Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Abstract

SummaryCholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either El Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the El Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. 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 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.

The Type 4 Prepilin Peptidases Comprise a Novel Family of Aspartic Acid Proteases

“…Strain JM290 is JM109 carrying pTrc99A/EpsI (6)His (19). Strain MK90 is K38 carrying p3Z-A, which provides tcpA under the control of a T7 promoter, and pGP1-2, which provides the heat-inducible T7 RNA polymerase that drives transcription of the tcpA on p3Z-A (22 (21), was constructed by the cloning of a 980-bp EcoRI/NsiI fragment containing tcpJ into the EcoRI and PstI sites in the polylinker of pUC18.…”

“…Plasmid p3Z-A was constructed by the cloning of the 1-kilobase pair DraI tcpA fragment into the SmaI site of the Promega expression vector, pGEM3Z (22). Plasmid pJM294 is a pACYC184 derivative containing vcpD (19). Plasmid pRTG7H3 is a tcpA-expressing plasmid (23).…”

“…Chemical protease inhibition studies are consistent with this finding. The mutational analysis was extended to a second TFPP of V. cholerae, VcpD, which is required for toxin secretion (19). Taken together, the results lead to the conclusion that members of the TFPP family are bilobed aspartic acid proteases and due to their complete insensitivity to pepstatin should be regarded as a novel family of non-pepsinlike acid proteases.…”

“…The VcpD TFPP is responsible for processing type 4 prepilins such as those involved in mannose-sensitive pilus formation and processes the extracellular protein secretion type 4 prepilin-like proteins required for the secretion of toxin and other enzymes by V. cholerae. VcpD can also cleave TcpA prepilin, although the cognate TFPP for TcpA is TcpJ (19). Wild-type, D147A, and D212A forms of VcpD expressed from plasmid pJM294 derivatives were introduced into E. coli JM109 carrying the tcpA-expressing plasmid pRTH3G7.…”

Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway

“…CT is transported in a two-step process, which first involves the translocation of the individual subunits across the cytoplasmic membrane via the Sec pathway (8). In the periplasmic compartment, the subunits assemble into the hexameric AB 5 complex, which is subsequently translocated across the outer membrane via the type II secretion pathway, encoded by the extracellular protein secretion genes (epsC-epsN) and vcpD (pilD) (9)(10)(11)(12)(13). This pathway is also responsible for extracellular secretion of other potential virulence factors that include hemagglutinin͞protease (HA͞protease), chitinase, neuraminidase, and lipase (11,14).…”