Persistent diarrhea is a major cause of increased morbidity and mortality among children in the developing world and patients with HIV infection. Although the effects of persistent diarrhea are clear, the causes are less so. Enteroaggregative Escherichia coli (EAggEC) are important etiologic agents of persistent diarrhea, yet apparently only among children who are already malnourished. In these populations, the tandem synergistic effect of the persistent diarrhea and the malnutrition leads to a particularly malignant outcome. The pathogenetic mechanisms of Eaggec diarrhea are not yet understood, however, several potential mechanisms have been implicated. Accumulated evidence suggests that the fundamental mechanism of EAggEC pathogenicity is small bowel mucosal colonization, featuring bacteria embedded in a thick mucus blanket, followed by the elaboration of a cytotoxin with enterotoxic properties. The mechanism of intestinal colonization appears to be adherence by one of several fimbrial colonization factors, which mediate adherence to epithelial cells and mucus, as well as aggregative adherence of the bacteria to each other. The mucus-bacterial layer takes on the appearance of a biofilm, which quite plausibly, may serve as a persistent barrier of the absorption of nutrients. The cytotoxic effects appear to be due to the elaboration of a 108 kDa heat-liable protein, which is plasmid-encoded and partially neutralizable with anti-cholera toxin antibodies. Such damage to absorptive cells may further decrease the ability of the intestine to absorb nutrients, perpetuating malnutrition. This proposal seeks to continue basic studies of EAggEC pathogenesis. The experiments proposed herein will focus on one particular prototype strain, 042, which has been shown to cause diarrhea in adult volunteers.
The aims of the proposal are 1) To characterize EAggEC adherence to the intestinal mucosa.
This aim will encompass continued study of EAggEC fimbriae as well as exploration of mucus interaction and its role in nutrient absorption. 2) To elucidate the role of the EAggEC plasmid-associated cytotoxin (PAC). Cytotoxicity appears to be an integral part of the interaction of EAggEC with the intestinal mucosa.
This aim will include the cloning and sequencing of the gene encoding the PAC, the construction of PAC(-) mutants and the study of the effect of the PAC on human intestinal cells and explants. 3) To determine the role of EAggEc biofilm in malabsorption. The investigators hypothesize that the thick biofilm induced on the surface of the intestine may impair nutrient absorption, leading to malnutrition and persistent diarrhea. Using a rabbit model, they will measure the kinetics of radiolabeled nutrient absorption and assess the role of various virulence factors in affecting absorption. Successful pursuit of the aims of this grant will result in advances in the understanding of EAggEc as well as mechanisms of persistent diarrhea and malabsorption.
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