this process.