An official website of the United States government.
The proposed work will focus on this critical virulence determinant by addressing the following questions:<ol>
<li> How is the BFP assembled on the bacterial surface? How is its expression regulated in vivo? </li>
<LI>How does it function to confer virulence? </li></ol>
<P>
Enteropathogenic Escherichia coli (EPEC) are a significant cause of childhood diarrhea. EPEC infection of the small intestine involves adherence of the organism to the epithelial surface, reorganization of the underlying cytoskeleton, and in human volunteer studies, has been shown to require the entero-adherent factor (EAF) plasmid locus that codes for the expression of the bundle-forming pilus (BFP). BFP is a filamentous colonizing factor that belongs to the Type IV family of pilus proteins.</P>
<P>
BFP biogenesis will be studied using genetic, biochemical and ultrastructural methods. Knockout mutations of each of 12 bfp gene cluster cistrons will be prepared to elucidate their role in biogenesis and pilus function. Antisera to each BFP biogenic protein will be used to determine its subcellular location, and immunoprecipitation, chemical cross-linking and thin-layer immunogold electron microscopy experiments will be conducted to co-localize biogenic proteins to the hypothesized "assembly complex". Small bowel biopsies and aspirates from EPEC-infected volunteers will be studied by reverse PCR to determine if, when and where in vivo transcriptional expression of bfpA occurs and the identification of the responsible environment cues. bfpT, a separate EAF plasmid locus that transcriptionally activates bfpA expression, will be similarly studied, including experiments to determine if a bfpT knockout mutant is virulent in human volunteers. Genes that regulate bfpT expression will be sought using a bfpT-CAT fusion reporter plasmid and an EPEC expression library. </p>
<P>
To learn more about the pathogenic function of BFP, bfp gene cluster mutants will be sought that: <br>
(a) produce morphologically normal pili, but do not adhere; or <br>
(b) do not produce pili, but retain adherence function.</p>
<P>
These mutants will be biochemically characterized and studied for virulence in human volunteers. Morphological evidence for BFP adherence function will also be sought using BFP-specific antisera for immunogold scanning electron microscopic studies of EPEC-infected tissue culture cells and small bowel biopsies. </p>
<P>
These studies seek to provide new information about the biogenesis and pathogenic function of Type IV pili in general and of BFP in particular. Because BFP is a proven virulence determinant, these studies might also lead to new strategies for the prevention of EPEC infections in children. </p>