Probing the mechanism of VPAHPND extracellular proteins toxicity purified from Vibrio parahaemolyticus AHPND strain in germ-free Artemia test system

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease caused by virulent strain of Vibrio parahaemolyticus (VPAHPND) that results in significant economic losses to shrimp aquaculture. The V. parahaemolyticus encodes deadly toxins (VPAHPND toxins) that are responsible for shrimp mortality during AHPND. Therefore, to better understand the toxicity mechanism of VPAHPND toxins, we have used the gnotobiotic Artemia franciscana model to determine the toxicity of recombinant PirA(VP) and PirB(VP) toxins in vivo. Subsequently, the study was validated by VPAHPND ECP30, ECP10 and ECP3 concentrated with 30, 10 and 3 kDa amicon filters to establish the toxicity towards brine shrimp larvae. It was found that recombinant PirB(VP) is more toxic to brine shrimp larvae as compared to PirA(VP). Moreover, the survival of brine shrimp larvae challenged with a mixture of PirA(VP) and PirB(VP) toxins decreased similar to 2-fold as compared to PirB(VP) toxin and similar to 3-fold as compared to PirA(VP) toxin, as anticipated, as these 2 toxins seem to form an active complex. The study also confirms that VPAHPND ECP10 and ECP3, comprising PirA(VP) and PirB(VP) toxins, exhibits toxic effect to brine shrimp larvae. The ECP30, lacking PirA(VP), showed reduced toxicity. The VP(AHPND)ECP10 and ECP3 (containing PirA(VP )and PirB(VP) toxins, along with other proteins) were more toxic than their respective equivalent amounts of pure toxins, suggesting that VPAHPND ECP10 and ECP3 contains additional, but uncharacterized, toxins. Hence, our study provides substantial evidence that toxicity of V. parahaemolyticus AHPND strains is mediated by VPAHPND ECP comprised of PirA(VP) and PirB(VP) (mostly) and other ECP or toxins produced by the bacterium.