Treatment of Microsporidium Nosema bombycis Spores with the New Antiseptic M250 Helps to Avoid Bacterial and Fungal Contamination of Infected Cultures without Affecting Parasite Polar Tube Extrusion.

Microsporidia are a group of widespread eukaryotic spore-forming intracellular parasites of great economic and scientific importance. Since microsporidia cannot be cultured outside of a host cell, the search for new antimicrosporidian drugs requires an effective antiseptic to sterilize microsporidian spores to infect cell lines. Here, we show that a new polyhexamethylene guanidine derivative M250, which is active against fungi and bacteria at a concentration of 0.5-1 mg/L, is more than 1000 times less effective against spores of the microsporidium Nosema bombycis, a highly virulent pathogen of the silkworm Bombyx mori (LC50 is 0.173%). Treatment of N. bombycis spores that were isolated non-sterilely from silkworm caterpillars with 0.1% M250 solution does not reduce the rate of spore polar tube extrusion. However, it completely prevents contamination of the Sf-900 III cell culture medium by microorganisms in the presence of antibiotics. The addition of untreated spores to the medium results in contamination, whether antibiotics are present or not. Since 0.1% M250 does not affect spore discharging, this compound may be promising for preventing bacterial and fungal contamination of microsporidia-infected cell cultures.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth.

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite ß-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

Laboratory Cultivation of Vairimorpha (Nosema) ceranae (Microsporidia: Nosematidae) in Artificially Infected Worker Bees.

Nosemosis type C is a dangerous and widespread disease of the adult European honey bee Apis mellifera and is caused by the spore-forming intracellular parasite Vairimorpha (Nosema) ceranae. The search for new ways of therapy for this disease is complicated due to the seasonal availability of V. ceranae-infected insects as well as the lack of a developed system for the pathogen's cultivation. By carrying out trials which used different infectious dosages of the parasite, spore storage protocols, host age, and incubation temperatures, we present a simple, safe, and efficient method of V. ceranae propagation in artificially infected worker bees in the laboratory. The method is based on feeding the groups of adult worker bees with microsporidian spores and insect maintenance in plastic bottles at 33 °C. The source of the spores originated from the cadavers of infected insects from the previous round of cultivation, in which the infective spores persist for up to six months. An analysis of five independent cultivation rounds involving more than 2500 bees showed that the proposed protocol exploiting the dosage of one million spores per bee yielded over 60 million V. ceranae spores per bee, and most of the spore samples can be isolated from living insects.

Heterologous expression of scFv fragment against Vairimorpha (Nosema) ceranae hexokinase in Sf9 cell culture inhibits microsporidia intracellular growth.

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 (VcHK) with a representativeness of about 5 million bacterial transformants. Two variants of VcHK-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 ß-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 VcHK-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.

Construction and heterologous overexpression of two chimeric proteins carrying outer hydrophilic loops of Vairimorpha ceranae and Nosema bombycis ATP/ADP carriers.

Microsporidia Nosema bombycis and Vairimorpha ceranae cause destructive epizootics of honey bees and silkworms. Insufficient efficiency of the antibiotic fumagillin against V. ceranae, its toxicity and the absence of effective methods of N. bombycis treatment demand the discovery of novel strategies to suppress infections of domesticated insects. RNA interference is one such novel treatment strategy. Another one implies that the intracellular development of microsporidia may be suppressed by single-chain antibodies (scFv fragments) against functionally important parasite proteins. Important components of microsporidian metabolism are non-mitochondrial, plastidic-bacterial ATP/ADP carriers. These membrane transporters import host-derived ATP and provide the capacity to pathogens for energy parasitism. Here, we analyzed membrane topology of four V. ceranae and three N. bombycis ATP/ADP transporters to construct two fusion proteins carrying their outer hydrophilic loops contacting with infected host cell cytoplasm. Interestingly, full-size genes of N. bombycis transporters may be derived from the Asian swallowtail Papilio xuthus genome sequencing project. Synthesis of the artificial genes was followed by overexpression of recombinant proteins in E. coli as insoluble inclusion bodies. The gene fragments encoding the loops of individual transporters were also effectively expressed in bacteria. The chimeric antigens may be used to construct immune libraries or select microsporidia-suppressing scFv fragments from synthetic, semisynthetic, naïve and immune antibody libraries. A further expression of such antibodies in insect cells may increase their resistance to microsporidial infections.

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

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.

Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.

Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.

Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

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/ß-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/ß-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.

Immunolocalization of an alternative respiratory chain in Antonospora (Paranosema) locustae spores: mitosomes retain their role in microsporidial energy metabolism.

