This project encompasses the structural study of Escherichia coli heat- labile enterotoxin (LT). LT is a member of a class of bacterial enterotoxins which are directly responsible for diseases of varying severity. The closely related enterotoxin from Vibrio cholerae (cholera toxin) produces a severe diarrheal disease which may result in death within hours; the milder infectious diarrhea produced by LT itself is rarely life-threatening in the developed world, but is a major cause of infant death in the third world. The annual incidence worldwide is estimated to be as high as 650 million cases, producing up to 800,000 deaths annually.
Specific aims of this proposal include elucidation of the mode of binding to the cell membrane receptor, of the determinants for specific recognition of target proteins in the infected cell, and of the catalytic mechanism of the enzymatically active A subunit. These structural questions are to be explored via high resolution x-ray diffraction protein structure determination and analysis of both native and engineered variants of LT complexed with model oligosaccharides and substrate analogues. Long-term goals of this project are: (l) to guide design of drugs and vaccines effective against enterotoxigenic diseases by providing a structural explanation for the biological function and activity of these proteins. LT and cholera toxin are 80% homologous in sequence, exhibit similar mechanisms of subunit assembly, are immunologically cross-reactive, bind to G-M1 gangliosides as membrane receptors, and share a common mechanism of action after incorporation into the cell. Therefore studies on LT are expected to be directly relevant to cholera as well. (2) to utilize the remarkable ability of LT to stimulate the mucosal immune system by designing prototype vaccines based on the incorporation of foreign epitopes into the LT structure. Proposed designs include incorporation of epitopes derived from influenza virus hemagglutinin and incorporation of epitopes derived from the malarial parasite Plasmodium falciparum.
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