SummaryVibrio vulnificus causes acute cell death and a fatal septicaemia. In this study, we show that contact with host cells is a prerequisite to the acute cytotoxicity. We screened transposon mutants defective in the contact-dependent cytotoxicity. Two mutants had insertions within two open reading frames in a putative RTX toxin operon, the rtxA1 or rtxD encoding an RTX toxin (4701 amino acids) or an ABC type transporter (467 amino acids). An rtxA1 mutation resulted in a cytotoxicity defect, which was fully restored by in trans complementation. The expression of RtxA1 toxin increased after host cell contact in a timedependent manner. The RtxA1 toxin induced cytoskeletal rearrangements and plasma membrane blebs, which culminated in a necrotic cell death. RtxA1 colocalized with actin and caused actin aggregation coinciding with a significant decrease in the F/G actin ratio. The RtxA1 toxin caused haemolysis through pore formation (radius 1.63 nm). The rtxA1 deletion mutant was defective in invading the blood stream from ligated ileal loops of CD1 mice. The rtxA1 null mutation resulted in over 100-fold increase in both intragastric and intraperitoneal LD 50s against mice. Overall, these results show that the RtxA1 toxin is a multifunctional cytotoxin and plays an essential role in the pathogenesis of V. vulnificus infections.

Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescentphase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methylaccepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and The expression of virulence determinants in bacteria is known to be regulated by various environmental and host factors (38). During host-parasite interactions, many novel genes that not expressed during in vitro growth have been demonstrated to be coordinately regulated or stimulated by host factors encountered in vivo (20). The usefulness of the information concerning virulence expression gained from in vitro studies is therefore incomplete in relation to in vivo bacterial pathogenesis.Vibrio vulnificus, an opportunistic pathogen, experiences a dramatic environmental change during its infection process. V. vulnificus is an estuarine bacterium that preferentially affects individuals who are heavy drinkers of alcohol and patients with underlying hepatic diseases and other immunocompromised conditions. The pathogen frequently causes fatal septicemia with a rapid progress, resulting in a mortality rate of more than 50% within a few days. The putative virulence factors of V. vulnificus reported so far include a hemolysin (15), a protease (29), phospholipase A2 (55), siderophores (53), and capsular polysaccharides (61a). We reported that the ToxRS system of V. vulnificus, a transmembrane signal-transducing transcription activator, regulated the expression of the hemolysin gene vvhA (32). The ToxRS system was reported to play an important role in regulating in vivo virulence gene expression during V. cholerae infection in a mouse model (33). However, whether the V. vulnificus ToxRS system plays an important role in regulating in vivo virulence gene expression during infection needs further study. V. vulnificus, while infecting the susceptible hosts, passes through gastric acidity, experiences an abrupt pH increase in the duodenum, receives bile secretion, invades into intestinal mucosa, and eventually enters the bloodstream where the pathogen multiplies. During this complicated infection process, V. vulnificus should be able to sense changes in the environmental parameters in the host milieu. The changing signals are likely relayed to specific genes by cognate signal transduction systems, resulting in the expression of specific virulence factors (33). Virulence factors required for in vivo survival and growth of V. vulnificus are ex...

SummaryVibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicaemia and necrotizing wound infections. We tested whether V. vulnificus produces signalling molecules (autoinducer 1 and/or 2) stimulating Vibrio harveyi quorum-sensing system 1 and/ or 2. Although there was no evidence for signalling system 1, we found that V. vulnificus produced a signalling activity in the culture supernatant that induced luminescence expression in V. harveyi through signalling system 2. Maximal autoinducer 2 (AI-2) activity was observed during mid-exponential to early stationary phase and disappeared in the late stationary phase when V. vulnificus was grown in heart infusion broth containing 2.5% NaCl. V. vulnificus showed increased signalling activity when it was cultured in the presence of glucose (0.5%) and at low pH (pH 6.0). From a cosmid library of V. vulnificus type strain ATCC 29307, we have identified the AI-2 synthase gene ( luxS Vv ) showing 80% identity with that of V. harveyi ( luxS Vh ) at the amino acid level. To investigate the pathogenic role of luxS Vv , a deletion mutant of the clinical isolate V. vulnificus MO6-24/O was constructed. The luxS Vv mutant showed a significant delay in protease production and an increase in haemolysin production. The decreased protease and increased haemolysin activities were restored to the isogenic wild-type level by complementation with the wild-type luxS Vv allele. The change in phenotypes was also complemented by logarithmic phase spent media produced by the wild-type bacteria. Transcriptional activities of the haemolysin gene ( vvhA ) and protease gene ( vvpE ) were also observed in the mutant using chromosomal P vvhA :: lacZ and P vvpE :: lacZ transcriptional reporter constructs: transcription of vvhA was increased and of vvpE decreased by the mutation. The mutation resulted in an attenuation of lethality to mice. Intraperitoneal LD 50 of the luxS Vv mutant increased by 10-and 750-fold in ferric ammonium citrate-non-overloaded and ferric ammonium citrateoverloaded mice respectively. The time required for the death of mice was also significantly delayed in the luxS Vv mutant. Cytotoxic activity of the organism against HeLa cells, measured by lactate dehydrogenase (LDH) release assay, was also decreased significantly by the mutation. Taken together, the V. vulnificus LuxS quorum-sensing system seems to play an important role in co-ordinating the expression of virulence factors.

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