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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