Enteropathogenic Escherichia coli (EPEC) are an important cause of diarrhea in infants. The long-term objectives of this project are to understand the pathogenesis of disease due to this organism and to develop diagnostic reagents and vaccine candidates for prevention of disease due to this pathogen. There are also many similarities between the pathogenesis of EPEC and the intestinal pathogenesis of enterohemorrhagic (Shiga toxin- producing) E. coli O157:H7 (EHEC) which have been responsible for many large outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) in the U.S. and elsewhere due to the ingestion of contaminated beef, water, apple cider, and other vehicles. Dr. Kaper has shown that there is genetic similarity between some intestinal virulence factors of EPEC and EHEC and information resulting from the proposed studies will also yield insights into the pathogenesis of EHEC. The pathognomonic intestinal histopathology associated with EPEC infection is the attaching and effacing (A/E) lesion where brush border microvilli are effaced, the bacteria are intimately attached to the epithelial cell membrane, and high concentrations of polymerized actin accumulate beneath the adherent bacteria. Previous work supported by this project has revealed that the A/E histopathology is encoded on a 35 kb pathogenicity island called LEE for Locus of Enterocyte Effacement. This pathogenicity island encodes a type III protein secretion system, and transfer of the LEE into E. coli K-12 confers the A/E phenotype upon this avirulent host strain. In the next period of support, Dr. Kaper proposes to further characterize the functions of genes contained within the LEE. There are four specific aims for the proposed studies: 1) Characterize heretofore cryptic genes of the LEE, particularly those genes potentially encoding secreted proteins; 2) Further characterize the type III secretion system encoded on the LEE; 3) Study the regulation of the LEE-encoded genes and the effect of the Per transcriptional activator; 4) Clone and characterize the gene(s) encoding the initial EPEC adhesin that is responsible for initial binding to human intestinal tissue cultured in vitro. The proposed experimental approach will use a combination of molecular genetics, cell biology, and animal studies to achieve a better understanding of how EPEC infects intestinal epithelial cells and causes disease.