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

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 ± 1.2U/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 ± 0.01mM and kcat 1726min-1 for glucose, and Km 0.39 ± 0.05mM and kcat 1976min-1 for ATP, at pH8.8. N. ceranae HK showed Km 0.3 ± 0.04mM and kcat 3293min-1 for glucose, and Km 1.15 ± 0.11mM and kcat 3732min-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.