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Parasitology Research

, Volume 118, Issue 6, pp 1811–1820 | Cite as

Cloning, expression, characterization, and immunological properties of citrate synthase from Echinococcus granulosus

  • Ning Wang
  • Hui Zhu
  • Jiafei Zhan
  • Cheng Guo
  • Nengxing Shen
  • Xiaobin Gu
  • Weimin Lai
  • Yue Xie
  • Xuerong Peng
  • Guangyou YangEmail author
Helminthology - Original Paper
  • 214 Downloads

Abstract

The larval stages of the tapeworm Echinococcus granulosus (Cestoda: Taeniidae) are the causative agent of cystic echinococcosis, one of the most important parasitic zoonoses worldwide. E. granulosus has a complete pathway for the tricarboxylic acid cycle (TCA), in which citrate synthase (CS) is the key enzyme. Here, we cloned and expressed CS from E. granulosus (Eg-CS) and report its molecular characterization. The localization of this protein during different developmental stages and mRNA expression patterns during H2O2 treatment were determined. We found that Eg-CS is a highly conserved protein, consisting of 466 amino acids. In western blotting assays, recombinant Eg-CS (rEg-CS) reacted with E. granulosus-positive sheep sera and anti-rEg-CS rabbit sera, indicating that Eg-CS has good antigenicity and immunoreactivity. Localization studies, performed using immunohistochemistry, showed that Eg-CS is ubiquitously expressed in the larva, germinal layer, and adult worm sections of E. granulosus. Eg-CS mRNA expression levels increased following H2O2 exposure. In conclusion, citrate synthase might be involved in the metabolic process in E. granulosus. An assessment of the serodiagnostic potential of rEg-CS based on indirect ELISA showed that, although sensitivity (93.55%) and specificity (80.49%) are high, cross-reactivity with other parasites precludes its use as a diagnostic antigen.

Keywords

Echinococcus granulosus Citrate synthase Tricarboxylic acid cycle Immunohistochemical localization mRNA expression 

Notes

Acknowledgements

We are grateful to Chunyan Li, Hongyu Song, and Ruiqi Hua for their help and suggestions. We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.

Authors’ contributions

NW and GYY conceived and designed the study and critically revised the manuscript. JFZ, CG, NXS, XBG, and WML performed the experiments, analyzed the data, and drafted the manuscript. XRP and YX conducted the data analysis. HZ and GYY revised the manuscript. All authors have approved the final manuscript.

Financial support

This research was funded by the National Natural Science Foundation of China [grant number 31672547].

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics statement

All animals included in this study were purchased from the Laboratory Animal Center of Sichuan Agricultural University. All procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals and with approval from the Animal Ethics Committee of Sichuan Agricultural University (Ya’an, China; Approval No. 2013–028).

