Elsevier

Fish & Shellfish Immunology

Volume 84, January 2019, Pages 341-351
Fish & Shellfish Immunology

Full length article
Expression of Macrobrachium rosenbergii lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) in Saccharomyces cerevisiae and evaluation of its immune function

https://doi.org/10.1016/j.fsi.2018.07.045Get rights and content

Highlights

  • M. rosenbergii LGBP was expressed in S. cerevisiae and its MV was about 45 kDa.

  • MIC and MBC of MrLGBP were ranged from 0.340 to 0.802 and 1.189 to 1.810.

  • MrLGBP can agglutinate one Gram-positive and four Gram-negative bacteria species.

  • MrLGBP enhanced enzyme activity and immunity genes expression level of prawns.

  • MrLGBP may improve the immunity of M. rosenbergii to resist V. parahaemolyticus.

Abstract

Pattern recognition proteins (PRPs) activate the innate immune system in invertebrates, and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) is an important PRP with various biological functions. Here, the open reading frame (ORF) of Macrobrachium rosenbergii LGBP (MrLGBP) was cloned into plasmid vector pHAC181, then integrated into downstream of the GAL1 promoter of Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination, followed by its expression in the yeast eukaryotic system. The resulting recombinant LGBP contained a 3 × HA-tag at its C terminus and had a molecular weight of about 45 kDa, as evaluated by western blot analysis. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were ranged from 0.340 to 0.802 and 1.189–1.810 μM, respectively. The recombinant MrLGBP protein agglutinated almost all tested bacteria except Bacillus thuringiensis and Staphylococcus aureus. These results revealed that this recombinant protein exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria. M. rosenbergii prawns were fed with the recombinant yeast strain MrLGBP for 1 month and challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. These prawns showed lower mortality and higher enzymatic activity and expression levels of immunity genes than did the control groups. All these results suggest that MrLGBP may play important roles in the innate immunity of crustaceans, and recombinant strain S. cerevisiae MrLGBP may be useful for the development of an effective immune feed additive in the future.

Introduction

The giant freshwater prawn, Macrobrachium rosenbergii, has commercial value and has been commercially cultured in China and other Southeast Asian countries [1,2]. Nonetheless, prawn culture has been restricted, owing to epidemic infectious diseases such as white tail disease (WTD), extra small virus (XSV) infection [3,4], and spiroplasma disease caused by a novel pathogen, spiroplasma MR-1008 [5,6]. As a consequence, there is a huge business loss in the local economy. Therefore, research on the immunity of this prawn is desirable for control of diseases and to ensure long-term survival of the crustacean culture industry.

Crustaceans, like other invertebrates, lack the adaptive immune system and rely entirely on their innate immune responses for protection against invading pathogens [7]. Pattern recognition proteins (PRPs) perform important functions in the innate immune response by recognizing cell wall components of pathogens, such as β-1,3-glucans (BGs), lipopolysaccharides (LPSs), and peptidoglycans (PGs), as non-self molecules and trigger cellular and humoral responses [8]. In crustaceans, several PRPs, including β-1,3-glucan–binding protein (BGBP), LPS-binding protein (LBP), PG-binding protein (PGBP), and LPS- and BG-binding protein (LGBP), which recognize and respond to microbial intruders, are reported to be involved in the activation of the prophenoloxidase (proPO) system [9]. In recent years, many of these PRPs have been studied. For instance, complementary (c) DNA of BGBP from white shrimp Litopenaeus vannamei has been cloned and characterized [10]. In addition, LGBP cDNA from the freshwater crayfish Pacifastacus leniusculus has been cloned and characterized [8], as have cDNAs from tiger shrimp Penaeus monodon [11], kuruma shrimp Marsupenaeus japonicus [12], blue shrimp Litopenaeus stylirostris [13], white shrimp L. vannamei [14], fleshy prawn Fenneropenaeus chinensis [15], and M. rosenbergii [16]. Nevertheless, studies on the expression and functions of PRPs in crustaceans are limited. Amparyup [17] has reported that PmLGBP functions as a PRP for LPS and BG in the proPO activation system of P. monodon. Chen [18] has found that LGBP binds to seaweed polysaccharides and activates the proPO system in the white shrimp L. vannamei, whereas Chaosomboon recently reported the LGBP protein from Fenneropenaeus merguiensis and its function as a pattern recognition receptor along with its broad specificity for diverse pathogens [19]. All these proteins have been mainly expressed in an Escherichia coli prokaryotic system; the use of a yeast system for protein expression is relatively rare.

In comparison with E. coli, yeast has an advanced heterologous-protein–folding pathway. In addition, the yeast signal sequence allows yeast cells to secrete proteins that are properly folded and processed. With the broad application of the industrial fermentation technology, yeast species have shown great advantages in the expression of clinically and industrially important proteins [20,21]. As a generally regarded as safe (GRAS) organism, Saccharomyces cerevisiae is widely used in the food and beverage industry and has been successfully employed to express a variety of exogenous eukaryotic proteins such as a hepatitis B vaccine, human insulin, human granulocyte colony-stimulating factor, and a human blood vessel inhibitor [22]. On the other hand, few reports have described the application of an S. cerevisiae eukaryotic system to the expression of crustacean LGBP.

