The role of Pm–fortilin in protecting shrimp from white spot syndrome virus (WSSV) infection

Abstract

Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca⁺⁺ binding domain. Pm–fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80–100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.

Introduction

White spot disease of shrimp caused by white spot syndrome virus (WSSV) is responsible for a major proportion of the diseases plaguing commercial shrimp ponds, and has resulted in high mortality and economic losses [1], [2], [3]. It is well known that shrimp, other crustaceans and invertebrates in general lack a truly adaptive immune response system and appear to rely on a variety of innate immune response systems to rapidly and efficiently recognize and destroy “non-self” materials [4]. It has been shown that shrimp that recover from viral infection contain a humoral neutralizing factor that can reduce the virus [5]. This led to the possibility of using an agent or immunostimulant to stimulate this humoral factor and enhance the shrimp defense mechanisms. Such applications have been reported against WSSV infection, for example, oral administration of lipopolysaccharide to Penaeus japonicus [6], [7], glucan to Penaeus monodon [8], [9], and fucoidan to P. monodon. Shrimp hemocyanin was also proved to be a useful antiviral agent against both DNA and RNA viruses [10]. Novel antimicrobial peptides have also been discovered that have potential uses to reduce mortality in aquaculture [11], [12], [13], [14], [15], [16]. WSSV envelope and structural proteins have been studied intensively as possible immunotherapeutic agents to prevent WSSV infections in Penaeus spp [17], [18], [19], [20], [21].

Recently, a number of genes that were involved in the shrimp immune system have been discovered and characterized [13], [22], [23], [24], [25], [26]. In our previous work, molecules in a signal transduction pathway, i.e., syntenin, translationally controlled tumor protein (TCTP)/fortilin and eukaryotic translation initiation factor 5A (eIF5A) were characterized and shown to display functions that protected WSSV-infected shrimp from death. In crustaceans, a translationally controlled tumor protein (TCTP) gene was identified in P. monodon and named by us as Pm–fortilin. The protein is well conserved with a Ca⁺⁺ binding domain, similar to that the fortilin genes reported from plants and animals [27]. Pm–fortilin was shown to have similar anti-apoptotic properties to their human homologs in other mammalian cells. Pm–fortilin protein was present at high levels during the onset of viral infections in P. monodon [28].

In this study, we explore the possibility of protecting black tiger shrimp from WSSV infection by the administration of a recombinant Pm–fortilin protein. We also performed a yeast two-hybrid screening assay on a P. monodon cDNA library, using Pm–fortilin as bait and found that fortilin interacts with a previously unknown protein, we have isolated named fortilin binding protein 1 (FBP1).