Elsevier

Fish & Shellfish Immunology

Volume 94, November 2019, Pages 398-406
Fish & Shellfish Immunology

Full length article
A new crustin is involved in the innate immune response of shrimp Litopenaeus vannamei

https://doi.org/10.1016/j.fsi.2019.09.028Get rights and content

Highlights

  • A new crustin gene (LvCrustinB) from pacific white shrimp Litopenaeus vannamei.

  • LvCrustinB is ubiquitously distributedand its expression is up-regulated by V. parahaemolyticus or WSSV infection.

  • LvCrustinB directly bind to polysaccharides, which included PGN, LTA, and LPS.

  • LvCrustinB is involved in antibacterial immune defense against V. parahaemolyticus in vivo.

Abstract

Crustin is an antimicrobial peptide (AMP) that plays a key role in the innate immunity of crustaceans. This study cloned a new crustin from Pacific white shrimp Litopenaeus vannamei, which we designated as LvCrustinB, using rapid amplification of cDNA ends (RACE). The full-length cDNA of LvCrustinB is 751 bp with an open reading frame (ORF) of 591 bp encoding a peptide of 196 amino acids that includes a putative signal sequence. LvCrustinB is a type II crustin that has a glycine-rich region and a single whey acidic protein domain (WAP) domain. The mRNA transcript of LvCrustinB was detected in all examined tissues and was found to be most abundantly expressed in the epithelium and muscle. The expression of LvCrustinB in hemocytes was significantly upregulated after L. vannamei was challenged with LPS, Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). When LvCrustinB was knocked down with RNAi, the mortality rate of L. vannamei significantly increased after V. parahaemolyticus or WSSV infection. Recombinant LvCrustinB was produced using Pichia pastoris GS115 and was shown to bind to 2 g-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and 2 g-negative bacteria (Escherichia coli and V. parahaemolyticus) via polysaccharides, which included PGN, LTA, and LPS. In vivo, the recombinant LvCrustinB remarkably protected L. vannamei from V. parahaemolyticus infection. These results suggest that LvCrustinB plays an important role in innate immunity and may be potentially utilized as antibacterial agents in shrimp.

Introduction

Unlike vertebrates, invertebrates largely rely on innate immunity to defend themselves from pathogenic invasion [1], which primarily involves mechanisms such as phagocytosis, encapsulation, clotting, and a variety of soluble antimicrobial peptides (AMPs) [2]. AMP systemic production has been extensively studied in insect innate immune response, which is regarded as the most important characteristic of humoral immunity [3,4]. AMPs act as frontline effectors of host defense against a wide range of microbes, including bacteria, fungi, and viruses [[5], [6], [7], [8]]. Multiple types of AMPs have been isolated from a wide variety of invertebrate phyla, including insects [9], ascidians [10,11], chelicerates [12], annelids [13], and mollusks [[14], [15], [16]]. Due to their small size, AMPs can be synthesized without elevated metabolic cost and can rapidly diffuse to infected sites. Moreover, because of their remarkable specificity for prokaryotes and their low toxicity for eukaryotic cells, many AMPs have been investigated and exploited as novel antibiotics [17].

Based on their important economic value, research on crustaceans and their innate immunity is highly valued. To date, several AMPs from crustaceans have been identified and characterized, including defensins, crustins, penaeidins, and anti-LPS factor [[18], [19], [20]]. Crustins are AMPs that are widespread among crustaceans. These humoral factors have diversity functions in the innate immunity in different species [1,4,21]. Crustins are cationic cysteine-rich AMPs that contain a glycine-rich, cysteine-rich, or proline-rich region at the N-terminus, and one or more whey acidic protein (WAP) domains at the C-terminus [[21], [22], [23]].

