The proposed research will continue the molecular genetic characterization of the F1845 fimbrial adhesin of a diarrhea-associated strain of Escherichia coli. Mutations will be introduced in the F1845 determinant of the clinical isolate C1845, and their effects on virulence will be assessed in animal models of enteric and urinary tract infections. The transcriptional regulation of F1845 expression will be characterized in detail. This characterization will involve determination of the number and sizes of transcripts and location of promoters employing the techniques of Northern hybridization, nuclease protection assays, and primer extension analysis. Identification of genes encoding regulatory proteins and sites of action of their products will involve nucleotide sequence analysis, trans-complementation experiments with recombinant constructs, and DNA binding studies involving DNaseI protection and gel mobility shift assays. The receptor binding domain of the F1845 fimbrial subunit protein will be located.
This aim will be initially approached by nucleotide sequence comparison of related fimbrial subunit genes, and by the construction of hybrid subunit genes from determinants with differing receptor specificities. Precise identification of receptor-binding domains will be accomplished by observing the effects of specific mutations generated in vitro, and by the ability of synthetic peptides and antibodies prepared against them to block adhesive functions of F1845. Evolutionary relationships among F1845-related-adhesin determinants will be studied by sequence comparisons and hybridization analysis; mechanisms of dissemination of these determinants among E.coli will be studied by a structural analysis of the junctions of the F1845 determinant with the E. coli chromosome, and by functional transposition assays. These studies will define the role of a newly defined family of E. coli adhesins in enteric and urinary tract disease, suggest potential preventative and therapeutic measures based on knowledge of receptor binding domains, and provide a clearer understanding of regulatory mechanisms and the molecular epidemiology of E. coli fimbrial adhesin determinants.
