Three toxins of V. cholerae that increase short circuit current in Ussing chambers have been identified. They include cholera toxin (CT), Zot (zonula occludens toxin) which acts by disrupting tight junctions, and Ace (accessory cholera enterotoxin), the subject of this proposal. The investigator has identified, cloned and sequenced the ace gene. Preliminary studies using crude toxin extracts in animal models indicated that Ace acts by increasing transcellular ion transport. Ace increased short- circuit current in Ussing chambers and caused fluid secretion in ligated rabbit ileal loops, characteristic of a classic enterotoxin. The predicted protein sequence of Ace shows striking similarity to the product of the cystic fibrosis bene. Based on activity and structural correlates, the investigator proposes to examine two hypotheses for the mechanism of action of Ace. The 'second messenger' model is that Ace binds to a receptor on the epithelial cell membrane and activates a second messenger which increases chloride secretion via an endogenous channel. The 'pore-forming' model is that Ace forms a new channel by inserting into the epithelial cell membrane.
The specific aims are to: 1) compare native and recombinant Ace by purifying and characterizing both proteins: 2) determine the presence and protective function of an immune response to Ace: 3) study the effects of Ace on cellular function to distinguish between the 'second messenger' and 'pore- forming' hypotheses of Ace activity; 4) identify protein domains contributing to Ace activity; and 5) study the role of each of the V. cholerae toxins in virulence by constructing isogenic chromosomal mutants. The investigator will use bacterial and molecular genetic and cell physiology methods to examine the mechanism(s) of action of Ace on the gastrointestinal epithelial cell. The long term objectives of the proposal are to enhance understanding of the role of Ace in cholera pathogenesis, to identify possible new mechanisms of action of toxins, and to determine how the toxins of V. cholerae interact to cause disease.
