We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.Vibrio vulnificus, a gram-negative estuarine bacterium, causes wound infections and septicemia in humans, mostly in immunocompromised people and those with underlying conditions such as hemochromatosis, liver cirrhosis, and alcoholism (2,3,5,23). Infection is usually acquired via direct contact or the gastrointestinal (GI) route; in both cases, skin lesions with ulcer and edema are common (5,12,32).Strains of V. vulnificus secrete a variety of products that have been implicated in bacterial virulence and pathogenesis, including capsular polysaccharide (34), cytolysin (7, 16), metalloprotease (protease) (15,19), phospholipases (31), and siderophores (25). The purified protease of V. vulnificus has been shown to increase vascular permeability and induce edema by activating the plasma kallikrein-kinin cascade (18,21,22) and to cause hypodermic hemorrhage (20). It also facilitates bacterial acquisition of iron by digesting heme proteins, transferrin and lactoferrin (24). The cytolysin, a pore-forming cytotoxin and hemolysin (13), is lethal to mice at a submicrogram level (26). It damages mast cells, resulting in release of histamine (36), and causes hypotension, tachycardia (14), and skin (9) and pulmonary (26) damage in animals. Collectively, the cytolysin and the protease are thought to be important for the pathogenesis of V. vulnificus. The presence of cytolysin in V. vulnificus-infected mice (10) and the detection of anticytolysin antibodies in sera from mice and a human that survived V. vulnificus disease (8) further support the role of cytolysin in disease development.Genes encoding the protease (4) and cytolysin (35) of V. vulnificus have been cloned, and isogenic mutants deficient in either gene product have been isolated by an allelic exchange technique (29, 33). Although purified cytolysin and protease exhibited a variety of biological activities that seemed to be detrimental to the animals, elimination of either factor did not attenuate the pathogenicity in mice, as assayed with several animal models (29,33). In fact, the protease-deficient (PD) mutant was even more virulent than the wild-type strain in mice challenged orally (29). As mentioned above, both the cytolysin and the protease are able to increase vascular permeability and cause tissue damage, which may enhance bacterial invasiveness. Therefore, deficiency in either factor may not be sufficient to reduce the bacterial virulence, since compensation of the other factor may occur. We therefore reasoned that elimination of both factors may be necessary for attenuation of bacterial virulence. To test this hypothesis, we generated a dou...