(Taken from application) 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 and causes reorganization of the underlying cytoskeleton. In human volunteer studies, EPEC virulence requires the entero-adherent factor (EAF) plasmid that includes the locus responsible for the elaboration of the filamentous colonizing factor BFP (bundle-forming pilus). BFP homologous to members of a family of bacterial virulence factors known as the Type IV pili. The proposed studies will determine: a) if BFP are virulence factors, and b) if the genes encoding these pili are expressed at the site of infection, i.e., the upper gastro-intestinal tract of humans. There are no known animal models for this diarrheagenic bacterial infection; therefore, human volunteers will be challenged with isogeneic variants of virulent EPEC strain B171-8. The variants that will be tested include: the wild type strain (B171-8); a knock-out mutant (B171-8 A) of the structural locus for BFP elaboration; and, a knock-out mutant (B171-8 T) of bfpT, another EAF-plasmid locus that encodes a trans-activating transcriptional regulator of bfpA, the gene encoding the structural subunit of BFP. The volunteers will be monitored clinically and the three groups compared with respect to stool volume and consistency and the presence and severity of fever, nausea, abdominal discomfort, or malaise. Aspirates and tissue biopsies will be obtained from EPEC-infected volunteers and studied using reverse transcriptase-PCR amplification of bfpA and bfpT mRNA. The in situ expression of these putative virulence genes will be monitored from jejunal, duodenal, and gastric samples, as well as the fecal specimens of these volunteers. The time course of expression of these genes will be monitored in different volunteers at 2, 4, 6, 8, 12, and 18 hours after ingestion of the bacteria. These studies seek to prove that BFP is a virulence determinant and to demonstrate that the regulated expression of the genes that code for this fimbriae respond to the environmental signals the bacteria encounter in the upper gastro-intestinal tract of humans. These studies may provide new information about the pathogenic function of Type IV pili in general and may lead to new strategies to prevent EPEC infections in children.
| Bieber, D; Ramer, S W; Wu, C Y et al. (1998) Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science 280:2114-8 |
