EPITOPE MAPPING OF BLACK-PIGMENTED BACTEROIDES FIMBRIAE

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The long-term objective of this study is to explore which fimbrial epitopes of black-pigmented Bacteroides mediate colonization to eukaryotic cell surfaces. Specifically, the fimbrial proteins from oral black-pigmented Bacteroides, including B. gingivalis, B. intermedius, and B. intermedius, and B. endodontalis, will be purified, and a library of monoclonal antibodies against their natural epitopes will be prepared. These monoclonal reagents will be tested for their ability to inhibit fimbrial attachment to epithelial cells. This will permit us to identify fimbrial epitopes of biological relevance. These epitopes will then be characterized with regard to their primary peptide structure and conformation. Next, the peptides of important regions of the fimbrial molecules will be synthesized and their immunogenicity with various carriers will be determined in an attempt to elicit antibodies which would inhibit fimbrial mediated epithelial attachment. The synthetic peptides will then be coupled to iodinatable heterobifunctional crosslinking agents to identify receptors on epithelial cells which mediate fimbrial attachment. Biochemical and immunochemical methods will be used with an emphasis on production and analysis of synthetic peptides. The functional assays include evaluation of the binding of black-pigmented Bacteroides to epithelial cells. The significance of these findings resides in the potential importance of fimbriae in the colonization of black-pigmented Bacteroides. Fimbriae are likely responsible for colonization by interacting with epithelial cells, other bacteria in coaggregates, and may be important in interacting with the host protective cells, such as neutrophils. Once these virulence-mediating functions have been elucidated, and the domains of the fimbriae responsible for these interactions identified, design of vaccines using synthetic peptides of critical domains could be considered. Thus, the studies outlined in this subproject could then provide the groundwork for a rational approach for immunization to modulate colonization of black-pigmented Bacteroides.