Covalent actin cross-linking by the Vibrio cholerae RTX toxin.

摘要

The Gram-negative bacterium Vibrio cholerae is the causative agent of cholera disease. The hallmark symptoms of cholera are profuse rice-water diarrhea and vomiting, which if untreated, can subsequently lead to extreme dehydration and death. V. cholerae inhabit estuarine environments and are transmitted to the human host through the consumption of contaminated food or water. Following ingestion of the bacteria, V. cholerae colonizes the upper intestine and exports enterotoxins to alter host cells and elicit disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization through the novel mechanism of covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The goal of this study was to mechanistically define the individual components of the actin cross-linking reaction---the actin cross-linking enzyme, the cytoskeletal actin substrate, and the cross-linked actin product. The region of the toxin responsible for cross-linking activity, the actin cross-linking domain (ACID), has been identified, and an in vitro actin cross-linking assay was developed with a fusion protein of the ACID and the N-terminal portion of Lethal Factor from Bacillus anthracis (LFNACD). It has been established that the ACD is a cross-linking enzyme that directly catalyzes the formation of covalent linkages between actin molecules in the absence of other host proteins. Mg 2+ and ATP are essential cofactors for the cross-linking reaction, and G-actin was determined to be the cytoskeletal substrate of the RTX toxin through the use of inhibitors of actin dynamics. In addition, the data presented in this work demonstrate that actin cross-linked both in vivo and in vitro can be purified and analyzed by mass spectrometry to biochemically characterize the cross-linked actin peptide.; Ongoing investigation of the actin cross-linking activity of the V. cholerae RTX toxin will further determine the enzymatic properties of the ACD, provide insight into potential interactions between the ACD and actin, and evaluate the contribution of covalent actin cross-linking to the pathogenesis of cholera disease.