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

Vibrio vulnificus expresses a number of potential virulence determinants th at may contribute to its ability to cause a severe and rapidly disseminatin g septicemia in susceptible hosts. We have cloned and characterized two gen es encoding products related to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV pe ptidase/N-methyltransferase (PilD) mutant of Pseudomonas aeruginosa Nifh a V. vulnificus genomic library. One of the genes (vvpD) encodes a protein ho mologous to PilD and other members of the type IV peptidase family that com pletely restores this activity in a P. aeruginosa mutant deficient in the e xpression of PilD. The other gene (vvpC) encodes a homolog of PilC from P. aeruginosa, where it is essential for assembly of type IV pill. Phenotypic characterization of a V. vulnificus vvpD mutant, constructed by allelic exc hange, showed that VvpD is required for the expression of surface pill, sug gesting that the pill observed on V. vulnificus are of the type IV class. T his mutant was also unable to secrete at least three extracellular degradat ive enzymes, and the localization of one of these (the cytolysin/hemolysin) to the periplasmic space indicates that these proteins are normally export ed 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 pill and/or extracellular secretion of severa l exoenzymes.