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We did a comparative analysis of the small subunit ribosomal DNA (rDNA) for two species of Microsporidia, Semenovaia chironomi and Neoperezia chironomi, both parasites of Chironomus plumosus (Diptera, Chironomidae). These two microsporidial species have been described previously on the basis of light and electron microscopic studies. The former species is dimorphic, producing both single diplokaryotic spores and uninucleate spores in sporophorous vesicles (SPVs) in packets of 16, while the latter species is monomorphic, disporoblastic, producing only uninucleate spores in SPVs. Based on their life cycles, S. chironomi and N. chironomi were assigned to two different families, Burenellidae and Neopereziidae. However, molecular analysis shows 96.7% sequence similarity for the small subunit rDNA between these two species. Remarkable similarities of the spore ultrastructure (mainly of the extrusion apparatus) justify a transfer of S. chironomi to Neoperezia, establishing a new combination, Neoperezia semenovaiae. Neoperezia belongs to Clade V, Class Aquasporidia sensu Vossbrinck and Debrunner-Vossbrinck (2005), and is in its spore ultrastructure similar to its closest relatives, namely Bryonosema, Schroedera, Pseudonosema, Trichonosema and Janacekia. We therefore conclude that similarities in spore ultrastructure reflect the phylogenetic relatedness of these Microsporidia, as opposed to the strikingly diverse life cycles., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, gammaCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.

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. [ABSTRACT FROM AUTHOR]

Microsporidia, a large group of fungi-related intracellular parasites, are characterized by drastically reduced metabolism. They possess genes encoding glycolysis components, and the glycerol–phosphate shuttle, but lack mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except α and β subunits of E1 enzyme of pyruvate dehydrogenase (PDH) complex. Here, we have expressed PDH subunits from the microsporidum Paranosema ( Antonospora) locustae in Escherichia coli. Western blot analysis with antibodies raised against recombinant proteins has revealed their specific accumulation in mature spores of P. locustae but not in the intracellular development stages. Two subunits were coprecipitated as a single heterooligomeric complex by anti-α or anti-β PDH antibodies. Ultracentrifugation of spore homogenate has shown the presence of PDH in the soluble fraction. Relocalization of the mitochondrial protein in microsporidial spore cytoplasm was confirmed by immunoelectron microscopy of ultrathin cryosections with affinity-purified anti-α PDH antibodies. On cryosections, parasite enzyme was found partly associated with the cytoplasmic side of ER and other intraspore membranes, suggesting that electrons might be transferred to any membrane acceptor and finally to oxygen in the parasite cell. [ABSTRACT FROM AUTHOR]

[Display omitted] • scFv library against microsporidia Vairimorpha ceranae hexokinase was constructed. • representativeness of constructed mouse immune library is 5 million transformants. • scFv-antibody inhibiting V. ceranae growth was selected by the library panning. • V. ceranae growth in Sf9 cell culture was confirmed by reverse transcription PCR. Secretion of hexokinase (HK) by microsporidia into infected cells suggests an important role for this enzyme for the intracellular development of parasites. To verify whether the expression of HK-specific antibodies in the host cell cytoplasm can suppress the growth of microsporidia, we constructed an immune library of recombinant scFv fragments against the enzyme of the honey bee pathogen Vairimorpha (Nosema) ceranae (Vc HK) with a representativeness of about 5 million bacterial transformants. Two variants of Vc HK-specific recombinant antibodies were selected by library panning and expressed in lepidopteran Sf9 cell line. Infecting of cells expressing two selected and control scFv fragments with V. ceranae spores was followed by their cultivation for 4 days. Analysis of parasite β-tubulin as well as spore wall protein SWP32 transcripts in infected cultures by reverse transcription PCR and real-time qPCR showed (1) V. ceranae growth in cells heterologous to bee pathogens, (2) its inhibition by one of the selected Vc HK-specific recombinant antibodies. The latter result once again emphasizes an important role of microsporidia hexokinases in their relationships with infected host cells and suggests further focusing on the mechanisms of such suppression, as well as on the search for new V. ceranae - inhibiting scFv fragments. [ABSTRACT FROM AUTHOR]