In this study, the important PRP-LGBP of M. rosenbergii (hereafter MrLGBP), was expressed in S. cerevisiae system. The open reading frame (ORF) of the MrLGBP gene was cloned into plasmid vector pHAC181, and the recombinant plasmid was integrated into downstream of the GAL1 promoter of S. cerevisiae strain GAL1-ScRCH1 using a homologous recombination technique. By means of d-galactose, the target protein was expressed in yeast cells, and tentative immune functions of the recombinant protein as well as its effect on the immunity of M. rosenbergii were determined. This study is intended to provide useful information and experimental basis for crustacean innate immunity to prevent and control aquatic diseases.

Section snippets

Experimental animals, microbial strains, and media

Healthy M. rosenbergii prawns (average body weight of 20 g) were obtained from a commercial farm in Wuxi, Jiangsu province of China. The prawns were maintained in flat-bottomed glass tanks (200 L) with aerated and filtered freshwater at 28 ± 1 °C in the laboratory. They were daily fed with a commercial prawn diet at a rate of 4% body weight for 2 weeks before experimentation.

Plasmid vector pHAC181 (constructed by our laboratory) was cultured in the Luria Bertani (LB) liquid medium (10 g/L

Cloning of MrLGBP cDNA and homologous recombination

The construction strategy of the S. cerevisiae expression system is described in Fig. 1A. Total RNA was extracted from M. rosenbergii hemolymph and amplified with an LGBP-specific primer pair. The amplified ORF of the target cDNA was 1098 bp (Fig. 1B) and encoded a predicted protein of 366 amino acid residues. After restriction enzyme digestion and T4 DNA ligase reaction, the cDNA fragment and vector pHAC181 were ligated and transfected into competent cells Trans1-T1. Positive transformants

Discussion

Invertebrates lack the ability to produce antibodies but have an efficient innate immune system to defend themselves against invading pathogens. PRPs perform an important function in invertebrates by activating the innate immune defense [8,9]. LGBP is one of the important PRPs that has various biological functions [33]. In this study, an S. cerevisiae eukaryotic system was used to express MrLGBP. M. rosenbergii LGBP cDNA ORF (1098 bp) was cloned into vector pHAC181, and the recombinant plasmid

Acknowledgements

This work was supported by grants from the Natural Science Foundation of Jiangsu province (Youth Fund) (No.BK20150147), Jiangsu provincial agricultural support project (No. BE2014306), the National Postdoctoral fund (No.2014M561571) and China Postdoctoral Science Fund Project (No. 2018M632255).

References (51)

  • M.S. Yeh et al.

    Molecular cloning and characterization of lipopolysaccharide-and β-1,3-glucan-binding protein from the giant freshwater prawn Macrobrachium rosenbergii and its transcription in relation to foreign material injection and the molt stage

    Fish Shellfish Immunol.

    (2009)
  • P.T. Amparyup et al.

    Pattern recognition protein binds to lipopolysaccharide and-β-1, 3-glucan and activates shrimp prophenoloxidase system

    J. Biol. Chem.

    (2012)
  • Y.Y. Chen et al.

    Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in white shrimp Litopenaeus vannamei

    Dev. Comp. Immunol.

    (2016)
  • A. Chaosomboon et al.

    Lipopolysaccharide-and β-1,3-glucan-binding protein from Fenneropenaeus merguiensis functions as a pattern recognition receptor with a broad specificity for diverse pathogens in the defense against microorganisms

    Dev. Comp. Immunol.

    (2017)
  • R. Verma et al.

    Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems

    J. Immunol.

    (1998)
  • G.P. Cereghino et al.

    Applications of yeast in biotechnology: protein production and genetic analysis

    Curr. Opin. Biotechnol.

    (1999)
  • A. Kapoor et al.

    Fungi biosorption-an alternative treatment option for heavy metal bearing wastewaters: a review

    Bioresour. Technol.

    (1995)
  • C.C. Liu et al.

    Function of an anti-lipopolysaccharide factor (ALF) isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection

    J. Invertebr. Pathol.

    (2014)
  • H. Yang et al.

    Purification and characterization of a calcium independent lectin (PjLec) from the haemolymph of the shrimp Penaeus japonicas

    Fish Shellfish Immunol.

    (2007)
  • H.P. Misra et al.

    The role of superoxide anion in the auto oxidation of epinephrine and a simple assay of superoxide dismutase

    J. Biol. Chem.

    (1972)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method

    Methods

    (2001)
  • P. Amparyup et al.

    Pattern recognition protein binds to lipopolysaccharide and beta-1, 3-glucan and activates shrimp prophenoloxidase system

    J. Biol. Chem.

    (2012)
  • D. Zhao et al.

    Molecular cloning and characterization of the lipopolysaccharide and beta-1, 3-glucan binding protein in Chinese mitten crab (Eriocheir sinensis)

    Comp. Biochem. Physiol. B

    (2009)
  • X. Zhang et al.

    Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) stimulates prophenoloxidase activating system in Chinese mitten crab (Eriocheir sinensis)

    Dev. Comp. Immunol.

    (2016)
  • A.I. Campa-Córdova et al.

    Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to b-glucan and sulphated polysaccharide

    Fish Shellfish Immunol.

    (2002)
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