Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

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Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3′-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

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