PATHOGENESIS OF ETEC INFECTIONS

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PROPOSAL (Adapted from the applicant's abstract): Enterotoxigenic Escherichia coli (ETEC) are a leading cause of infectious diarrhea worldwide, causing hundreds of thousands of deaths each year. There is presently no licensed vaccine to prevent infections caused by these organisms. Furthermore, many avenues of basic clinical investigation remain unexplored. The long-term objective of these studies is to discover viable candidates for ETEC vaccine development employing molecular pathogenesis studies to more fully understand the essential determinants of virulence. All prior vaccine and pathogenesis studies to date have focused on extrachromosomal (plasmid)-encoded virulence factors. The studies proposed here attempt to capitalize on the recent discovery of a pathogenicity island (PAI) in the chromosome of the prototypical ETEC strain H10407, located in the selC gene, the site of two previously described PAIs in other pathogenic E. coli. At least two genes encoded on the island, tia and DleoA, affect virulence in an animal model of infection and modulate the release of a known critical virulence factor, the heat-labile toxin (LT). Understanding the events which facilitate the export and delivery of this toxin to its target receptors on eukaryotic cells could prove valuable in the ultimate development of an effective vaccine. The studies proposed herein focus primarily on the role of the PAI in modulating the release of LT. The outlined experiments concentrate on a specific locus within the PAI that contains open reading frames (ORFs) encoding putative proteins with motifs common to bacterial secretion systems. These studies are directed at ascertaining the role of these genes in modulating the synthesis and export of LT. Finally, experimental challenge studies will examine the contribution of the PAI to virulence of ETEC in human infection. Specific Aims: 1. Construction of in-frame deletions in selected genes located on the PAI. In this aim, the focus will be on ORFs within a putative secretion element of the PAI. The resulting collection of isogeneic deletion mutants will then be used in subsequent experiments directed at their phenotypic characterization. 2. Phenotypic characterization of isogeneic deletion mutants. Each mutant will be examined in a number of assays to investigate the effects of the deletion on toxin synthesis, export, and delivery to eukaryotic target molecules. These experiments will be used to confirm the investigator's hypothesis that additional genes of the PAI, which together encode a multicomponent membrane transporter, are required to modulate LT release. 3. Subcellular localization and characterization of proteins encoded by virulence genes on the PAI. While assessing the impact of each mutation on virulence and toxin release, the individual proteins will be characterized on a molecular level with experiments to define their subcellular location. These studies will be needed to validate the present hypothetical model of the role played by these proteins as LT is released from the periplasmic space of ETEC. 4. Testing of an in-frame deletion mutant in a human experimental challenge model of ETEC infection. To examine the contribution of the PAI to virulence of ETEC H10407, the DleoA in-frame deletion mutant will be tested in humans.