Elsevier

Fish & Shellfish Immunology

Volume 80, September 2018, Pages 155-164
Fish & Shellfish Immunology

Full length article
Identification of a crustacean β-1,3-glucanase related protein as a pattern recognition protein in antibacterial response

https://doi.org/10.1016/j.fsi.2018.06.004Get rights and content

Highlights

  • A β-1,3-glucanase related protein (PcBGRP) was identified in Procambarus clarkii.

  • PcBGRP expression was induced by carbohydrates and bacteria challenge.

  • PcBGRP protected host from Aeromonas hydrophila infection.

  • PcBGRP recognized microbes and initiated the prophenoloxidase activating system.

Abstract

Prophenoloxidase (proPO) activating system is an important immune response for arthropods. β-1, 3-glucanase related protein (previously named as lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) in crustaceans) is a typical pattern recognition receptor family involved in the proPO activation by recognizing the invading microbes. In this study, we pay special attention to a bacteria-induced β-1,3-glucanase related protein from red swamp crayfish Procambarus clarkii, an important aquaculture specie in China. This protein, designated PcBGRP, was found a typical member of crustacean BGRP family with the glucanase-related domain and the characteristic motifs. PcBGRP was expressed in hemcoyes and hepatopancreas, and its expression could be induced by the carbohydrate and bacteria stimulants. The induction by lipopolysaccharide (LPS) and β-1,3-glucan (βG) was more significant than by peptidoglycan (PG). The response of PcBGRP to the native Gram-negative bacterial pathogen Aeromonas hydrophila was more obvious than to Gram-positive bacteria. Using RNA interference and recombinant protein, PcBGRP was found to protect crayfish from A. hydrophila infection revealed by the survival test and morphological analysis. A mechanism study found PcBGRP could bind LPS and βG in a dose-dependent manner, and the LPS recognizing ability determined the Gram-negative bacterium binding activity of PcBGRP. PcBGRP was found to enhance the PO activation both in vitro and in vivo, and the protective role was related to the PO activating ability of PcBGRP. This study emphasized the role of BGRP family in crustacean immune response, and provided new insight to the immunity of red swamp crayfish which suffered serious disease during the aquaculture in China.

Introduction

Pattern recognition receptors (PRRs) are essential for innate immunity, especially in invertebrates which do not have adaptive immunity and rely only on innate immunity to resist pathogen invasion [1]. Emerging evidences suggest the higher abundance of typical PRRs in invertebrates than in vertebrates [2]. These PRRs recognizes the conserved pathogen associated molecular patterns (PAMPs) exposed on the surface of microbes, mostly carbohydrate-related molecules, such as lipopolysaccharide (LPS) from Gram-negative bacteria, peptidoglycan (PG) from both Gram-negative and Gram-positive bacteria, and β-1,3-glucan (βG) from fungi. The recognition of pathogens led to the activation of innate immune responses, including the cell-mediated responses such as phagocytosis, encapsulation, nodule formation, and the humoral responses including the production of antimicrobial peptides, melanization, and so on [3,4].

Melanization, is a key immune response in invertebrates. It followed the activation of the prophenoloxidase (proPO) activating system. When the recognition of invading pathogens was accomplished by PRRs, a serial serine protease cascade would be initiated leading to the final activation of proPO activating enzyme which could specifically cleavage the inactive PO from zymogen to active form. The activated PO then catalyze two reactions to convert monophenol to o-diphenol, and then to o-quinone finally. These compounds could themselves converted into highly active othroquinone which would polymerizes into melanin. The final products and intermediates are harmful to the pathogens [5,6]. Deposition of melanin on the pathogen surface also associates with cellular responses by aiding hemocytes phagocytosis and encapsulation [7].

