Gene expression analysis and anti-WSSV property of antimicrobial peptides from the black tiger shrimp Penaeus monodon

Abstract

Antimicrobial peptides or AMPs are small peptides which play an important role in the innate immune system by defending against invading microorganisms. The crustin, lysozyme, antilipopolysaccharide factor (ALF), and penaeidin (PEN) are AMPs identified from the Penaeus monodon EST database (http://www.pmonodon.biotec.or.th). Analysis of the P. monodon AMP transcripts by semi-quantitative RT-PCR revealed that the expression of ALFPm3, ALFPm6 and PenmonPEN5 was significantly increased after shrimp were challenged with white spot syndrome virus (WSSV). In this study, we further characterized PenmonPEN5 for its role in anti-WSSV. Genomic organization of the Penmon PEN5 gene determined by PCR and genome walking revealed two exons interrupted by an intron, while the 5´ upstream sequence contained a putative promoter, TATA box, and regulatory sequences, three GATA, and two each of GATA-3, activator protein 1 (AP-1) and dorsal transcription factor binding. These cis-regulatory elements are reported to be involved in the transcription of several arthropods antimicrobial peptide genes. The PenmonPEN5 mRNA was mainly expressed in shrimp hemocytes and was up-regulated about 1.8 fold at 24h after challenge with WSSV as detected by quantitative real-time RT-PCR. The suppression of PenmonPEN5 transcript levels by RNA interference mediated gene silencing led to an increase of WSSV copy numbers about 1.9 fold. The recombinant PenmonPEN5 protein (rPenmonPEN5) over-expressed in the yeast Pichia pastoris exhibited antibacterial activity against Gram-positive bacteria, Micrococcus luteus and Aerococcus viridans. Incubation of the P. monodon hemocyte primary cell culture with the mixture of WSSV and rPenmonPEN5 inhibited the propagation of WSSV only at 6.25μM but not at higher concentration of the protein. Taken together, the results suggest a possible role of PenmonPEN5 in the shrimp’s antiviral immunity but its defense mechanism requires further investigation.