Elsevier

Fish & Shellfish Immunology

Volume 105, October 2020, Pages 186-194
Fish & Shellfish Immunology

Full length article
An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection

https://doi.org/10.1016/j.fsi.2020.06.046Get rights and content

Highlights

  • A novel serine proteases contain two ShK-domains from Eriocheir sinensis, EsShK-SP, was reported first time so far.

  • The expression of EsShK-SP was significantly up-regulated after Spiroplasma eriocheiris infection.

  • The over-expression of EsShK-SP increased the proPO transcription and PO activity to resist S. eriocheiris infection.

  • EsShK-SP RNAi decreased the proPO transcription and PO activity to promote S. eriocheiris infection.

Abstract

A novel serine protease contains two ShK-domain was found from the Chinese mitten crab Eriocheir sinensis (EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed that EsShK-SP was expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasma eriocheiris, the expression of EsShK-SP was significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC and S. eriocheiris stimulated the hemocytes of E. sinensis, the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number of S. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number of S. eriocheiris in the EsShK-SP silenced group was significantly increased compared to the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role during E. sinensis against S. eriocheiris through regulation of the proPO system.

Introduction

The Chinese mitten crab, Eriocheir sinensis, is a famous freshwater product in China. In recent years, the diseases of E. sinensis have become more and more serious, especially tremor disease (TD), one of the most destructive diseases in E. sinensis, has already resulted in great economic losses currently [1]. The pathogen of TD was identified and named Spiroplasma eriocheiris [2,3]. Invertebrates, including crabs, lack an adaptive immune system that plays a vital role during hosts defend against pathogens infection [4,5]. Therefore, it is essential to study the innate immune mechanisms of E. sinensis against S. eriocheiris to control TD.

Serine protease is an important family of proteolytic enzymes with serine as the active center [6]. Serine protease not only can hydrolyze proteins but also plays a crucial role in blood coagulation, molting, cellular immunity, and humoral immunity [7]. Melanization reaction, as one of the most effective methods of humoral immunity, is caused by activation of the REDOX and phenol oxidase (proPO) systems [[8], [9], [10], [11]]. The proPO system, one of the most important components in the innate immune system [12], can recognize and respond to lipopolysaccharides (LPS) or peptidoglycan (PGN) from bacteria, and β-1,3-glucan from fungi [10]. In the proPO system, serine protease acts as an upstream activator [12,13] to initiate a series of downstream reactions that kill the pathogen [9,[14], [15]]. In recent years, more and more researches have been done on serine protease in crustaceans, especially those related to immunity. After silencing the serine proteases EsTLSP-I and EsTLSP-II from E. sinensis, the expression of proPO and lipopolysaccharide-β-1,3-glucan binding protein (LGBP) were notably reduced, the copy number of S. eriocheiris and the mortality of crabs increased significantly [16]. After Penaeus monodon was stimulated by Vibrio parahaemolyticus, the expression level of a serine protease in hemolymph was significantly up-regulated. Moreover, the expression of serine protease, the PO activity decreased by 36% both at the transcriptional and protein levels after RNAi. Then the cells were stimulated with recombinantly expressed serine protease, PO activity, and serine protease activity were significantly up-regulated [17]. After stimulated by Listonella anguillarum, the expression levels of serine proteinase and proPO in the hemolymph of E. sinensis were up-regulated [18].

According to sequences, topological and functional similarities, serine protease can group into at least 72 different families, such as chymotrypsin, subtilisin, lactoferrin, and mucin [7]. EsShK-SP, a novel serine protease contain 2 ShK-domains from E. sinensis, is the first time reported so far, the function of it is still not clear. So, it is very essential to study its function, especially that relate to innate immune responses against S. eriocheiris. In this study, the characteristics and function of EsShK-SP in response to S. eriocheiris challenge was studied. Thus, this study provides useful information about a novel aspect of EsShK-SP in the innate immune response of E. sinensis against S. eriocheiris challenges.

