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


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.


Microsporidia Nosema bombycis Nosema ceranae Hexokinase Heterologous expression Enzyme assay 


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.


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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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