Genomic and AHPND pathogenesis characterization of a novel Vibrio parahaemolyticus strain carrying an ISVal1-like insertion in pirBvp.

Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio parahaemolyticus and other Vibrio spp. that harbor a pVA plasmid encoding PirA and PirB toxins within the Tn6264 composite transposon. The pirAvp and pirBvp genes are highly susceptible to mutations due to the transposition dynamics of Tn6264 and its ISVal1 element. Here, we characterized a novel AHPND-causing V. parahaemolyticus strain, ST110-8, featuring a mutant pirBvp due to an ISVal1-like insertion. Its complete genome was hybrid-assembled using short- and long-reads, revealing five antibiotic resistance genes, including blaCTX-M-55. The Tn6264 composite transposon on the pVA_ST110-8 plasmid showed a similar structure to Tn6264 in wild-type reference strains, except for the insertion of ISVal1-like in the pirBvp gene. With this insertion, a new composite transposon was formed by the ISVal1-like element and the right ISVal1 of Tn6264 and transposed itself to between the hipA and marR gene on chromosome 1, creating a second copy of the mutant pirBvp gene. RT-PCR confirmed the mRNA expression of both pirAvp and mutant pirBvp. However, Western blot analysis showed reduced PirA protein levels, while PirB was undetectable, consistent with structural modeling suggesting that the mutant PirB is more prone to proteolysis. Interestingly, immersion challenge bioassays showed that ST110-8 could still induce a mild AHPND, with an LD50 of 8.08 ± 0.24 × 105 CFU/ml, approximately 1.7 times more attenuated compared to the wild type AHPND strain XN89. These findings provide insights into AHPND toxin mechanisms and highlight the need to monitor PirB integrity in field surveillance.