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

Abstract

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND 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 VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND 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 rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

Characterization of toxicity and structure of PirABvc‐like proteins that are structurally almost identical to shrimp AHPND‐causing PirAB toxin

“…PirAB vc -like protein seemed to bind APN from the marine shrimp, H. gigas (Table 2). Moreover, a recent study found that L. vannamei APN1 in hemocytes functioned as a receptor mediating PirAB toxin penetration into hemocytes and may be involved in AHPND pathogenesis (Luangtrakul et al, 2021). The disagreement between this finding and our results is likely due to the different materials and methods used in the studies.…”

Expression of the AHPND Toxins PirAvp and PirBvp Is Regulated by Components of the Vibrio parahaemolyticus Quorum Sensing (QS) System

“…In particular, we suggested that the role of the PirA vp component is to recognize the glycan of its receptor on the host cells, after which PirB vp penetrates the cell membrane to form an unregulated channel, ultimately leading to critical cell damage [ 14 ]. Other reports have further shown that both PirA vp and PirB vp can bind directly to the receptor Lv APN1, and that PirB vp was translocated to the cytoplasm and nucleus of hemocytes [ 22 , 23 ], suggesting that the PirA vp and PirB vp toxins might be involved in other, additional pathogenic mechanisms. Until the present study, however, the mechanisms that might regulate expression of the pirA and pirB genes themselves had remained unknown.…”

Identification and Expression Analysis of an Interacting Protein (LvFABP) that Mediates Vibrio parahaemolyticus AHPND Toxin Action

“…The in-depth examinations of its interaction with AHPND toxin are also novel. A previous report described the involvement of LvAPN1 of hemocytes in AHPND pathogenesis as a VP AHPND toxin receptor mediating toxin penetration into hemocytes ( 28 ). Since the digestive system is considered the first barrier to VP AHPND infection and since the hepatopancreas is the main target organ with the most obvious symptoms, we believe that the identification of toxin interacting proteins in the digestive system, especially with the hepatopancreas, is significant for disease control.…”