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Heterologous overexpression of active hexokinases from microsporidia Nosema bombycis and Nosema ceranae confirms their ability to phosphorylate host glucose

  • Viacheslav V. DolgikhEmail author
  • Alexander A. Tsarev
  • Sergey A. Timofeev
  • Vladimir S. Zhuravlyov
Immunology and Host-Parasite Interactions - Original Paper

Abstract

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5–9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and kcat 1726 min−1 for glucose, and Km 0.39 ± 0.05 mM and kcat 1976 min−1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and kcat 3293 min−1 for glucose, and Km 1.15 ± 0.11 mM and kcat 3732 min−1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.

Keywords

Microsporidia Nosema bombycis Nosema ceranae Hexokinase Heterologous expression Enzyme assay 

Notes

Funding information

This work was supported by Russian Science Foundation (RSF 18-16-00054) and Russian Foundation of Basic Research (RFBR 18-34-00265mol_a).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

References

  1. Antoine M, Boutin JA, Ferry G (2009) Binding kinetics of glucose and allosteric activators to human glucokinase reveal multiple conformational states. Biochemistry 48:5466–5482CrossRefGoogle Scholar
  2. Ardehali H, Yano Y, Printz RL, Koch S, Whitesell RR, May JM, Granner DK (1996) Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves. J Biol Chem 271:1849–1852CrossRefGoogle Scholar
  3. Bradford M (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Cáceres AJ, Portillo R, Acosta H, Rosales D, Quiñones W, Avilan L, Salazar L, Dubourdieu M, Michels PA, Concepción JL (2003) Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi. Mol Biochem Parasitol 126:251–262CrossRefGoogle Scholar
  5. Chen T, Ning D, Sun H, Li R, Shang M, Li X, Wang X, Chen W, Liang C, Li W, Mao Q, Li Y, Deng C, Wang L, Wu Z, Huang Y, Xu J, Yu X (2014) Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme. PLoS One 9:e107940CrossRefGoogle Scholar
  6. Claeyssen E, Wally O, Matton DP, Morse D, Rivoal J (2006) Cloning, expression, purification, and properties of a putative plasma membrane hexokinase from Solanum chacoense. Protein Expr Purif 47:329–339CrossRefGoogle Scholar
  7. Cuomo CA, Desjardins CA, Bakowski MA, Goldberg J, Ma AT, Becnel JJ, Didier ES, Fan L, Heiman DI, Levin JZ, Young S, Zeng Q, Troemel ER (2012) Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Res 22:2478–2488CrossRefGoogle Scholar
  8. Dean P, Sendra KM, Williams TA, Watson AK, Major P, Nakjang S, Kozhevnikova E, Goldberg AV, Kunji ERS, Hirt RP, Embley TM (2018) Transporter gene acquisition and innovation in the evolution of microsporidia intracellular parasites. Nat Commun 9:1709CrossRefGoogle Scholar
  9. Didier ES, Weiss LM (2008) Overview of microsporidia and microsporidiosis. Protistology 5:243–255Google Scholar
  10. Doehlert DC (1989) Separation and characterization of four hexose kinases from developing maize kernels. Plant Physiol 89:1042–1048CrossRefGoogle Scholar
  11. Dolgikh VV (2000) Activities of enzymes of carbohydrate and energy metabolism of the intracellular stages of the microsporidian, Nosema grylli. Protistology 1:87–91Google Scholar
  12. Dolgikh VV, Sokolova JJ, Issi IV (1997) Activities of enzymes of carbohydrate and energy metabolism of the spores of the microsporidian, Nosema grylli. J Eukaryot Microbiol 44:246–249CrossRefGoogle Scholar
  13. Ferguson S, Lucocq J (2018) The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases. Microbiologyopen 27:e00696CrossRefGoogle Scholar
  14. Harris MT, Walker DM, Drew ME, Mitchell WG, Dao K, Schroeder CE, Flaherty DP, Weiner WS, Golden JE, Morris JC (2013) Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase. Antimicrob Agents Chemother 57:3731–3737CrossRefGoogle Scholar
  15. Heinz E, Hacker C, Dean P, Mifsud J, Goldberg AV, Williams TA, Nakjang S, Gregory A, Hirt RP, Lucocq JM (2014) Plasma membrane-located purine nucleotide transport proteins are key components for host exploitation by microsporidian intracellular parasites. PLoS Pathog 10:e1004547CrossRefGoogle Scholar
  16. Huang Y, Zheng S, Mei X, Yu B, Sun B, Li B, Wei J, Chen J, Li T, Pan G, Zhou Z, Li C (2018) A secretory hexokinase plays an active role in the proliferation of Nosema bombycis. PeerJ 6:e5658CrossRefGoogle Scholar
  17. Katinka MD, Duprat S, Cornillot E, Méténier G, Thomarat F, Prensier G, Barbe V, Peyretaillade E, Brottier P, Wincker P, Delbac F, El Alaoui H, Peyret P, Saurin W, Gouy M, Weissenbach J, Vivarès CP (2001) Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 414:450–453CrossRefGoogle Scholar