References

  1. Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ (2008) Ethanol catabolism in Corynebacterium glutamicum. J Mol Microbiol Biotechnol 15:222–233PubMedGoogle Scholar
  2. Bloxham DP, Parmelee DC, Kumar S, Wade RD, Ericsson LH, Neurath H, Walsh KA, Titani K (1981) Primary structure of porcine heart citrate synthase. Proc Natl Acad Sci USA 78:5381–5385PubMedGoogle Scholar
  3. Bloxham DP, Parmelee DC, Kumar S, Walsh KA, Titani K (1982) Complete amino acid sequence of porcine heart citrate synthase. Biochemistry 21:2028–2036PubMedGoogle Scholar
  4. Cabrera G, Cabrejos ME, Morassutti AL, Cabezón C, Orellana J, Hellman U, Zaha A, Galanti N (2008) DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts. J Cell Physiol 216:498–506PubMedGoogle Scholar
  5. Cheng TL, Liao CC, Tsai WH, Lin CC, Yeh CW, Teng CF, Chang WT (2009) Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase. J Cell Biochem 107:1002–1015PubMedGoogle Scholar
  6. Chinta SJ, Rane A, Yadava N, Andersen JK, Nicholls DG, Polster BM (2009) Reactive oxygen species regulation by AIF- and complex I-depleted brain mitochondria. Free Radic Biol Med 46:939–947PubMedPubMedCentralGoogle Scholar
  7. Deplazes P, Rinaldi L, Alvarez Rojas CA, Torgerson PR, Harandi MF, Romig T, Antolova D, Schurer JM, Lahmar S, Cringoli G (2017) Global distribution of alveolar and cystic echinococcosis. Adv Parasitol 95:315–493PubMedGoogle Scholar
  8. Eckert J, Gemmell MA, Meslin FX, Pawłowski ZS (2001) WHO/OIE manual on echinococcosis in humans and animals: a public health problem of global concern. Int J Parasitol 31:1717–1718Google Scholar
  9. Evans CT, Owens DD, Sumegi B, Kispal G, Srere PA (1988) Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase. Biochemistry 27:4680–4686PubMedGoogle Scholar
  10. Garcia HH, Del Brutto OH (2005) Neurocysticercosis: updated concepts about an old disease. Lancet Neurol 4:653–661PubMedGoogle Scholar
  11. Garcia HH, Moro PL, Schantz PM (2007) Zoonotic helminth infections of humans: echinococcosis, cysticercosis and fascioliasis. Curr Opin Infect Dis 20:489–494PubMedGoogle Scholar
  12. Giordano A, Calvani M, Petillo O, Grippo P, Tuccillo F, Melone MA, Bonelli P, Calarco A, Peluso G (2005) tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1. Cell Death Differ 12:603–613PubMedGoogle Scholar
  13. Guo ZH, Kubo M, Kudo M, Nibe K, Horii Y, Nonaka N (2011) Growth and genotypes of Echinococcus granulosus found in cattle imported from Australia and fattened in Japan. Parasitol Int 60:498–502PubMedGoogle Scholar
  14. Guo C, Wang Y, Huang X, Wang N, Yan M, He R, Gu XB, Xie Y, Lai WM, Jing B (2017) Molecular cloing and bioinformatics analysis of lactate dehydrogenase from Taenia multiceps. Parasitol Res 116:2845–2852PubMedPubMedCentralGoogle Scholar
  15. Hsieh CL, Huang CN, Lin YC, Peng RY (2007) Molecular action mechanism against apoptosis by aqueous extract from guava budding leaves elucidated with human umbilical vein endothelial cell (HUVEC) model. J Agric Food Chem 55:8523–8533PubMedGoogle Scholar
  16. Hu D, Song X, Xie Y, Zhong X, Wang N, Zheng Y, Gu XB, Wang T, Peng XR, Yang GY (2015) Molecular insights into a tetraspanin in the hydatid tapeworm Echinococcus granulosus. Parasit Vectors 8:311PubMedPubMedCentralGoogle Scholar
  17. Irshadullah M, Rani M (2011) Comparative studies on the biochemical composition and polypeptide profiles of the cyst walls from sterile and fertile hydatid cysts of Echinococcus granulosus from buffalo host. Helminthologia 48:88–93Google Scholar
  18. Jenkins D, Romig T, Thompson R (2005) Emergence/re-emergence of Echinococcus spp.—a global update. Int J Parasitol 35:1205–1219PubMedGoogle Scholar
  19. Khan AH, El-Buni AA, Ali MY (2001) Fertility of the cysts of Echinococcus granulosus in domestic herbivores from Benghazi, Libya, and the reactivity of antigens produced from them. Pathog Glob Health 95:337–342Google Scholar
  20. Leducq R, Gabrion C (1992) Developmental changes of Echinococcus multilocularis metacestodes revealed by tegumental ultrastructure and lectin-binding sites. Parasitol 104:129–141Google Scholar
  21. Lee SJ, Kwon CH, Kim YK (2009) Alterations in membrane transport function and cell viability induced by ATP depletion in primary cultured rabbit renal proximal tubular cells. Korean J Physiol Pha 13:15–22Google Scholar
  22. Li T, Ito A, Pengcuo R, Sako Y, Chen X, Qiu D, Xiao N, Craig PS (2011) Post-treatment follow-up study of abdominal cystic echinococcosis in tibetan communities of northwest Sichuan Province, China. PLoS Negl Trop Dis 5:e1364PubMedPubMedCentralGoogle Scholar
  23. Linossier MT, Dormois D, Perier C, Frey J, Geyssant A, Denis C (1997) Enzyme adaptations of human skeletal muscle during bicycle short-sprint training and detraining. Acta Physiol 161:439–445Google Scholar
  24. Liu Q, Yu L, Han XF, Fu Q, Zhang JX, Tang H, Zhao SY (2000) Cloning and tissue expression analysis of the human citrate synthase cDNA. Acta biol exp sin 33:207–214Google Scholar
  25. Maríngarcía J, Goldenthal MJ, Damle S, Pi Y, Moe GW (2009) Regional distribution of mitochondrial dysfunction and apoptotic remodeling in pacing-induced heart failure. J Card Fail 15:700–708Google Scholar
