Vibrio vulnificus is a pathogen that causes both severe necrotizing wound infections and life-threatening food-borne infections. Food-borne infection is particularly lethal as the infection can progress rapidly to primary septicemia resulting in death from septic shock and multiorgan failure. In this study, we use both bioluminescence whole animal imaging and V. vulnificus bacterial colonization of orally infected mice to demonstrate that the secreted multifunctional-autoprocessing RTX toxin (MARTX_Vv) and the cytolysin/hemolysin VvhA of clinical isolate CMCP6 have an important function in the gut to promote early in vivo growth and dissemination of this pathogen from the small intestine to other organs. Using histopathology, we find that both cytotoxins can cause villi disruption, epithelial necrosis, and inflammation in the mouse small intestine. A double mutant deleted of genes for both cytotoxins was essentially avirulent, did not cause intestinal epithelial tissue damage, and was cleared from infected mice by 36 hours by an effective immune response. Therefore, MARTX_Vv and VvhA seem to play an additive role for pathogenesis of CMCP6 causing intestinal tissue damage and inflammation that then promotes dissemination of the infecting bacteria to the bloodstream and other organs. In the absence of these two secreted factors, we propose that this bacterium is unable to cause intestinal infection in humans. Vibrio vulnificus causes disease both by infection of wounds from seawater and by consumption of contaminated foods, especially oysters. Wound infection results in necrotizing fasciitis and edema in extremities with mortality of ∼25% as the incidence of septicemia is low. Contaminated food consumption by contrast can lead to highly invasive infections that progress rapidly from an intestinal infection to primary septicemia. Case-fatality rates are ≥50%, with rates as high as 100% in individuals who receive no antibiotic therapy. The aim of this study is to elucidate virulence mechanisms of food-borne infection of the most highly virulent strains of V. vulnificus. We developed a novel intragastric infection model for a highly virulent clinical isolate from Korea in which we can observe the bacterial load in live mice and applied this to study of wild type and strains genetically altered to delete genes for two secreted cytotoxins. Using this model, we show that both the multifunctional-autoprocessing RTX toxin (MARTX_Vv) and the cytolysin VvhA contribute to rapid in vivo growth of bacteria and that the presence of these factors directly correlates with mouse mortality. These exotoxins are then directly linked to intestinal damage and inflammation.

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.

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.

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. In the present study, DNA targets of SmcR, a Vibrio vulnificus LuxR homologue, were selected from a random pool of DNA fragments by using a cycle selection procedure consisting of in vitro DNA-SmcR interaction, purification of SmcR-DNA complexes, and PCR amplification of SmcR-bound DNA. The amplified DNA fragments were cloned and analyzed separately by electrophoretic mobility shift assay to verify the specific binding of SmcR to the DNA. The DNA sequences bound by SmcR were determined by DNase I footprinting, and alignment of the resulting 29 sequences revealed a 22-bp consensus SmcR-binding sequence, 5′-TTATTGATWWRWTWNTNAATAA-3′ (where W represents A or T, R is G or A, and N is any nucleotide), with an 8-bp (TTATTGAT) inverted repeat. The consensus sequence revealed greater efficiency for the binding of SmcR than the SmcR-binding sequence previously identified within P_vvpE. Mutational analysis demonstrated that the 9th and 10th bases from the center are the most essential for SmcR binding. A genome-wide search using the consensus sequence predicted that at least 121 genes are under the control of SmcR, and 10 of these newly identified SmcR regulon members were verified as being regulated by SmcR in V. vulnificus as well as in vitro. The consensus sequence and newly identified genes should be of use for elucidating the regulatory mechanism of SmcR and provide further insight into the role of the quorum sensing in V. vulnificus pathogenesis.

The Vibrio vulnificus aphB mutant was significantly less virulent than the wild type and was impaired in motility and adherence to host cells. Microarray analysis revealed that AphB of V. vulnificus (AphB _Vv ) influences the expression of over 10% of the V. vulnificus genome. The combined results indicated that AphB _Vv is a global regulator contributing to the pathogenesis of V. vulnificus .