Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in developing countries and the most important cause of traveler's diarrhea. Effective vaccines for ETEC are not currently available. ETEC intestinal colonization is a crucial first step in the infection process, and it is believed to be mediated by species-specific pili. Longus (long pilus), a recently identified type IV pili (T4P) and one of the most prevalent pili described in human ETEC strains, has structural similarity and protein sequence homology to the well known virulence-associated T4Ps expressed by important human bacterial pathogens. The long-term goals of this project are to identify the role of Longus in ETEC colonization of the human gut and to develop vaccine candidates to prevent ETEC diarrhea. I hypothesize that Longus contributes to colonization of human intestine by binding to cells or non-cell surfaces, and that Longus-based vaccines, capable of inducing anti-Longus antibodies, will block ETEC colonization and prevent disease. To test this hypothesis I propose the following specific aims: 1) Identify the genes necessary for Longus expression and evaluate Longus-gene variation, 2) Evaluate Longus-mediated adherence and colonization of to human intestinal surfaces, and 3) Test immunogenicity of Longus in a mouse model. I will first clone the genes necessary for Longus expression, evaluate gene variation among worldwide isolates, and construct a Longus structural subunit mutant essential for the evaluation of Longus phenotypes. Wild type and mutants will be compared for cell adherence to human intestinal cells, and in biofilm formation assays. ETEC colonization will be evaluated by using a novel human intestine explant-Severe combined immunodeficiency (SCID) mouse model. The explant will be infected with ETEC and evaluated for colonization by colony count and histology. Longus immunogenicity and cross-reactivity with longus variants will be tested on sera from immunized mice with Longus-expressing attenuated Salmonella. Demonstration that Longus mediates binding to cells and biofilm formation will greatly contribute to our knowledge on Longus function. The development of a human explant/SCID mouse model will have crucial implications for the understanding ETEC colonization at the molecular level. Information that Longus is immunogenic across variants and that anti-Longus antibodies may block colonization and prevent disease will facilitate the construction of effective ETEC vaccines.
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