Microsporidia are a group of fungus-related intracellular parasites with severely reduced metabolic machinery. They lack canonical mitochondria, a Krebs cycle, and a respiratory chain but possess genes encoding glycolysis enzymes, a glycerol phosphate shuttle, and ATP/ADP carriers to import host ATP. The recent finding of alternative oxidase genes in two clades suggests that microsporidial mitosomes may retain an alternative respiratory pathway. We expressed the fragments of mitochondrial chaperone Hsp70 (mitHsp70), mitochondrial glycerol-3-phosphate dehydrogenase (mitG3PDH), and alternative oxidase (AOX) from the microsporidium Antonospora (Paranosema) locustae in Escherichia coli. Immunoblotting with antibodies against recombinant polypeptides demonstrated specific accumulation of both metabolic enzymes in A. locustae spores. At the same time comparable amounts of mitochondrial Hsp70 were found in spores and in stages of intracellular development as well. Immunoelectron microscopy of ultrathin cryosections of spores confirmed mitosomal localization of the studied proteins. Small amounts of enzymes of an alternative respiratory chain in merogonial and early sporogonial stages, alongside their accumulation in mature spores, suggest conspicuous changes in components and functions of mitosomes during the life cycle of microsporidia and the important role of these organelles in parasite energy metabolism, at least at the final stages of sporogenesis.

Heterologous expression of pyruvate dehydrogenase E1 subunits of the microsporidium Paranosema (Antonospora) locustae and immunolocalization of the mitochondrial protein in amitochondrial cells.

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 alpha and beta subunits of E(1) 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-alpha or anti-beta 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-alpha 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.

Evolution of the endoplasmic reticulum and the Golgi complex.

By analyzing the morpho-physiological features of the Golgi complex, its relationship with the endoplasmic reticulum in different species, and the molecular machineries involved in intracellular transport, we conclude that; (1) all eukaryotic cells have either Golgi complexes or remnants thereof; (2) all eukaryotic cells have a large minimal set of proteins that are involved in intracellular transport; and (3) several indispensable molecular machines are always present in secreting eukaryotic cells. Using this information, our data about mechanisms of intra-Golgi transport and phylogenetic analysis of several molecular machines, we propose a model for the evolution of the Golgi complex and the endoplasmic reticulum.

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function.

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.

Pyruvate-converting activity in the spores of the microsporidian genus Paranosema (Antonospora).

Microsporidia, a large group of fungi-related protozoa with an obligate intracellular lifestyle, are characterized by a drastically reduced cell machinery and a unique metabolism. These parasites possess genes encoding glycolysis components and glycerol-phosphate shuttle, but lack typical mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except for two subunits of the E(1) enzyme of the pyruvate dehydrogenase complex. This study demonstrates that in spite of the above, destroyed spores of the microsporidian Paranosema (Antonospora) grylli and P. locustae deplete pyruvate content in the incubation medium. This activity is sensitive to heat, proportionally distributed between the soluble and the insoluble fractions and does not depend on additional ions or cofactors.

Immunocytochemical identification of the major exospore protein and three polar-tube proteins of the microsporidia Paranosema grylli.

Microsporidia are intracellular eukaryotic parasites that can infect a wide range of animal hosts with several genera causing opportunistic infections in immunodeficient patients. Their spore wall and their unique extrusion apparatus, which has the form of a long polar tube, confer resistance of these parasites against the environment and during host-cell invasion. In contrast to parasites of vertebrates, the spore-wall and polar-tube proteins of many microsporidia species still remain to be characterized, even though a great number of microsporidia infect invertebrates. Here, we have identified one spore-wall protein and three polar-tube proteins of the microsporidia Paranosema grylli that infects the cricket Gryllus bimaculatus. Incubation of intact spores with an alkaline-saline solution resulted in the selective extraction of a major 40 kDa protein. A wash of the discharged (or destroyed) spores with SDS and the following solubilization of their polar tubes with 50-75% 2-mercaptoethanol extracted a major protein of ca. 56 kDa. When the polar tubes were solubilized in the presence of SDS, two additional proteins of 46 and 34 kDa were extracted. Antibodies specific for these extracted proteins were generated and isolated by incubation of immune sera with the protein bands that had been transferred to nitrocellulose. Western blotting demonstrated the cross-reactivity of the anti-p46 and anti-p34 antibodies. Immuno-electron microscopy with the anti-p40 antibody revealed specific decoration of the microsporidia exospore. The 56, 46 and 34 kDa proteins were characterized as polar-tube components due to the clear antibody labeling of the polar filament.