Most crustins are divided into three groups based on their structures (types I–III) [21]. Type I crustins are reported to contain a signal sequence in the N-terminal and a WAP domain in C-terminal, as well as a region harboring cysteine-rich residues in the middle. Type II crustins possess not only a cys-rich region but also a long gly-rich domain of approximately 40–80 aa adjacent to the signal region [21]. Type III crustins are a group of WAP domain-containing proteins that lack both the Gly-rich domain of type II crustin molecules and the Cys-rich region present in type I crustin and II crustin [2]. However, some new types of crustin-like proteins, including double WAP domain-containing proteins (type IV) [[24], [25], [26], [27]] and insect crustin-like proteins with an extra aromatic amino acid-rich region between the cysteine-rich domain and WAP domain (type V) [28], have also been discovered in invertebrates. Approximately 50 crustin-like genes have been found in 20 different crustacean species, including some penaeid shrimp [21].

The Pacific white shrimp, Litopenaeus vannamei, an important commercial crustacean that is naturally distributed along the Pacific coasts of Central and South America, has become the primary species currently cultured in Pacific rim countries [29]. Due to the rapid expansion of the aquaculture industry, world shrimp production has increased in recent decades. However, industrial development has been severely affected by outbreaks of viral and bacterial diseases [30,31]. Under these circumstances, investigating the mechanisms of immune defense against diseases may be beneficial to shrimp culture [31,32].

In this study, a new crustin gene named LvCrustinB from shrimp L. vannamei was cloned and characterized. LvCrustinB, which is a type II crustin, possesses a long Gly-rich domain between the signal peptide and the WAP domain. Upregulation of LvCrustinB mRNA was observed when shrimp were challenged with lipopolysaccharide (LPS), Vibrio parahaemolyticus, and white spot syndrome virus (WSSV). Higher mortality caused by V. parahaemolyticus or WSSV was also observed when LvCrustinB was knocked down. Recombinant LvCrustinB (rLvCrustinB) was proven to be effective in reducing the mortality of shrimp challenged with V. parahaemolyticus in vivo. These observations indicate that the LvCrustinB plays an important role in the defense mechanism of L. vannamei.

Section snippets

cDNA cloning

A sequence predicted to encode a crustin was obtained from the shrimp L. vannamei transcriptome [33], and specific primers were designed to clone the LvCrustinB gene (Table 1). Total RNA was extracted from L. vannamei tissues (hemocytes, hepatopancreas, gills, stomach, intestine, and epithelia) using TRIzol (Invitrogen, USA). The full-length sequence of LvCrustinB was amplified using the SMARTer RACE cDNA Amplification kit (Clontech, Japan) according to the manufacturer's protocol by a two-step

Characteristics of LvCrustinB

The full-length cDNA of LvCrustinB was 751 bp, containing a 5′ terminal untranslated region (UTR) of 34 bp, a 3′ UTR of 126 bp within a poly (A) tail, and an open reading frame (ORF) of 591 bp (GenBank No. MK593455) (Fig. 1). The ORF of LvCrustinB encoded a protein of 196 amino acids, and its calculated molecular mass is approximately 19.9 kDa. The LvCrustinB protein contained a putative signal peptide (residues 1–17), suggesting it could be a secreted protein. The LvCrustinB peptide sequence

Discussion

Disease prevention remains the key to shrimp culture. To prevent shrimp disease, it is essential to study their immune mechanisms. In this study, a new AMP gene from L. vannamei, named LvCrustinB, was cloned and identified. LvCrustinB contains a characteristic WAP structure and a glycine-rich region (32 glycine residues). Based on the presence or absence of structural domains lying at the N-terminal region, Smith et al. [21] classified crustins into three sub-groups, designated as types I−III.

Funding information

This research was supported by the Science and Technology Program of Guangzhou City of China (No. 201804020013), the Science and Technology Planning Project of Guangdong Province (No. 2018B020204001), the Guangxi Key Research and Development Program (No. AB18221115), the Guangxi Prawn Industry Innovation Team (nycytxgxctd-14-05), the Guangxi Natural Science Fund (No. 2015GXNSFAA139067), and the Guangxi Aquatic Animal Husbandry Science and Technology Popularization and Application Project (No.

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    These authors contributed equally to this work.

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