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.

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.

Structure-Activity Relationship of Phytotoxic Natural 10-Membered Lactones and Their Semisynthetic Derivatives.

Ten-membered lactones (nonenolides) demonstrate phytotoxic, antimicrobial, and fungicidal activity promising for the development of natural product-derived pesticides. The fungus Stagonospora cirsii is able to produce phytotoxic stagonolides A (1), J (2), K (3) and herbarumin I (4) with high yield. The aim of this study was to create a set of structurally related nonenolides and to reveal the structural features that affect their biological activity. Stagonolide A (1) and C-7 oxidized stagonolide K (11) showed the highest phytotoxicity in leaf puncture assay and agar seedlings assay. The oxidation of C-7 hydroxyl group (as in 1, acetylstagonolide A (10) and (11) led to the manifestation of toxicity to microalgae, Bacillus subtilis and Sf9 cells regardless of the configuration of C-9 propyl chains (R in 1 and 10, S in 11). C-7 non-oxidized nonenolides displayed none or little non-target activity. Notably, 7S compounds were more phytotoxic than their 7R analogues. Due to the high inhibitory activity against seedling growth and the lack of side toxicity, mono- and bis(acetyl)- derivatives of herbarumin I were shown to be potent for the development of pre-emergent herbicides. The identified structural features can be used for the rational design of new herbicides.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid.

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

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.

Antibodies raised against a Sunn bug (Eurygaster integriceps Put.) recombinant protease, rGHP3p2, can inhibit gluten-hydrolyzing activity.

Sunn pest or Sunn bug, Eurygaster integriceps Put., salivary gland proteases are responsible for the deterioration of wheat flour quality during dough mixing, resulting from gluten hydrolysis. These proteases are highly heterogeneous and show low sensitivity to most types of proteinaceous inhibitors, meaning that such inhibitors cannot be used to prevent gluten damage. The present study describes the generation of a specific peptide antibody, raised against the active center of the recombinant gluten-hydrolyzing protease (GHP3). The recombinant protein, encoding two repeats of the GHP3 sequence element involved in forming the S4 pocket and binding of substrate at position P4, was designed and expressed in Escherichia coli. The antibodies raised to this recombinant protein showed inhibitory activity against the GHP3 protease. The results indicate that it is possible to design specific antibodies to inhibit wheat-bug gluten-hydrolyzing proteases.

Changes in antifungal defence systems during the intermoult period in the Colorado potato beetle.

Susceptibility to the fungus Metarhizium robertsii and changes in host defences were evaluated in different stages of the intermoult period (4-6 h, 34-36 h and 84-86 h post moult in IV larval instars) of the Colorado potato beetle. A significant thickening of the cuticle during larval growth was accompanied by decreases in cuticle melanization, phenoloxidase activity and epicuticular hydrocarbon contents (C28-C32). At the same time, a decrease in the conidial adhesion rate and an increase in resistance to the fungus were observed. In addition, we recorded significant elevation of the encapsulation rate and total haemocyte counts in the haemolymph during the specified period. The activity of detoxification enzymes decreased in the haemolymph but increased in the fat body during larval growth. No significant differences in the fatty acid content in the epicuticle were observed. The role of developmental disorders in susceptibility to entomopathogenic fungi is also discussed.

Infection of Chorthippus loratus (Orthoptera: Acrididae) with Liebermannia sp. (Microsporidia) in South-Western Russia.

Chorthippus loratus collected in Krasnodar Territory in 2017 was infected at 15% rate with a microsporidium possessing ovocylindrical binucleate spores, 2.6 × 1.2 µm in size. SSU RNA gene typing (Genbank accession # MH396491) showed its allocation to the genus Liebermannia. Degenerate primers based upon largest subunit RNA polymerase II (RPB1) sequences of closest relatives allowed amplifying the respective gene fragment of Liebermannia sp. (# MH396492). The present finding indicates worldwide distribution of the Liebermannia genus and parasitism in hosts with nonoverlapping geographic ranges (representing Neotropical versus Palearctic fauna), while previous observations were restricted to Acridoidea endemic for South America.

Discovery of a novel microsporidium in laboratory colonies of Mediterranean cricket Gryllus bimaculatus (Orthoptera: Gryllidae): Microsporidium grylli sp. nov.

A microsporidium was found in a Mediterranean cricket Gryllus bimaculatus from a pet market in the UK and a lab stock at the Moscow Zoo (originating from London Zoo). The spores were ovoid, uninucleate, 6.3 × 3.7 µm in size (unfixed), in packets by of 8, 16, or 32. The spores were easily discharged upon dessication or slight mechanical pressure. The polar tube was isofilar, with 15-16 coils arranged in 1-2 rows. The polaroplast was composed of thin lamellae and occupied about one third of the spore volume. The endospore was 200 nm thick, thinning over the anchoring disc. The exospore was thin, uniform, and with no ornamentation. Phylogenetics based upon small subunit ribosomal RNA (Genbank accession # MG663123) and RNA polymerase II largest subunit (# MG664544) genes placed the parasite at the base of the Trachipleistophora/Vavraia lineage. The RPB1 locus was polymorphic but similar genetic structure and identical clones were found in both isolates, confirming their common geographic origin. Due to in insufficient ultrastructural data and prominent divergence from described species, the parasite is provisionally placed to the collective taxon Microsporidium.

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.

Interactions of two insect pathogens, Paranosema locustae (Protista: Microsporidia) and Metarhizium acridum (Fungi: Hypocreales), during a mixed infection of Locusta migratoria (Insecta: Orthoptera) nymphs.

Locusta migratoria nymphs were fed Paranosema locustae spores and/or surface-treated with Metarhizium acridum 3 (assay 1), 6 (assay 2) or 9 days (assay 3) post microsporidia application (p.m.a.). These three dates corresponded to the key phases of P. locustae development: (a) mass proliferation, (b) transition to sporogenesis and (c) onset of spore maturation, respectively. As a result, locust mortality due to mixed treatment increased slower, equally and faster, as compared to mortality expected from the combination of two pathogens in assays 1-3, respectively. However, a statistically significant difference in survival times was observed only in assay 3, indicating that only at the phase of spore maturation microsporidia drastically increase locust susceptibility to fungal infection. Analysis of perished nymphs showed that fungal treatment 3 days p.m.a. impeded development of microsporidia. Fungal sporulation on locust cadavers was not affected by co-occurring microsporidiosis.

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.

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.