The overall aim of this project is to study the molecular mechanisms of the adherence of bacteria to mammalian cells. For this purpose the type 1 fimbria-medicated attached of Escherichia coli and other Enterobacteriaceae will be studied in detail as a paradigm of bacterial adhesion and colonization. Specific efforts will be directed toward: (1) the isolation and purification of the D-mannose binding adhesin (FimH) of E. coli type 1 fimbriae to homogeneity, (2) characterization of the receptor binding properties of the purified FimH adhesin, (3) determining the structural features of FimH that cause it to recognize D-mannose-containing receptors on eukaryotic cells, (4) determining the frequency and range of the distribution of FimH and its gene sequences among type 1 fimbriated members of the family Enterobacteriaceae, and (5) studying the antiadhesive properties of polyclonal and monoclonal antibodies against various genera and species of Enterobacteriaceae in vitro and in vivo in a mouse model of ascending pyelonephritis. The D- mannose binding adhesin will be isolated from FimH overproducing recombinant E. coli transformed with the mutant clone pUT2004 of type 1 fimbriae. The FimH produced by this mutant in the form of fimbriosomes will be isolated from culture supernatants by absorption to and elution from mannan containing yeast cells or by ultrafiltration and gel filtration on columns of Sepharose 4B. The purified FimH in monomeric form or in various sages of polymerization will be assayed for kinetics, D-mannose specificity and affinity of binding to purified D-mannose- containing type 1 fimbrial receptor isolated from guinea pig erythrocytes and immobilized on microtiter plates. The sugar structural specificity of binding will be tested by the inhibition of E. coli or type 1 fimbrial-induced yeast cell or guinea pig erythrocyte agglutination by various D-mannose-containing glycosides and oligosaccharides. Attempts will be made to complement FimH mutants of one species of Enterobacteriaceae with the unaltered gene products of another encoded by a compatible plasmid. Various strains and species of this family will be probed with monoclonal and polyclonal anti-FimH antibodies and its products. Mice will be injected with monoclonal and polyclonal antibodies against intact FimH and synthetic peptides thereof and then challenged intravesically with type 1 fimbriated E. col to determine the protective activity of FimH. These studies should lead to the development of new vaccines against many serious gram-negative bacillary infections in humans.
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