Characterization of components of type IV pili and type II extracellular secretion in Vibrio vulnificus and determination of their role in virulence

摘要

Vibrio vulnificus expresses a number of cell-associated and extracellular products that may be involved in its ability to cause invasive disease in mamrnalian and marine vertebrate hosts. We have cloned and characterized four genes encoding productsrelated to components of the type IV pilus biogenesis and general secretory (type II) pathways by complementation of a type IV prepilin peptidase (PilD) mutant of Pseudomonas aeruginosa with a V. vulnificus genomic library. One of the genes (pilD) encodes a protein homologous to PilD and other members of the type IV prepilin peptidase family, which completely restores this activity in a P. aeruginosa mutant deficient in the expression of PilD. The other genes (pilA-C) encode homologs of type IV pilus biogenesis components. These include the type IV pilin protein subunit (PilA) and two proteins essential for assembly of type IV pili (PilB and PilC). Phenotypic characterization of V. vulnificus with a mutation in pilD showed that PilD is required for trie expression of surface pili. This mutant is also unable to secrete at least three extracellular degradative enzymes; the localization of one of these (cytolysin) to the periplasmic space indicates that these proteins are normally exported via the typeII secretion pathway. Loss of PilD results in a significant decrease in Chinese hamster ovary cell cytotoxicity, adherence to HEp-2 cells, and virulence in a mouse model. Capsule formation and serum resistance are not affected in the PilD mutant, indicating that in addition to capsule, virulence of V. vulnificus requires type IV pili and/or extracellular secretion of degradative enzymes.