IDENTIFICATION OF THE VIBRIO-CHOLERAE TYPE-4 PREPILIN PEPTIDASE REQUIRED FOR CHOLERA-TOXIN SECRETION AND PILUS FORMATION

Cholera toxin secretion is dependent upon the extracellular protein se cretion apparatus encoded by the eps gene locus of Vibrio cholerae. Al though the eps gene locus encodes several type four prepilin-like prot eins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepili n peptidase from the V. cholerae genomic database by virtue of its hom ology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plas mid disruption or deletion of this peptidase gene in either El Tor or classical V. cholerae O1 biotype strains results in a dramatic decreas e in cholera toxin secretion. in the case of the El Tor biotype mutant s, surface expression of the type 4 pilus responsible for mannose-sens itive haemagglutination is abolished. The cloned V. cholerae peptidase processes either Epsl or MshA preproteins when cc-expressed in E. col i. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepillin peptidase required for pilus assembly and cytolysin secret ion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin pep tidase required for pilus assembly and cholera toxin secretion has bee n designated VcpD.