FUNCTIONAL CHARACTERIZATION OF IMMUNE-RELATED PROTEINS FROM BLACK TIGER SHRIMP Penaeus monodon IN RESPONSE TO PATHOGEN INFECTION

Abstract

Shrimp immunity comprises of various effector molecules that are expressed and function in response to various stimuli including pathogens. In this research, two immune-related proteins; Anti lipopolysaccharise factor isoform 3 (ALFPm3) and Viral responsive protein 15 (PmVRP15) were characterized. The ALFPm3 has previously been shown to exhibit very active antimicrobial activity against a broad range of Gram-positive and Gram-negative bacteria, fungi and virus. The propose of this research is to study the effects of treating ALFPm3 on membrane of Vibrio harveyi. The recombinant ALFPm3 protein was found to localize on the V. harveyi cells in vivo, followed by inducing membrane permeabilization and leakage of cytoplasmic components. Membrane disruption and damage, bleb and pore formation, and the leakage of cytoplasmic contents were all clearly observed by scanning and transmission electron microscopy. Our results suggested that ALFPm3 effectively kills bacteria through bacterial membrane permeabilization mechanism. Other immune-related protein with unknown function, namely PmVRP15, is of interest among the highly up-regulated gene identified in hemocyte of White spot syndrome virus (WSSV)-infected shrimp. RNAi-mediated silencing of PmVRP15 gene in WSSV-infected shrimp resulted in a significant decreased in the expression of WSSV genes including ie-1 (immediate early stage), wsv477 (early stage) and vp28 (late stage) by 83.5%, 85.5% and 94.8%, respectively. The significant decrease in the cumulative mortality of WSSV-infected shrimp following PmVRP15 knockdown suggested its important role in viral propagation. The interaction between PmVRP15 and WSSV proteins was identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation (Co-IP). WSV399 protein was identified as a PmVRP15 binding protein. Immunobloting analysis (in vitro) and immunoelectron microscopy (in vivo) identified WSV399 as the tegument protein. Moreover, the regulation of PmVRP15 gene expression was studied. About 2 kbp of 5’ flanking promoter sequences of PmVRP15 gene were identified by genome walking technique. Promoter deletion assay identified (-525/-428) and (-287/-209) nucleotide positions as repressor and activator binding sites, respectively. The computational analysis and site directed mutagenesis revealed that the repressor binding site (-525/-428) is regulated by interferon regulatory factor (IRF) and activator binding sites in (-287/-209) region are regulated by octamer transcription factor (Oct-1) and nuclear factor of activated T cells (NFAT).