  18. Kopetzki E, Entian KD (1985) Glucose repression and hexokinase isoenzymes in yeast. Isolation and characterization of a modified hexokinase PII isoenzyme. Eur J Biochem 146:657–662CrossRefGoogle Scholar
  19. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298CrossRefGoogle Scholar
  20. Moore B, Zhou L, Rolland F, Hall Q, Cheng WH, Liu YX, Hwang I, Jones T, Sheen J (2003) Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 300:332–336CrossRefGoogle Scholar
  21. Moreno F, Herrero P (2002) The hexokinase 2-dependent glucose signal transduction pathway of Saccharomyces cerevisiae. FEMS Microbiol Rev 26:83–90CrossRefGoogle Scholar
  22. Morris MT, DeBruin C, Yang Z, Chambers JW, Smith KS, Morris JC (2006) Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-amino-acid C-terminal tail. Eukaryot Cell 5:2014–2023CrossRefGoogle Scholar
  23. Neary CL, Pastorino JG (2010) Nucleocytoplasmic shuttling of hexokinase II in a cancer cell. Biochem Biophys Res Commun 394:1075–1081CrossRefGoogle Scholar
  24. Nwagwu M, Opperdoes FR (1982) Regulation of glycolysis in Trypanosoma brucei: hexokinase and phosphofructokinase activity. Acta Trop 39:61–72Google Scholar
  25. Petit T, Diderich JA, Kruckeberg AL, Gancedo C, Van Dam K (2000) Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae. J Bacteriol 182:6815–6818CrossRefGoogle Scholar
  26. Pino MF, Kim KA, Shelton KD, Lindner J, Odili S, Li C, Collins HW, Shiota M, Matschinsky FM, Magnuson MA (2007) Glucokinase thermolability and hepatic regulatory protein binding are essential factors for predicting the blood glucose phenotype of missense mutations. J Biol Chem 282:13906–13916CrossRefGoogle Scholar
  27. Racagni GE, Machado de Domenech EE (1983) Characterization of Trypanosoma cruzi hexokinase. Mol Biochem Parasitol 9:181–188CrossRefGoogle Scholar
  28. Reinke AW, Balla KM, Bennett EJ, Troemel ER (2017) Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins. Nat Commun 8:14023CrossRefGoogle Scholar
  29. Roth EF Jr (1987) Malarial parasite hexokinase and hexokinase-dependent glutathione reduction in the Plasmodium falciparum-infected human erythrocyte. J Biol Chem 262:15678–15682Google Scholar
  30. Saito T, Maeda T, Nakazawa M, Takeuchi T, Nozaki T, Asai T (2002) Characterisation of hexokinase in Toxoplasma gondii tachyzoites. Int J Parasitol 32:961–967CrossRefGoogle Scholar
  31. Schmiedl A, Breitling F, Winter C, Queitsch I, Dübel S (2000) Effect of engineered cysteines on yield, solubility and activity in various recombinant antibody formats expressed in E. coli. J Immunol Methods 242:101–114CrossRefGoogle Scholar
  32. Senderskiy IV, Timofeev SA, Seliverstova EV, Pavlova OA, Dolgikh VV (2014) Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes. PLoS One 9:e93585CrossRefGoogle Scholar
  33. Simon LM, Nagy M, Ábrahám M, Boross L (1985) Comparative studies on soluble and immobilized yeast hexokinase. Enzym Microb Technol 7:275–278CrossRefGoogle Scholar
  34. Sols A, De La Fuente G, Villarpalasi C, Asensio C (1958) Substrate specificity and some other properties of baker’s yeast hexokinase. Biochim Biophys Acta 30:92–101CrossRefGoogle Scholar
  35. Sun M, Liao S, Zhang L, Wu C, Qi N, Lv M, Li J, Lin X, Zhang J, Xie M, Zhu G, Cai J (2016) Molecular and biochemical characterization of Eimeria tenella hexokinase. Parasitol Res 115:3425–3433CrossRefGoogle Scholar
  36. Tokarev YS, Timofeev SA, Malysh JM, Tsarev AA, Ignatieva AN, Tomilova OG, Dolgikh VV (2018) Hexokinase as a versatile molecular genetic marker for microsporidia. Parasitology 15:1–7.  https://doi.org/10.1017/S0031182018001737 CrossRefGoogle Scholar
  37. Tsaousis AD, Kunji ER, Goldberg AV, Lucocq JM, Hirt RP, Embley TM (2008) A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi. Nature 453:553–556CrossRefGoogle Scholar
  38. Vossbrinck CR, Debrunner-Vossbrinck BA (2005) Molecular phylogeny of the microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitol 52:131–142CrossRefGoogle Scholar
  39. Williams BA, Elliot C, Burri L, Kido Y, Kita K, Moore AL, Keeling PJ (2010) A broad distribution of the alternative oxidase in microsporidian parasites. PLoS Pathog 6:e1000761CrossRefGoogle Scholar
  40. Wiredu BD, Jaroenlak P, Prachumwat A, Williams TA, Bateman KS, Itsathitphaisarn O, Sritunyalucksana K, Paszkiewicz KH, Moore KA, Stentiford GD, Williams BAP (2017) Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia. Environ Microbiol 19:2077–2089CrossRefGoogle Scholar
  41. Yu Y, Zhang H, Guo F, Sun M, Zhu G (2014) A unique hexokinase in Cryptosporidium parvum, an apicomplexan pathogen lacking the Krebs cycle and oxidative phosphorylation. Protist 165:701–714CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Laboratory of Microbiological ControlAll-Russian Institute for Plant ProtectionPushkinRussia

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