Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection

Abstract

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...

The Biology of Vibrio vulnificus

“…An exception may be the toxin, which several studies have recently shown to be essential to virulence in mouse models (57,58,59,60,61).…”

The Makes Caterpillars Floppy (MCF)-Like Domain of Vibrio vulnificus Induces Mitochondrion-Mediated Apoptosis

“…1) (19,21,22). This 376-aminoacid domain shares homology with internal domains of other large bacterial toxins, including Photorhabdus Makes Caterpillar Floppy toxins Mcf1 and Mcf2 and Pseudomonas fluorescens FitD toxin (24,25).…”

“…Among the eight potential effector domains carried by MARTX Vv toxins of various isolates (21)(22)(23), a ubiquitous effector domain carried by all clinical biotype 1 V. vulnificus strains and by biotype 2 strains that infect eels is the Makes Caterpillars Floppy-like (MCF Vv ) domain ( Fig. 1) (19,21,22).…”

Monoclonal Antibodies against Vibrio vulnificus RtxA1 Elicit Protective Immunity through Distinct Mechanisms

“…However, a potential limitation for the application of antiserum against VvPS as a protective agent would be the existence of a large number of serotypes and carbotypes of VvPS, which could limit the cross-reactivity of anti-VvPS serum (45,59,60). The use of MAbs against RtxA1-C, the C-terminal region of V. vulnificus RtxA1/MARTX Vv , could avoid this potential concern because the C-terminal region of V. vulnificus RtxA1/MARTX Vv is highly conserved (12,14,15,41). In support of this, three therapeutic MAbs (21RA, 24RA, and 47RA) against the V. vulnificus M06-24/O strain recognized both intact and processed RtxA1/MARTX Vv products in different clinical and environmental strains (ATCC 29307, CMCP6, YJ016, and Evn1) ( Fig.…”

“…In the culture supernatants of CMCP6 and YJ016, an additional band with an apparent molecular mass of 170 kDa was also recognized by the three MAbs, presumably due to toxin variants with different arrangements of effector domains generated by V. vulnificus rtxA1 gene recombination (Fig. 5A) (41). Consistent with the data obtained with the three cross-reactive MAbs, RtxA1-C-specific antiserum detected the intact RtxA1/MARTX Vv protein (ϳ500 kDa) and the smaller bands (ϳ130 and/or ϳ170 kDa) from all strains tested, indicating that these smaller bands could be cleavage products of the intact RtxA1/MARTX Vv protein ( Fig.…”