β-1,3-glucanase related protein (BGRP) is a typical PRR family which have been identified widely from insects and crustaceans. The member of BGRP family were differently named based on the different recognizing properties, such as lipopolysaccharide and β-1,3-glucan binding protein (LGBP), β-1,3-glucan binding protein (BGBP), Gram-negative bacteria binding protein (GNBP), and so on. A conserved region of this family is a β-glucanase like domain (GLU domain) which retain the ligand binding ability [8]. Insect BGRPs usually contain an N-terminal cysteine-rich domain which can strongly binds to pathogen-originated carbohydrates. However, the crustacean BGRPs usually contain the GLU domain only. In addition, the crustacean proteins usually possess the cell adhesion motif which may be important for the interaction with cell surface.

BGRP family was found essential for proPO activation in decapod crustaceans. The LGBP from freshwater crayfish Pacifastacus leniusculus was the first identified BGRP family member in crustaceans. It was purified from hemocytes, and was found to bind lipopolysaccharide, laminarin and curdlan, and to activate the proPO activating system [9]. The function of LGBP from the black tiger shrimp Penaeus monodon was revealed using the recombinant protein. It was found to share a similar binding spectrum and activating ability to the freshwater crayfish orthrolog [10]. Recently, a LGBP from Chinese mitten crab Eriocheir sinensis was found to bind microorganisms including bacteria and fungi in addition to the carbohydrate ligands. Knockdown of its expression led to higher mortality rate and more bacterial counts than the control group after artificial infection [11]. The binding ability, expression profiles, immune functions of crustacean BGRP family were also studied in Marsupenaeus japonicus, Fennerepenaeus chinensis, and F. indicus [[12], [13], [14], [15]].

We observed the inducible expression of a BGRP family member when analyzing the transcriptomic sequencing data of the red swamp crayfish tissues which were infected by Aeromonas hydrophila, a native pathogen of red swamp crayfish. The full length sequence of this protein which was designated as PcBRGP was obtained for bioinformatics analysis to reveal the sequence characteristics. Recombinant protein was generated to study the function both in vitro and in vivo. RNA interference by double strand RNA (dsRNA) injection was also performed to uncover the role of PcBRGP. The possible mechanism how PcBGRP was involved in the antibacterial response was also investigated. Results showed that PcBGRP was essential for the crayfish immune response against the native Gram-negative pathogen. This study provided new insight into the antibacterial immunity of red swamp crayfish, which is becoming an important aquaculture specie of great economic value but suffers serious disease during the aquaculture at the same time in China.

Section snippets

Animals and reagents

Healthy red swamp crayfish with the weight about 5 g were purchased from a market in Jinan, Shandong, China, cultured in laboratory at 16 °C, and fed with shrimp daily. LPS from Escherichia coli O55:B5, PG from Staphylococcus aureus, and βG from Saccharomyces cerevisiae were purchased from Sigma. l-DOPA was purchased from Kayon (Shanghai, China). The bacterial strains, including S. aureus AS1.89, Micrococcus luteus SDMCC210058, E. coli DH5α, Pseudomonas aeruginosa, Vibrio anguillarum

PcBGRP was identified as a new member of crustacean BGRP family

The open reading frame of PcBGRP was 1107 bp long, encoding a polypeptide of 368 amino acid residues. The signal peptide consisted of 16 residues, while the mature peptide consisted of 342 residues. The predicted molecular mass and isoelectric point of the mature peptide were 40.3 kDa and 4.49, respectively. A typical glycoside hydrolase family 16 domain (GLU domain) was found from the 81st and the 274th residues by online SMART prediction. Two putative integrin recognition motif (RGD) from the

Discussion

BGRP family have been proven as an important kind of PRRs in invertebrates, especially in crustaceans and insects [9,17,18]. The members of this family were diversely designated GNBP, BGBP or LRBP due to their different binding abilities. A universal region of this family is a β-glucanase similar domain which is present from bacteria, fungi to metazoans. PcBGRP contained a typical β-glucanase like domain in which the two polysaccharides recognizing motifs (PsBM and βGRM) were conserved. This

Acknowledgement

This work was supported financially by the National Natural Science Foundation of China (Grant Nos. 31622058 & 31302217), the Young Scholars Program of Shandong University (YSPSDU, Grant No. 2015WLJH26) and the Fundamental Research Funds of Shandong University (Grant No. 2015TB018).

References (23)

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