Hemocyanin of Litopenaeus vannamei agglutinates Vibrio parahaemolyticus AHPND (VPAHPND) and neutralizes its toxin

Abstract

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VP_AHPND), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VP_AHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VP_AHPND infection and in response to challenge with partially purified toxin of VP_AHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VP_AHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VP_AHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VP_AHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VP_AHPND. Injecting the purified native hemocyanin along with VP_AHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VP_AHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VP_AHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VP_AHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10^-6 M. Our results indicated that upon VP_AHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VP_AHPND and also neutralization of VP_AHPND secreted toxin via direct interacting with the PirA protein.

Keywords: Hemocyanin; Shrimp immunity; Toxin neutralization; Vibrio parahaemolyticus AHPND.