Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/ N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.Vibrio vulnificus, a halophilic, estuarine bacterium, is an opportunistic human pathogen capable of causing fatal primary septicemias or necrotizing wound infections (60). Primary septicemia usually occurs in susceptible hosts following ingestion of raw seafood, mainly shellfish, which are often naturally colonized by the organism. Wound infections are usually acquired through introduction of the organism into preexisting or new wounds, often during handling of shellfish harvested from waters where the organism is present. Host risk factors that contribute to susceptibility include liver disorders such as cirrhosis and alcoholic liver disease, hematological conditions such as hemochromatosis, and compromised immune status (60).A number of individual bacterial surface factors and extracellular proteins have been implicated in the virulence and pathogenesis of V. vulnificus, including an exopolysaccharide capsule, lipopolysaccharide, metalloprotease, cytolysin, phospholipase, and siderophores (reviewed in reference 60). Construction of specific mutations in genes controlling expression of these cell-associated and extracellular factors and analysis of virulence in animal models have shown that only the exopolysaccharide capsule, the presence of a functional flagellar biogenesis system, and the siderophore to acquire iron from transferrin are essential virulence determinants (28,74,77). A determination of the in vivo role of other factors, which in general have potent in vitro activities, has remained elusive (16,49,72). Increasingly, experimental evidence suggests that virulence of V. vulnificus is multifactorial (60) and that the coordinate regulation and expression of several virulence factors are likely controlled...