1 SDS-polyacrylamide gel electrophoresis of purified fimbriae and autoradiograph of iodinated fimbriae

1 SDS-polyacrylamide gel electrophoresis of purified fimbriae and autoradiograph of iodinated fimbriae. with fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain name is uncovered on the surface of fimbriae. Amyloid b-Peptide (1-40) (human) Our results suggest that the amino-terminal domain name corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain name of fimbriae. plays an important role in the initiation and progression of periodontal disease. has been shown to attach to and invade oral epithelial cells in vitro (22, 26). Intracellular invasion of human epithelial cells is usually a key pathogenic property for a number of bacterial species (4C6). For these events, bacteria bind to epithelial cell membranes and induce a series of biochemical changes (27) that involve the induction of protein kinase activity (24, 25). A variety of cell surface structures have been postulated to play roles in conversation with host cells (2), and the initial binding of to target cells appears to be fimbriae mediated. Recent reports from several laboratories have shown that fimbriae bind to saliva-coated hydroxyapatite (13), human erythrocytes (17, 18), monocytes and macrophages (19), epithelial cells (7, 11, 16, 30), and gingival fibroblasts (8). The afimbriated mutants of have been shown to Amyloid b-Peptide (1-40) (human) possess diminished capacity for adherence to oral epithelial cells in vitro (7, 16, 30). In addition, antifimbrial monoclonal antibodies have been shown to block the adhesion of to human buccal epithelial cells in an in vitro assay (11). The above studies strongly implicate the major fimbrial protein FimA in adherence to these mammalian cells. Recent studies from Ogawa et al. (21) have shown that synthetic peptides of fimbrillin corresponding to its binding domains (amino acid residues 1 to 20, 69 to 80, and 171 to 181) inhibited the binding of fimbriae to gingival fibroblasts. Fimbriae have also been reported to induce the expression of inflammatory cytokines in human gingival fibroblasts and mouse peritoneal macrophages, strongly suggesting that fimbriae are crucial in bacterial interactions with the host gingival tissue (9). fimbriae can induce protein kinase-mediated phosphorylation of a signaling protein in mouse peritoneal macrophages and in human monocytes (15, 20). Since FimA appears to be key in the binding to and possible invasion of epithelial cells, we attempted to elucidate the important region(s) of fimbrillin that are involved in the attachment process. To this end, we have mapped the surface regions of the fimbrillin molecule by utilizing antipeptide antibodies against synthetic peptides corresponding to the FimA sequence (3). The selection of peptides was based on surface predictions incorporating three different parameters: hydrophilicity, convenience, and mobility (23). Antipeptide antibodies that reacted with native fimbriae, thereby suggesting that this corresponding peptides are surface uncovered, were used as inhibitors of binding to epithelial cells. The peptides synthesized are outlined in Table ?Table1.1. Peptide synthesis and preparation of peptide conjugates were carried out by a method described earlier (14). The composition and sequence of peptides were confirmed by amino acid analysis on a Beckman Amyloid b-Peptide (1-40) (human) model 121MB sequencer, and the amino acid sequence was confirmed by an automated stepwise procedure on an Applied Biosystems model 477A sequencer. TABLE 1 Sequence of synthetic peptides corresponding to the segments of 42C6149C6869C9081C9899C1102561 was produced in one-half-strength (18 mg/ml) brain heart infusion broth, supplemented with 5 mg of yeast extract per ml and buffered at a pH of 7.4. Cells were then incubated for 2 days in an anaerobic chamber (85% N2, 10% H2, 5% CO2). Fimbrial preparation and iodination. fimbriae were purified by the procedure of Sojar et al. (29), and purity was confirmed by a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (12) (Fig. ?(Fig.1A).1A). Purified fimbriae were iodinated using the chloramine T method (10) with the following modification. Ten micrograms of purified fimbriae was labeled with 0.5 Amyloid b-Peptide (1-40) (human) mCi of sodium iodine-125 (Amersham Pharmacia Biotech Inc., N.J.) in 0.5 M PBS, pH 7.2, in the presence of 10 l of chloramine T SIRT1 (1 mg/ml) for 60 s. Adding 20 l of sodium metabisulfite (2 mg/ml) terminated iodination. After termination,.