Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor

Acute hepatopancreatic necrosis disease (AHPND) caused by PirAB(VP)-producing strain of Vibrio parahaemolyticus, VP(AHPND), has seriously impacted the shrimp production. Although the VP(AHPND) toxin is known as the VP(AHPND) virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VP(AHPND) toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VP(AHPND) or the partially purified VP(AHPND) toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VP(AHPND) bacteria in the stomach after VP(AHPND) toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirA(VP) and rPirB(VP) toxins, supporting the notion that silencing of LvAPN1 prevented the VP(AHPND) toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VP(AHPND) toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VP(AHPND) toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.