Functional characterization of hemocyte homeostasis associated protein, HHAP in two species of crustaceans

Abstract

Hemocyte homeostasis-associated protein (PmHHAP) has been reported as a viral responsive protein that plays an important role in controlling hemocyte homeostasis in shrimp Penaeus monodon. In this study, the role of PmHHAP in regulating apoptosis in shrimp was investigated. In vivo gene silencing of PmHHAP could induce the high level of apoptosis in shrimp hemocytes and altered the expression of apoptosis-related genes particularly PmCasp and PmCaspase. Moreover, PmHHAP was able to bind to PmCasp and the rPmHHAP protein could decrease apoptosis in the rPmCasp-treated hemocytes cells. These results indicated that PmHHAP is an anti-apoptosis protein that regulates hemocyte homeostasis by inhibiting apoptosis in P.monodon. Yeast two-hybrid and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein of white spot syndrome virus, WSSV134. The viral protein WSSV134 is a late protein of WSSV which is initially expressed after 24-hour post infection (hpi) and is likely involved in viral propagation. Co-silencing of PmHHAP and WSSV134 prior to WSSV infection showed significant increase of caspase activity, which was higher than silencing only PmHHAP or WSSV134, suggesting that these two proteins might work concordantly to control apoptosis during WSSV infection. Suppression subtractive hybridization (SSH) of PmHHAP silenced shrimp hemocytes identified several viral responsive genes as well as hemocyte homeostasis-related genes such as the transcription factor ATF-β, cathepsin L, lactate dehydrogenase, ferritin and inhibitor of apoptosis (IAP), some of which should be selected for further investigation. In addition, a homolog of HHAP in crayfish Pacifastacus leniusculus with 47% similarity to PmHHAP was functional characterized. PlHHAP was detected in all examined tissues but highly expressed in intestine and gill. Surprisingly, PlHHAP was found to play important role in bacterial but not viral defense in contrast to PmHHAP. PlHHAP transcript remained unchanged upon WSSV infection but dramatically increased upon bacterial Aeromonas hydrophila B1 challenge. Furthermore, suppression of PlHHAP resulted in increasing the number of bacteria in intestine but did not affected hemocyte apoptosis or the expression of genes (CHF, astakine1, astakine2, and IGFBP7) involved in crayfish hematopoiesis and hemocyte homeostasis suggested that HHAP from different crustacean species have different functions in defense against invading pathogens.