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.

Role of the posterior vacuole in Spraguea lophii (Microsporidia) spore hatching.

Microsporidia constitute a large group of obligate intracellular protozoan parasites that inject themselves into host cells via the extrusion apparatus of the infective spore stage. Although the injection process is poorly understood, its energy source is thought to reside in the posterior vacuole that swells significantly during spore firing. Here we report the presence and localisation of the key peroxisomal enzymes catalase and acyl-CoA oxidase (ACOX) within the posterior vacuole of Spraguea lophii (Doflein, 1898) spores. Western blot analyses show that these enzymes discharge out of the spore and end up in the medium external to the extruded sporoplasms. The presence of a catalase enzyme system in the Microsporidia was first made evident by the detection of significant levels of molecular oxygen in the medium containing discharging spores in the presence of hydrogen peroxide. Catalase was visualised in inactive, activated, and discharged spores using alkaline diaminobenzidine (DAB) on glutaraldehyde-fixed cells. The position of these enzymes within the extrusion apparatus before and during spore discharge support the Lom and Vávra model that postulates discharge occurs by an eversion process. In addition to these enzymes, spores of S. lophii contain another characteristic peroxisomal component, the very long chain fatty acid (VLCFA) nervonic acid. A sizeable decrease in nervonic acid levels occurs during and after spore discharge. These data indicate that nervonic acid is discharged from the spore into the external medium during firing along with the catalase and ACOX enzymes.

Investigations into microsporidian methionine aminopeptidase type 2: a therapeutic target for microsporidiosis.

The Microsporidia have been reported to cause a wide range of clinical diseases particularly in patients that are immunosuppressed. They can infect virtually any organ system and cases of gastrointestinal infection, encephalitis, ocular infection, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles such as albendazole are active against many species of Microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin, ovalicin and their analogues have been demonstrated to have antimicrosporidial activity in vitro and in animal models of microsporidiosis. Fumagillin has also been demonstrated to have efficacy in human infections due to E. bieneusi. Fumagillin is an irreversible inhibitor of methionine aminopeptidase type 2 (MetAP2). Homology cloning employing the polymerase chain reaction was used to identify the MetAP2 gene from the human pathogenic microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Brachiola algerae and E. bieneusi. The full-length MetAP2 coding sequence was obtained for all of the Encephalitozoonidae. Recombinant E. cuniculi MetAP2 was produced in baculovirus and purified using chromatographic techniques. The in vitro activity and effect of the inhibitors bestatin and TNP-470 on this recombinant microsporidian MetAP2 was characterized. An in silico model of E. cuniculi MetAP2 was developed based on crystallographic data on human MetAP2. These reagents provide new tools for the development of in vitro assay systems to screen candidate compounds for use as new therapeutic agents for the treatment of microsporidiosis.