The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice

Abstract

Vibrio vulnificus expresses a number of potential virulence determinants that may contribute to its ability to cause a severe and rapidly disseminating septicemia in susceptible hosts. We have cloned and characterized two genes encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV peptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein homologous to PilD and other members of the type IV peptidase family that completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pili. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exchange, showed that VvpD is required for the expression of surface pili, suggesting that the pili observed on V. vulnificus are of the type IV class. This mutant was also unable to secrete at least three extracellular degradative enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally exported via the type II secretion pathway. Loss of VvpD resulted in significant decreases in CHO cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance were not affected in the vvpD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of several exoenzymes.

FIG. 1

Genetic organization and partial restriction map of the 4.8-kb PstI fragment showing restriction sites used for cloning various constructs. Genes encoded on the fragment are shown as boxes. The ▾ symbol indicates the insertion site for the Ω fragment. Complementation of P. aeruginosa pilD and pilC mutants, indicated by restoration of PO4 phage sensitivity, is shown on the right. The vvpD probe derived from the 1-kb SalI fragment used in Southern blots is shown as a bar at the bottom.

FIG. 2

Comparison of amino acid sequences of V. vulnificus VvpC (Vv VvpC) with homologs from P. aeruginosa (Pa PilC) and A. hydrophila (Ah TapC). Differences in the C7184 and MO6-24 VvpC sequences are shaded. The alignment was generated by using the Pileup program of the Genetics Computer Group (Madison, Wis.).

FIG. 3

Comparison of amino acid sequences of V. vulnificus VvpD (Vv VvpD) with homologs from P. aeruginosa (Pa PilD) and A. hydrophila (Ah TapD). The conserved cysteine and glycine residues shown to be involved in leader peptidase and N-methyltransferase activities are shaded. The alignment was generated by using the Pileup program of the Genetics Computer Group.

FIG. 4

Southern blot comparison of various restriction digests of V. vulnificus chromosomal DNA, using vvpD as a probe. Lanes: A, PstI; B, PstI-HindIII; C, SalI. Positions of molecular weight markers are indicated on the left.

FIG. 5

Sodium dodecyl sulfate–Tricine–15% polyacrylamide gel showing in vitro activity of VvpD on P. aeruginosa prepilin substrate. Membranes were prepared from V. vulnificus strains carrying the plasmids indicated. Lanes: A, prepilin substrate only; B, C7184(pMMB67HE.cam); C, C7184D₁₂Ω(pMMB67HE.cam); D, C7184D₁₂Ω(pRPD1). Positions of molecular weight markers are shown on the left in kilodaltons.

FIG. 6

Electron micrographs of V. vulnificus C7184 strains showing surface pili (indicated by arrows). (A) C7184; (B) C7184D₁₂Ω; (C) C7184D₁₂Ω(pRPD1). Bar = 200 nm.

FIG. 7

Electron micrographs of V. vulnificus C7184 (A) and C7184D₁₂Ω (B) stained with ruthenium red. Bar = 200 nm.