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.

Fig. 1.

Schematic diagrams of MARTX toxins. The gray and blue regions represent regions conserved in all MARTX family toxins that are required for translocation and autoprocessing of the central effector region. The color-coded centrally located effector domains are different in each MARTX toxin and are described further in the text. (A) MARTX_Vv variants from V. vulnificus Biotype 1 strains. Variants are referred to in the text by the first letter of the strain in which they were first recognized. (B) MARTX variants that probably served as donors in recombination events leading to the emergence of M-, D-, and O-type MARTX_Vv toxins.

Fig. 2.

Neighbor-joining tree of all strains used in this study derived from a 10-locus MLST CLUSTALW alignment. A similar MLST profile was derived from the V. cholerae N16961 complete genome and was used as a divergent sequence to root the tree. Shapes as described in the legend refer to the effector domain region of the rtxA1 gene determined by nucleotide sequencing. Filled shapes are human isolates; open shapes are market oyster isolates. Bootstrap confidence values from 1,000 replicates are shown at branch points. Asterisks at 99–645 DP-C4 and NSV 5829 indicate these sequences include point mutations that introduce a stop codon in the ORF. Detailed strain descriptions are given in Table S1.

Fig. 3.

Alignment of the portions of the rtxA1 gene from MO6-24/O and CMCP6 that encode the Mcf effector domain and the CPD. (A) A 1,519-bp deletion could not be identified because of 73 unmatched nucleotides in the MO6-24/O that do not derive from any part of the absent pmt locus. (B and C) Alignment of unmatched sequence from MO6-24/O and neighbor-joining tree of a 593-bp nucleotide sequence representing the 73 nucleotides plus 260 nucleotides on each side with potential rtxA donor sequences from V. anguillarum and the V. vulnificus plasmid pR99 and with other V. vulnificus strains more closely related to the donor sequences (note the differences in nucleotide polymorphisms in strains from different lineages). Boxes in the tree indicate the MLST lineage to which the strain is assigned (Fig. 2). Bootstrap confidence values from 1,000 replicates are shown at branch points. The rtxA gene sequence from X. bovienii, which also has an mcf–cpd junction in its rtxA gene (11), was used to root tree. (D) Schematic representation of how novel variants arose by recombination with V. anguillarum and plasmid rtxA genes.

Fig. 4.

(A) Neighbor-joining tree of a 1,308-bp region corresponding to the acd of rtxA genes from various species and plasmids. Bootstrap confidence values from 1,000 replicates are shown at the branch points. (B) Anti-actin Western blots of HeLa cell lysates after incubation with indicated bacterial strains for indicated times. M, monomeric actin; D, dimeric actin; T, trimeric actin.