  26. Monteiro KM, de Carvalho MO, Zaha A, Ferreira HB (2010) Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host. Proteomics 10:1985–1999PubMedGoogle Scholar
  27. Orrenius S (2007) Reactive oxygen species in mitochondria-mediated cell death. Drug Metab Rev 39:443–455PubMedGoogle Scholar
  28. Paredes R, Godoy P, Rodríguez B, García MP, Cabezón C, Cabrera G, Jiménez V, Hellman U, Sáenz L, Ferreira A (2011) Bovine (Bos taurus) humoral immune response against Echinococcus granulosus and hydatid cyst infertility. J Cell Biochem 112:189–199PubMedGoogle Scholar
  29. Rana SV (2008) Metals and apoptosis: recent developments. J Trace Elem Med Biol 22:262–284PubMedGoogle Scholar
  30. Siu PM, Donley DA, Bryner RW, Alway SE (2003) Citrate synthase expression and enzyme activity after endurance training in cardiac and skeletal muscles. J Appl Physiol 94:555–560PubMedGoogle Scholar
  31. Smyth JD, Mcmanus DP (1989) The physiology and biochemistry of cestodes. Cambridge University Press, CambridgeGoogle Scholar
  32. Song XJ, Yan M, Hu DD, Wang Y, Wang N, Gu XB, Yang GY (2016) Molecular characterization and serodiagnostic potential of a novel dithiol glutaredoxin 1 from Echinococcus granulosus. Parasit Vectors 9(1):456PubMedPubMedCentralGoogle Scholar
  33. Song XJ, Hu DD, Yan M, Wang Y, Wang N, Gu XB, Yang GY (2017) Molecular characteristics and serodiagnostic potential of dihydrofolate reductase from Echinococcus granulosus. Sci Rep 7(1):514PubMedPubMedCentralGoogle Scholar
  34. Spotin A, Majdi MMA, Sankian M, Varasteh A (2012) The study of apoptotic bifunctional effects in relationship between host and parasite in cystic echinococcosis: a new approach to suppression and survival of hydatid cyst. Parasitol Res 110:1979–1984PubMedGoogle Scholar
  35. Suissa M, Suda K, Schatz G (1984) Isolation of the nuclear yeast genes for citrate synthase and fifteen other mitochondrial proteins by a new screening method. EMBO J 3:1773–1781PubMedPubMedCentralGoogle Scholar
  36. Thompson RA, McManus DP (2002) Towards a taxonomic revision of the genus Echinococcus. Trends Parasitol 18:452–457PubMedGoogle Scholar
  37. Torgerson PR, Macpherson CN (2011) The socioeconomic burden of parasitic zoonoses: global trends. Vet Parasitol 182:79–95PubMedGoogle Scholar
  38. Torgerson PR, Brecht D, Nicolas P, Niko S, Lee WA, Fumiko K, Rokni MB, Zhou XN, Fèvre EM, Banchob S (2015) World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: a data synthesis. PLoS Med 12:e1001920PubMedPubMedCentralGoogle Scholar
  39. Turčeková Ľ, Šnábel V, Dudiňák V, Gašpar V, Dubinský P (2009) Prevalence of cystic echinococcosis in pigs from Slovakia, with evaluation of size, fertility and number of hydatid cysts. Helminthologia 46:151–158Google Scholar
  40. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344PubMedPubMedCentralGoogle Scholar
  41. Wang N, Zhong X, Song X, Gu X, Lai W, Xie Y, Peng XR, Yang GY (2017) Molecular and biochemical characterization of calmodulin from Echinococcus granulosus. Parasit Vectors 10:597PubMedPubMedCentralGoogle Scholar
  42. Wang N, Zhan JF, Cheng G, Li CY, Shen NX, Gu XB, Xie Y, Peng XR, Yang GY (2018) Molecular characterisation and functions of Fis1 and Pdcd6 genes from Echinococcus granulosus. Int J Mol Sci 19(9):2669PubMedCentralGoogle Scholar
  43. Wiegand G, Remington SJ (1986) Citrate synthase: structure, control, and mechanism. Annu Rev Biophys Biophys Chem 15:97–117PubMedGoogle Scholar
  44. Wu MD, Yan M, Xu J, Liang YQ, Gu XB, Jing B, Lai WM, Peng XR, Yang GY (2018) Expression, tissue localization and serodiagnostic potential of Echinococcus granulosus leucine aminopeptidase. Int J Mol Sci 19(4):1063PubMedCentralGoogle Scholar
  45. Zheng H, Zhang W, Zhang L, Zhang Z, Li J, Lu G, Zhu YQ, Wang YZ, Huang Y, Liu J (2013) The genome of the hydatid tapeworm Echinococcus granulosus. Nat Genet 45:1168–1175PubMedPubMedCentralGoogle Scholar
  46. Zinsser VL, Moore CM, Hoey EM, Trudgett A, Timson DJ (2013) Citrate synthase from the liver fluke Fasciola hepatica. Parasitol Res 112:2413–2417PubMedGoogle Scholar
  47. Zischka H, Larochette N, Hoffmann F, Hamöller D, Jägemann N, Lichtmannegger J, Jennen L, Müllerhöcker J, Roggel F, Göttlicher M (2008) Electrophoretic analysis of the mitochondrial outer membrane rupture induced by permeability transition. Anal Chem 80:5051–5058PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Ning Wang
    • 1
    • 2
  • Hui Zhu
    • 2
  • Jiafei Zhan
    • 1
  • Cheng Guo
    • 1
  • Nengxing Shen
    • 1
  • Xiaobin Gu
    • 1
  • Weimin Lai
    • 1
  • Yue Xie
    • 1
  • Xuerong Peng
    • 3
  • Guangyou Yang
    • 1
    Email author
  1. 1.Department of Parasitology, College of Veterinary MedicineSichuan Agricultural UniversityChengduChina
  2. 2.College of BioengineeringSichuan University of Science and EngineeringZigongChina
  3. 3.Department of Chemistry, College of Life and Basic ScienceSichuan Agricultural UniversityYa’anChina

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