Section snippets

Gene cloning and sequence analysis of EsShK-SP

Part of the EsShK-SP gene sequence was obtained based on the previous transcriptome data of E. sinensis and the corresponding primers were designed based on this sequence. After the hemolymph of E. sinensis was extracted, the total RNA was extracted by using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's protocol. Then the 3′ and 5’ -RACE product was obtained according to the SMARTer RACE Kit. Finally, the full length of EsShK-SP was obtained by the 3′ and 5′-RACE sequence

Sequence analysis of EsShK-SP

The full-length cDNA of EsShK-SP is 1927 bp, which consists of a 1260 bp ORF, a 105-bp 5′-noncoding region (UTR), and a 562 bp 3′-UTR. The ORF region encodes 419 amino acids (AA) containing a 32 AA signal peptide. There are four domains in EsShK-SP, the transmembrane domain consisting of 23 AA, the ShK1 domain consisting of 39 AA, the ShK2 domain consisting of 27 AA and Tryp-SPC domain consisting of 235 AA using SMART program analysis. The predicted molecular mass of the mature protein is

Discussion

E. sinensis plays an important role in the freshwater aquaculture industry in China. In recent years, the disease of it is becoming more and more serious, especially the TD, which caused by S. eriocheiris, has brought huge economic losses to the aquaculture industry [2,22]. To protect crabs from epidemics, plenty of researches had been done on the innate immune mechanisms of E. sinensis against bacteria and viruses [23]. Due to the lack of a truly adaptive immune response system, invertebrates,

CRediT authorship contribution statement

Xiaohui Cao: Methodology, Formal analysis, Writing - original draft. Yinyue Lu: Investigation. Jiyun Li: Investigation. Xiaoli Xia: Investigation. Qi Gao: Investigation. Wei Gu: Methodology, Formal analysis, Writing - original draft. Wen Wang: Supervision. Qingguo Meng: Methodology, Formal analysis, Writing - original draft.

Acknowledgments

The current work was supported by grants from the National Key Research and Development Project of China (Grant No. 2019YFC1605800), the National Natural Science Foundation of China (NSFC Nos. 31870168) and the Modern Fisheries Industry Technology System Project of Jiangsu Province (Grant No. JFRS-01).

References (35)

Cited by (5)

  • Ubiquitin-modified proteome analysis of Eriocheir sinensis hemocytes during Spiroplasma eriocheiris infection

    2022, Fish and Shellfish Immunology
    Citation Excerpt :

    As an indispensable part of innate immune system, the proPO system only exists in invertebrates and assumes an essential role in the host cell nonself-recognition system [52]. After the pathogens recognized by host cell surface pattern recognition proteins (PRPs), proPO system would be activated and transform proPO into the PO, which helped the host cell against the foreign pathogens [53,54]. The proPO system of E. sinensis was one of the main immune response against the S. eriocheiris infection.

  • Effects of the interaction between a clip domain serine protease and a white spot syndrome virus protein on phenoloxidase activity

    2022, Developmental and Comparative Immunology
    Citation Excerpt :

    Expression of Clip-SPs at the transcriptional level has been reported in many tissues. However, several studies in different organisms revealed that Clip-SPs were highly expressed in hemocytes, such as FmClipSP of F. merguiensis, PmPPAE1 and PmPPAE2 of P. monodon (Charoensapsri et al., 2009, 2011), PmClipSP2 (Amparyup et al., 2013), PmSnake of P. monodon (Monwan et al., 2017), prophenoloxidase activating enzyme (ppA) of Pacifastacus leniusculus (Wang et al., 2001), PlcSP of P. leniusculus (Guo et al., 2017), Sp-SP3 and Sp-SP5 of S. paramamosain (Wei et al., 2019), EsShK-SP of E. sinensis (Cao et al., 2020), PAP-1 in Manduca sexta, the tobacco hornworm (Gupta et al., 2005) and PAP in flies (Jiang and Kanost, 2000). FmClipSP was primarily expressed in hemocytes and the intestine.

  • Proteomic analysis of Eriocheir sinensis hemocytes in response to hypoxia stress

    2021, Aquaculture Reports
    Citation Excerpt :

    Once pathogens recognized by host cell receptor (LGBP or BGBP), the proPO system would converts proPO into the active form (PO) and then help the host cell eliminate the invading pathogen (Vargas-Albores and Yepiz-Plascencia, 2000). The proPO system of E. sinensis participated in the host cell against many pathogens infection, such as Vibrio anguillarum, Aeromonas hydrophila, Vibro alginolyticus and Spiroplasma eriocheiris (Jia et al., 2018; Hou et al., 2020; Cao et al., 2020). Except pathogens could activate the E. sinensis proPO system, hypoxia stress also could significantly increase the crab hemocytes PO activity (Song et al., 2021).

View full text