Vibrio vulnificus is a marine bacterium causing serious septicemia and wound infection in humans. It produces an RTX toxin that can lyse a variety of cells and is important for virulence in mice. In this study, we explored the role of RTX in pathogenesis by characterizing an RTX-deficient mutant. This mutant showed an ∼2-log reduction in virulence for mice infected by various routes. Survival of the mutant at the infection site and subsequent spread into the bloodstream were impaired. In mice pretreated with cyclophosphamide to deplete the neutrophils, both the virulence and survival at the infection site of this mutant were enhanced. This mutant was further shown to be more readily cleared from the macrophage-rich mouse peritoneal cavity and phagocytosed by murine macrophages. These findings suggest that the RTX of V. vulnificus is required for bacterial survival during infection by protecting the organism from phagocytosis.

Previous work suggested that a metalloprotease, Vvp, may be a virulence factor of Vibrio vulnificus, which causes severe wound infection and septicemia in humans. To determine the role of Vvp in pathogenesis, we isolated an isogenic protease-deficient (PD) mutant of Vibrio vulnificus by in vivo allelic exchange. This PD mutant was as virulent as its parental strain in mice infected intraperitoneally and was 10-fold more virulent in mice infected via the oral route. Furthermore, the PD mutant was indistinguishable from its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin. These data suggest that Vvp is not essential for virulence in the mouse. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twofold higher than that of the wild-type strain and remained for a much longer period. The higher cytolysin activity of the PD mutant may be associated with the enhanced virulence in mice infected via the oral route.Vibrio vulnificus, a halophilic gram-negative marine bacterium, causes infectious diseases in humans, especially in people who are immunocompromised or have underlying conditions such as hemochromatosis, liver cirrhosis, or alcoholism (3,4,5,29). It mainly produces two types of disease: primary sepsis via gastrointestinal infection, and wound infection. In either case, skin lesions with ulcer and edema are observed (13,15,36,37).This organism produces a few extracellular products implicated in bacterial virulence and pathogenesis, including cytolysin, protease, phospholipases, and siderophores (17,18,24,38). Only a single extracellular protease (designated Vvp) has been identified (16,24). This protease has been shown to increase vascular permeability and edema through activating the Hageman factor-plasma kallikrein-kinin cascade (25,26,27). It can also facilitate iron acquisition by the organism by digesting heme proteins, transferrin, and lactoferrin (31). Recently, Miyoshi et al. reported that the purified protease is able to cause hypodermic hemorrhage in guinea pigs (28). Based on these observations, Vvp was thought to be important in bacterial growth and disease developments.We have previously cloned the vvp gene (6), which encodes a protease with its amino acid composition and the N-terminal sequence derived from the deduced amino acid sequence identical to those of the purified Vvp (16). To further determine the role of Vvp in pathogenesis by genetic approaches, we isolated a protease-deficient (PD) mutant by introducing an in-frame deletion to the vvp gene of a clinical V. vulnificus strain using an allelic exchange technique. By comparing the pathogenicity of this PD mutant with that of its parental strain, we would be able to determine whether the protease is an important virulence factor. Here we report the characterization of this PD mutant with respect to (i) virulence in mice, (ii) ability to invade the blood stream from the peri...

Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificus virulence in mice.

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...

Cytotoxicity is an important virulence determinant in the pathogenesis of Vibrio vulnificus, and two cytotoxins, RTX (encoded by rtxA1) and cytolysin/hemolysin (encoded by vvhA), have been identified in this organism. We showed that the quorum-sensing regulator LuxO controlled the cytotoxicity of this organism: a ⌬luxO mutant exhibited low cytotoxicity, whereas a constitutively activated luxO mutant, luxO(D47E), remained highly cytotoxic. The cytotoxicity of the ⌬luxO mutant was restored when smcR, a Vibrio harveyi luxR homologue repressed by luxO, was further deleted. SmcR then was shown to repress the expression of both rtxA1 and vvhA. A DNA library of V. vulnificus was screened in Escherichia coli for clones that upregulated vvhA in the presence of SmcR, and hlyU, which has been shown to positively regulate rtxA1 and vvhA, was identified. We demonstrated that SmcR repressed the expression of hlyU and bound to a region upstream of hlyU in V. vulnificus. The deletion of hlyU resulted in the loss of cytotoxicity and reduced cytolysin/hemolysin production in the ⌬smcR mutant. The ⌬smcR ⌬hlyU mutant regained cytotoxicity and cytolysin/hemolysin activity when hns, which has been shown to repress the transcription of rtxA1 and interfere with hlyU, was further removed. Collectively, our data suggest that SmcR mediates the regulation of cytotoxicity by quorum-sensing signaling in V. vulnificus by repressing hlyU, an activator of rtxA1 and vvhA.Vibrio vulnificus, a Gram-negative marine bacterium, is an opportunistic pathogen causing septicemia and wound infection in humans; it has a high mortality rate, particularly in those who suffer from chronic liver diseases or are immunocompromised. This organism produces two major cytotoxins, cytolysin/hemolysin (encoded by vvhA) and RTX (repeats in toxin; encoded by rtxA1), that are implicated in its virulence (19,29). The cytolysin/hemolysin, which is an extracellular product, is lethal for mice at submicrogram levels. It lyses erythrocytes, increases vascular permeability (resulting in extensive extracellular edema in guinea pig skin), damages capillary endothelial cells, and causes mild inflammatory cell infiltration (7,8). The RTX toxin, which forms pores on cell membranes only after the contact of the bacterium with the host cell (12, 13), is required for V. vulnificus virulence in mice by promoting bacterial colonization at the infection site and subsequent invasion into the bloodstream (12, 15).Several regulators of vvhA and rtxA1 have been identified (1, 19). HlyU, which was identified by in vivo-induced antigen technology and is essential for V. vulnificus virulence in mice (11,19), is required for the expression of vvhA and rtxA1 and binds directly to a region upstream of the operon where rtxA1 is located (19,20).Quorum-sensing (QS) signaling, which is widely used by bacteria to communicate with each other, regulates the virulence genes in a variety of microorganisms, including Vibrio species (23, 30). In Vibrio cholerae, the signals transduced from at least three QS ...