IDENTIFICATION OF RECEPTORS OF PIRAB^VP BINARY TOXIN RELEASED BY Vibrio parahaemolyticus CAUSING ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) IN Penaeus vannamei SHRIMP

Figure 1. Interaction of PirAB^Vp toxin with APN-1-5 isoforms. PirAB^Vp was indicated by red color. APNs were indicated by blue color.

Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio parahaemolyticus , an enteric pathogen carrying a large plasmid which encodes a binary toxin, PirAB^Vp . The tertiary structure of PirAB^Vp toxin shows similarity to Cry^Bt toxin produced by Bacillus thuringiensis that causes lethal infection in insects. In insect, aminopeptidase N (APN) is one of the receptors of Cry^Bt toxin, but the receptors in shrimp foregut and hepatopancreas that bind to PirAB^Vp remain unknown. Upon mining NCBI database, five isoforms of insect homologs of APN in shrimp (Penaeus vannamei ) were identified. Similar to insect APNs, shrimp APNs contain a transmembrane domain, a Cry Binding Region (CBR) and two signature amino acid sequence motifs. Phylogenetic analyses showed that crustacean APNs are similar to insect APNs but form a separate clade. In silico analysis of the tertiary structure of PirAB^Vp and predicted tertiary structure of shrimp APNs revealed all APN isoforms displayed the binding affinity to PirAB^Vp. Interestingly, following an experimental challenge of Specific Pathogen Free (SPF) P. vannamei shrimp with AHPND-causing V. parahaemolyticus ,  none of the five  APN transcripts were detected in the  foregut, and four out of five APN isoforms, APN-1, -2, -4 and -5 were detected by qRT-PCR in the hepatopancreas tissue.

The mRNA  expression  of  APN-4 and -5  were significantly higher in an AHPND susceptible than in a tolerant shrimp line. Modelling of proposed biologically functional PirAB^Vp toxin and APN interactions coupled with the mRNA expression data suggest while APNs serve as receptors of binary toxin in hepatopancreas,  and  in the gut PirAB^Vp toxin likely binds to other receptors. In addition, lower mRNA expression of APN-4 and -5 in hepatopancreas of AHPND-tolerant line suggests a reduction in toxin uptake by epithelial cells in hepatopancreatic tubule may contribute to AHPND resistance. Collectively, these findings shed new insights on the molecular mechanisms of PirAB^Vp toxin involved in V. parahaemolyticus pathogenesis in shrimp.