The long-term goal of this project is to understand the role of host macromolecules in bacterial colonization of the mucosal surfaces of the oropharynx. We will focus principally on the role salivary molecules play in the modulation of adherence of type 1 fimbriated gram negative bacteria frequent agents of nosocomial pneumonia) to mucosal epithelial cells. We will purify type 1 fimbrial-binding glycoproteins (FBGs) from saliva by routine chromatographic procedures. We will also test sample of known salivary glycoproteins obtained from other investigators in the field. The FBGs will be characterized by amino acid analysis, two- dimensional SDS polyacrylamide gel electrophoresis, peptide mapping, and/or immunological crossreactivity. Oligosaccharides will be characterized by chemical and lectin-binding assays and, where warranted, by mass spectrometry and nuclear magnetic resonance spectrometry. The specific antibody probes generated will be used to assay for the concentration of the various molecules in saliva. Purified FBGs will be radiolabeled and used to determine the specificity, affinity and number of binding sites on type 1 fimbriated E. coli and FN. Appropriate controls will include E. coli mutants which lack or overproduce the 29 kDa mannose-binding adhesin and other species which express type 1 adhesins. Attention will be given to the influence of pH, ionic strength and divalent cations. Competitive inhibition studies using FN will be used to further characterize these interactions. Radiolabeled FBGs will also be used in binding studies to determine the specificity, affinity and number of binding sites on epithelial cells. Attention will be given to the influence of pH, ionic strength, divalent cations and to the effects of saliva and FN on this interaction. Since the interaction of FBGs with type 1 fimbriae may be mediated through the oligosaccharide portion of the glycoprotein, we will study the effects of glycosidases on the interaction of E. coli with the purified FBGs and determine whether exposure of saliva to these glycosidases exposes additional FBGs. We will use an animal model to study the hypothesis that the increased gram negative colonization following trauma is due, at least in part, to changes in salivary or buccal cell-associated molecules. Samples of epithelial cells and saliva will be assayed for: 1) changes in E. coli (or other gram negative bacteria) adherence using standard adherence assays, 2) changes in levels of FBGs, Fn or Fn fragments on buccal cells and in saliva using immuno-fluorescence, Western blot analysis and quantitative immunological assays, and 3) changes in the levels of FBG- or Fn- degrading activity using will add significantly to the information needed to understand important aspects of bacterial pathogenesis in the oropharyngeal cavity.
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