Expression, localization and preliminary evaluation of diagnostic value of Echinococcus granulosus phosphoglycerate mutase

doi:10.12140/j.issn.1000-7423.2020.02.010

Abstract

Abstract:

Objective To clone and express Echinococcus granulosus phosphoglycerate mutase (EgPGAM), analyze its immunoreactivity and distribution in protoscoleces, hydatid cysts and adult worms, and preliminarily evaluate the diagnostic value.Methods Protoscolex RNA was extracted and reverse transcribed to cDNA, from which EgPGAM gene was amplified by PCR and sequenced. The physicochemical properties, conserved domains, antigen epitopes and homology of EgPGAM were analyzed by bioinformatics softwares. Mega 5.0 software was used to analyze the characteristics of the sequence, and the phylogenetic tree was constructed with the neighbor joining method. The EgPGAM gene was inserted into the pET32a plasmid, and transferred into Escherichia coli BL21(DE3) for induction of expression. The expressed proteins were purified by nickle column, and further analyzed by SDS-PAGE for verifying the expression products, and Western blotting for immunoreactivity. The distribution of EgPGAM in protoscoleces, hydatid cysts and adults was analyzed by immunofluorescence assay. The diagnostic value of EgPGAM was evaluated by indirect enzyme-linked immunosorbent assay(ELISA).Results The EgPGAM gene is 756 bp in length, encoding 251 amino acids. The encoded protein has a relative molecular weight Mr about 28 000, an isoelectric point of 8.29, containing no signal peptide or transmembrane region. EgPGAM protein contained 12 highly-conserved amino acid sites, 3 conserved catalytic sites, one substrate-binding site and 7 B epitopes. The EgPGAM had 99%, 94%, 78%, 57%, 45% and 4% amino-acid sequence homology with PGAMs of E. multilocularis, Taenia solium, Clonorchis sinensis, Caenorhabditis elegans, Homo sapiens and Ovis aries, respectively. SDS-PAGE showed that EgPGAM was highly expressed in E. coli BL21(DE3), mainly in soluble form. Western blotting showed reactions of EgPGAM with serum samples from E. granulosus-infected sheep. Immunofluorescence assay revealed that the EgPGAM was localized mainly on the tegument and hooks of protoscoleces, the germinal layer of the hydatid cyst and the cyst wall parenchyma of adults. The sensitivity and specificity of EgPGAM-based indirect ELISA were 55.6%(15/27) and 86.4% (89/103), respectively.Conclusion EgPGAM was widely distributed in the larvae and adult worms of E. granulosus. The recombinant EgPGAM had apparent immunoreactivity, but with low sensitivity in the indirect ELISA test. Therefore, EgPGAM is not a suitable candidate of diagnostic antigen for echinococcosis.

Key words: Echinococcus granulosus, Phosphoglycerate mutase, Prokaryotic expression, Immunofluorescence localization, Enzyme-linked immunosorbent assay

CLC Number:

R383.33

Hong-yu SONG, Yu-qing LIANG, Rui-qi HUA, Yuan SHI, Ai-guo YANG, Li GUO, Dong-bo YUAN, Yue XIE, Guang-you YANG. Expression, localization and preliminary evaluation of diagnostic value of Echinococcus granulosus phosphoglycerate mutase[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(2): 194-201.

