Vibrio vulnificus is a food-borne bacterial pathogen associated with 1% of all food-related deaths, predominantly because of consumption of contaminated seafood. The ability of V. vulnificus to cause disease is linked to the production of a large cytotoxin called the "multifunctional-autoprocessing RTX" (MARTX Vv ) toxin, a factor shown here to be an important virulence factor by the intragastric route of infection in mice. In this study, we examined genetic variation of the rtxA1 gene that encodes MARTX Vv in 40 V. vulnificus Biotype 1 strains and found four distinct variants of rtxA1 that encode toxins with different arrangements of effector domains. We provide evidence that these variants arose by recombination either with rtxA genes carried on plasmids or with the rtxA gene of Vibrio anguillarum. Contrary to expected results, the most common rtxA1 gene variant in clinical-type V. vulnificus encodes a toxin with reduced potency and is distinct from the toxin produced by strains isolated from market oysters. These results indicate that an important virulence factor of V. vulnificus is undergoing significant genetic rearrangement and may be subject to selection for reduced virulence in the environment. This finding would imply further that in the future on-going genetic variation of the MARTX Vv toxins could result in the emergence of novel strains with altered virulence in humans.phylogeny | RTX | oyster T he gram-negative bacterial pathogen Vibrio vulnificus inhabits coastal waters including the US Gulf Coast. The pathogen causes gastroenteritis, primary septicemia, and necrotizing fasciitis and can be difficult to treat. The bacteria are particularly rapid growers in vivo and are highly invasive; thus death can occur as quickly as 24-48 h after ingestion (1, 2). In the United States from 1998-2008, 985 cases of V. vulnificus infection resulting in 91 deaths were reported to the Centers for Disease Control. Of these cases, 60% were caused by food-borne illness, particularly associated with the consumption of raw seafood (3). In fact, V. vulnificus accounts for 1% of all food-related deaths in the United States (4). Numerous studies recently have described a V. vulnificus multifunctional-autoprocessing RTX toxin (MARTX Vv ) as having a major impact on virulence in mice (5-8). Toxins of the MARTX family are very large composite toxins. The 5,206-amino acid MARTX Vv toxin as found in the two sequenced strains of V. vulnificus, Korean isolate CMCP6 and Taiwanese isolate YJ016 (9, 10), are 99% identical. Similar to all MARTX toxins, the majority of MARTX Vv is comprised of conserved repeat regions at the N and C termini that are proposed to form a pore in the eukaryotic cell plasma membrane for translocation of the central portion of the secreted toxin to the cytosol (11). The central region includes a conserved cysteine protease domain (CPD) that is required for inositol hexakisphosphate-induced autoprocessing. After translocation, autoprocessing at leucine residues in unstructured regions between the effector do...

Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.