Identification and characterization of a repeat-in-toxin gene cluster in Vibrio anguillarum

Vibrio anguillarum is the causative agent of vibriosis in fish. Hemolysins of V. anguillarum have been considered virulence factors during infection. One hemolysin gene, vah1, has been previously identified but does not account for all hemolytic activity. The mini-Tn10Km mutagenesis performed with a vah1 mutant resulted in a hemolysin-negative mutant. The region surrounding the mutation was cloned and sequenced, revealing a putative rtx operon with six genes (rtxACHBDE), where rtxA encodes an exotoxin, rtxC encodes an RtxA activator, rtxH encodes a conserved hypothetical protein, and rtxBDE encode the ABC transporters. Single mutations in rtx genes did not result in a hemolysin-negative phenotype. However, strains containing a mutation in vah1 and a mutation in an rtx gene resulted in a hemolysin-negative mutant, demonstrating that the rtx operon is a second hemolysin gene cluster in V. anguillarum M93Sm. Reverse transcription-PCR analysis revealed that the rtxC and rtxA genes are cotranscribed, as are the rtxBDE genes. Additionally, Vah1 and RtxA each have cytotoxic activity against Atlantic salmon kidney (ASK) cells. Single mutations in vah1 or rtxA attenuate the cytotoxicity of V. anguillarum M93Sm. A vah1 rtxA double mutant is no longer cytotoxic. Moreover, Vah1 and RtxA each have a distinct cytotoxic effect on ASK cells, Vah1 causes cell vacuolation, and RtxA causes cell rounding. Finally, wild-type and mutant strains were tested for virulence in juvenile Atlantic salmon. Only strains containing an rtxA mutation had reduced virulence, suggesting that RtxA is a major virulence factor for V. anguillarum.