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References 33

[1]	Rialch A, Raina OK, Tigga MN , et al. Evaluation of Echinococcus granulosus recombinant EgAgB8/1, EgAgB8/2 and EPC1 antigens in the diagnosis of cystic echinococcosis in buffaloes[J]. Vet Parasitol, 2018,252:29-34.
[2]	Deplazes P, Rinaldi L, Alvarez RCA , et al. Global distribution of alveolar and cystic echinococcosis[J]. Adv Parasitol, 2017,95:315-493.
[3]	Song XJ, Hu DD, Yang GY . Problems and challenges in prevention and control of animal hydatid disease[J]. Chin J Zoono, 2015,31(3):264-271. (in Chinese)
	( 宋星桔, 胡丹丹, 杨光友 . 动物棘球蚴病防控面临的问题与挑战[J]. 中国人兽共患病学报, 2015,31(3):264-271.)
[4]	Wu WP, Wang H, Wang Q , et al. A nationwide sampling survey on echinococcosis in China during 2012-2016[J]. Chin J Parasitol Parsite Dis, 2018,36(1):1-14. (in Chinese)
	( 伍卫平, 王虎, 王谦 , 等. 2012-2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(1):1-14.)
[5]	Xiao S, Feng J, Yao M . Effect of antihydatid drugs on carbohydrate metabolism of metacestode of Echinococcus granulosus[J]. Chin Med J, 1995,108(9):682-688.
[6]	Xiao SH, Feng JJ, Guo HF , et al. Effects of mebendazole on glucose, glycogen, lactic acid, and lactate dehydrogenase in Echinococcus granulosus cyst wall[J]. Acta Pharmacol Sin, 1993,14(1):42-45.
[7]	Wang N, Zhu H, Zhan JF , et al. Cloning, expression, characterization, and immunological properties of citrate synthase from Echinococcus granulosus[J]. Parasitol Res, 2019,118(6):1811-1820.
[8]	Wu MD, Yan M, Xu J , et al. Molecular characterization of triosephosphate isomerase from Echinococcus granulosus[J]. Parasitol Res, 2018,117(10):3169-3176.
[9]	Mcmanus DP, Smyth JD . Intermediary carbohydrate metabolism in protoscoleces of Echinococcus granulosus (horse and sheep strains) and Echinococcus multilocularis[J]. Parasitology, 1982,84(2):351-366.
[10]	Fothergill-Gilmore LA, Watson HC . Phosphoglycerate mutases[J]. Biochem Soc Trans, 1990,18(2):190-193.
[11]	Jedrzejas MJ . Structure, function, and evolution of phosphoglycerate mutases: comparison with fructose-2,6-bisphosphatase, acid phosphatase, and alkaline phosphatase[J]. Prog Biophys Mol Biol, 2000,73(2):263-287.
[12]	Fothergill-Gilmore LA, Watson HC . The phosphoglycerate mutases[J]. Adv Enzymol Relat Areas Mol Biol, 1989,62(1):227-313.
[13]	Fraser HI, Kvaratskhelia M, White MF . The two analogous phosphoglycerate mutases of Escherichia coli[J]. Febs Lett, 1999,455(3):344-348.
[14]	Wang F, Li RH, Yin GR . Research progress in phosphoglycerate mutase[J]. Chin J Schisto Control, 2012,24(3):353-357. (in Chinese)
	( 王芬, 李润花, 殷国荣 . 磷酸甘油酸变位酶研究进展[J]. 中国血吸虫病防治杂志, 2012,24(3):353-357.)
[15]	Zhang YH, Foster JM, Kumar S , et al. Cofactor-independent phosphoglycerate mutase has an essential role in Caenorhabditis elegans and is conserved in parasitic Nematodes[J]. J Biol Chem, 2004,279(35):37185-37190.
[16]	Song LX, Xu ZB, Yu XB , Molecular cloning and characterization of a phosphoglycerate mutase gene from Clonorchis sinensis[J]. Parasitol Res, 2007,101(3):709-714.
[17]	Zhou Y, Lin JJ, Yao LX , et al. Cloning, expressing and characterizing of a phosphoglycerate mutas gene of Schistosoma japonncum[J]. Chin J Biotechnol, 2008,24(9):1550-1555. (in Chinese)
	( 周岩, 林矫矫, 姚利晓 , 等. 日本血吸虫磷酸甘油酸变位酶SjPGAM基因的克隆、表达及功能分析[J]. 生物工程学报, 2008,24(9):1550-1555.)
[18]	Blader IJ, Manger ID, Boothroyd JC . Microarray analysis reveals previously unknown changes in Toxoplasma gondii-infected human cells[J]. J Biol Chem, 2001,276(26):24223-24231.
[19]	Margaret OA, Craig B , Anette R. cDNA microarray analysis of host-pathogen interactions in a porcine in vitro model for Toxoplasma gondii infection[J]. Infect Immun, 2006,74(7):4254-4265.
[20]	Djikeng A, Raverdy S, Foster J , et al. Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei[J]. Parasitol Res, 2007,100(4):887-892.
[21]	Wiederschain GY . The ELISA guidebook[J]. Biochemistry Moscow, 2009,74(9):1058.
[22]	Huang X, Chen L, Yang YD , et al. Expression, tissue localization and serodiagnostic potential of Taenia multiceps acidic ribosomal protein P2[J]. Parasite Vector, 2015,8(1):613.
[23]	Li CY, Lan JC, Luo L , et al. Characterisation and preliminary evaluation diagnostic value of a serpin from Dirofilaria immitis[J]. Chin J Prev Vet Med, 2019,41(1):52-58. (in Chinese)
	( 李春燕, 兰景超, 罗娌 , 等. 犬恶丝虫丝氨酸蛋白酶抑制剂特性分析与诊断价值的初步评价[J]. 中国预防兽医学报, 2019,41(1):52-58.)
[24]	Teichmann A, Vargas DM, Monteiro KM , et al. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage[J]. J Proteome Res, 2015,14(4):1700-1715.
[25]	Zhang WB, Mcmanus DP . Recent advances in the immunology and diagnosis of echinococcosis[J]. Fems Immunol Med Microbiol, 2006,47(1):24-41.
[26]	Simsek S, Koroglu E . Evaluation of enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot for immunodiagnosis of hydatid diseases in sheep[J]. Acta Trop, 2004,92(1):17-24.
[27]	Jeyathilakan N, Basith SA, John L , et al. Development and evaluation of flow through technique for diagnosis of cystic echinococcosis in cattle[J]. Vet Arhiv, 2010,80(5):549-559.
[28]	Wu MD, Song XJ, Yan M , et al. Cloning, expression and preliminary evaluation diagnostic value of Echinococcus granulosus cystatin gene[J]. Chin J Animal Vet Sci, 2018,49(3):572-579. (in Chinese)
	( 吴茂迪, 宋星桔, 闫敏 , 等. 细粒棘球绦虫半胱氨酸蛋白酶抑制剂基因的克隆、表达及诊断价值的初步评价[J]. 畜牧兽医学报, 2018,49(3):572-579.)
[29]	Wu MD, Yan M, Xu J , et al. Expression, tissue localization and serodiagnostic potential of Echinococcus granulosus leucine aminopeptidase[J]. Int J Mol Sci, 2018,19(4):1063.
[30]	Wang N, Zhong XQ, Song XJ , et al. Molecular and biochemical characterization of calmodulin from Echinococcus granulosus[J]. Parasite Vector, 2017,10(1):597.
[31]	Song XJ, Hu DD, Yan M , et al. Molecular characteristics and serodiagnostic potential of dihydrofolate reductase from Echinococcus granulosus[J]. Sci Rep, 2017,7(1):514.
[32]	Song XJ, Yan M, Hu DD , et al. Molecular characterization and serodiagnostic potential of a novel dithiol glutaredoxin 1 from Echinococcus granulosus[J]. Parasite Vector, 2016,9(1):456.
[33]	Pan W, Shen YJ, Han XM , et al. Transcriptome profiles of the protoscoleces of Echinococcus granulosus reveal that excretory-secretory products are essential to metabolic adaptation[J]. PLoS Negl Trop Dis, 2014,8(12):e3392.