Treatment of Microsporidium 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.

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

Nosemosis type C is a dangerous and widespread disease of the adult European honey bee and is caused by the spore-forming intracellular parasite . The search for new ways of therapy for this disease is complicated due to the seasonal availability of -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 propagation in artificially infected worker bees in the laboratory.

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

Traditional sanitation practices remain the main strategy for controlling infections caused by microsporidia . 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 ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth.

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.

Energy metabolism and its evolution in Microsporidia and allied taxa.

The reduction and specialization of the energy metabolism system is a common trait in the evolution of intracellular parasites. One group of fungi-related parasites, the Microsporidia, seems to have developed this trait far more than other eukaryotes. As an extreme adaptation for a parasitic lifestyle, some of them have completely lost the ability to synthesize ATP, relying heavily upon the metabolic processes of host cells to ensure their own development and reproduction.

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.

Expression of spider toxin in entomopathogenic fungus Lecanicillium muscarium and selection of the strain showing efficient secretion of the recombinant protein.

Beta/delta-agatoxin-1 of spider Agelena orientalis was expressed in entomopathogenic fungus Lecanicillium muscarium. To ensure secretion of the recombinant product by the fungus, the signal secretory peptide of the Metarhizium anisopliae Mcl1 protein was inserted into the sequence. For detection of the recombinant product and selection of transformants, the toxin sequence was also fused with eGFP at the C-terminus.

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.

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.

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.

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.

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.

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.

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.

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.