More than 8 million Americans per year (mostly women) suffer from a urinary tract infection (UTI), most of which are caused by Escherichia coli. Adhesive P pili, which facilitate colonization and disease by binding to receptors in the urinary tract are produced by most uropathogenic strains of' E. coli. Genes important in P pilus biogenesis (papA-K) are linked in an operon. P pilus assembly depends on the periplasmic PapD chaperone and the outer membrane PapC usher. Similar chaperone/usher pathways are required for the assembly of over 30 adhesive organelles in diverse pathogenic bacteria. An advanced genetic system will be blended with X ray crystallography, biochemistry, and high resolution electron microscopy to elucidate the structure, function, and mechanism of action of the chaperone/usher pathway and to study how this pathway is linked to the expression of new genes critical in pathogenesis. PapD binds to subunits to facilitate their folding and import into the periplasmic space. Site directed mutagenesis studies combined with new structural information and an in vitro folding assay will identify critical chaperone-subunit contacts and the mechanism of PapD-facilitated subunit folding. The molecular mechanism by which PapD prevents premature subunit interactions in the periplasm will be elucidated by determining the function of the subunit surfaces that are capped by PapD, in the quaternary interactions of the pilus. The molecular basis by which the usher facilitates chaperone dissociation and the translocation of subunits across the outer membrane will be studied in in vivo and in vitro reconstitution assays. PapC will be incorporated into unilamellar liposomes and used to reconstitute pili from purified chaperone-subunit complexes. These studies will allow a detailed dissection of pilus biogenesis. Subunits are siphoned OFF pathway in the absence of PapD, resulting in their misfolding and aggregation which generates a signal that activates the Cpx two component signal transduction pathways. OFF pathway subunits are produced during pilus biogenesis and may increase upon microbial colonization of the urinary tract. The role of the Cpx pathway in regulating the expression of pili and other virulence factors in response to microbial attachment will be determined. In conclusion, pilus biogenesis will be used as a model system to study common themes in bacterial pathogenesis, namely, the protein folding, secretion, and assembly of virulence factors and the link between these processes and signal transduction pathways. This work will lead to the development of novel anti-microbial therapeutics and vaccines.
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