Isolation and function analysis of apolipoprotein A-I gene response to virus infection in grouper

Highlights

• Ec-ApoA-I is expressed in all tested tissues.

• Ec-ApoA-I was up-regulated in the liver after challenge with bacterial and virus.

• Ec-ApoA-I possesses bacteriolyticactivity and antiviral activity.

• Ec-ApoA-I is distributed in both the cytoplasm and the nucleus.

Abstract

Apolipoproteins, synthesized mainly in liver and intestine and bounded to lipids, play important roles in lipid transport and uptake through the circulation system. In this study, an apolipoprotein A-I gene homologue was cloned from orange-spotted grouper Epinephelus coioides (designed as Ec-ApoA-I) by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-ApoA-I was comprised of 1278 bp with a 792 bp open reading frame (ORF) that encodes a putative protein of 264 amino acids. Quantitative real-time PCR (qPCR) analysis revealed that Ec-ApoA-I was abundant in liver and intestine, and the expression in liver was significantly (P < 0.01) up-regulated after the stimulation of LPS, Poly(I:C), Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). Recombinant Ec-ApoA-I (rEc-ApoA-I) was produced in Escherichia coli BL21 (DE3) expression system exhibited bacteriolyticactivity against Microcococcus lysodeikticus and Aeromonas hydrophila. Intracellular localization revealed that Ec-ApoA-I distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. Overexpression of Ec-ApoA-I in grouper Brain (GB) cells could inhibit the replication of SGIV. These results together indicated that Ec-ApoA-I perhaps is involved in the responses to bacterial and viral challenge.

Introduction

Fishes have a number of constitutive and inducible innate immune responses to defend against infection, many of which are similar to those of mammals. Some of the different soluble mediators include: pro-inflammatory cytokines, complement, and antimicrobial peptides [1]. Teleosts possess many of the same pro-inflammatory cytokines and complement components as mammals. Compared to mammals, teleosts possess a smaller number of antimicrobial peptides [2]. However, many species of fishes have other innate defense molecules such as natural antibodies [3], [4], apolipoproteins [5], [6], lysozyme [7], [8], [9], larger nonpeptide antimicrobial compounds such as squalamine [10], and other cationic steroidal derivatives [11].

Apolipoproteins, the protein component of various plasma lipoproteins, synthesized mainly in liver and intestine and bounded to lipids, play important roles in lipid transport and uptake through the circulation system [12]. A great deal of attention had been focused on lipoproteins and apolipoproteins in humans ever since the relationship between specific lipoproteins and cardiovascular disease became apparent [13]. However, little information has been reported to apolipoproteins in lower vertebrates [13]. Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum and participates in the reverse transport of cholesterol from tissues to the liver for excretion as the major activator of lecithin: cholesterol acyltransferase (LCAT) [14]. Since most fish utilize lipids as the major energy source in contrast to mammals which mainly use carbohydrates [15], lipid metabolism appears more important for homeostasis maintenance in fish than that in homeotherms [16].

Several other functions have been attributed to apoA-I. ApoA-I has a variety of immune-related properties including inhibition of bacterial endotoxin, antiviral activity, and inhibition of inflammatory cytokines. Mammalian ApoA-I demonstrates direct antimicrobial activity against a variety of viruses and bacteria [2], [7], [17], [18], [19], [20], [21], [22], [23], [24], [25]. For example, human ApoA-I has antiviral activity against Herpes Simplex Virus (HSV), human immunodeficiency virus (HIV), and xenotropic murine virus [21], [22], [23]. Teleostean ApoA-I isolated from carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), Channel Catfish (Ictalurus punctatus) and striped bass (Morone saxatilis) demonstrates in vitro antibacterial activity [2], [18], [24], [25].

Groupers, Epinephelus sp., are widely cultured in China and Southeast Asian countries. The emergence of bacterial and viral pathogens, including Vibrio alginolyticus, iridovirus and nervous necrosis virus, caused heavy economic losses in grouper aquaculture [26], [27], [28]. For example, the gram-negative bacterium, V. alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing grouper aquaculture, which has caused substantial economic losses globally [28]. Further, Singapore grouper iridovirus (SGIV) was isolated from diseased groupers, which belonged to family Iridoviridae [26], [29]. SGIV caused serious systemic diseases and resulted in more than 90% mortality in grouper either in fish farm or challenge experiments. The virus infection is characterized as hemorrhage and enlargement of the spleen of infected fish [29].

In the present study, the molecular characteristics of grouper ApoA-I, the tissue distribution, and expression patterns were investigated. The recombinant Ec-ApoA-I protein and mouse anti-Ec-ApoA-I were obtained. The intracellular localization and the antiviral activity were also studied. These present studies will help us for better understanding of its innate immune mechanisms in the anti-bacterial or anti-virus response of fishes.