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TheVibrio cholerae genome contains a 5.4-kbpil gene cluster that resembles theAeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designatedpilABCD andyacE , based on the nomenclature of related genes fromPseudomonas aeruginosa andEscherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and thepilA andpilD genes are 100% conserved. ThepilA gene encodes a putative type IV pilus subunit. However, deletion ofpilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating thatpilA does not encode the primary subunit of a pilus essential for these processes. ThepilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of thepilD gene showed thatpilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. SomepilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented intrans , indicating that the defect may not be directly due to a loss ofpilD . Taken together, these data demonstrate the effectiveness of theV. cholerae genome project for rapid identification and characterization of potential virulence factors.

Genetic Characterization of a New Type IV-A Pilus Gene Cluster Found in Both Classical and El Tor Biotypes of Vibrio cholerae