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

Abstract

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.

Fig. 1.

Western blot assay of A. locustae proteins. (A) Samples of spore homogenate cleared by centrifugation at 100 × g for 10 min (Sp) and stages of intracellular development (meronts and sporonts) ruptured by sonication (St) were equalized in protein concentration, and 20 μg was analyzed by immunoblotting. This experiment has clearly demonstrated specific accumulation of metabolic enzymes in microsporidial spores. To estimate protein diffusion from destroyed spores, an aliquot of crude debris (Db) resuspended in TS equal to the volume of homogenate was analyzed. (B) Double band recognized by anti-Hsp70 Abs represents two different proteins because serial dilution of anti-mitHsp70 immune serum up to 1:40,000 has resulted in drastic weakening of upper major band, whereas 66-kDa protein has remained specifically stained. (C) Specific decoration of 66-kDa protein by anti-mitHsp70 Abs depleted against major parasite protein of 75 kDa has confirmed the existence of unique epitopes in mitHsp70 polypeptide chain. Protein samples of Sp, St, and Db were prepared as described in the legend to panel A. (D) To analyze intrasporal localization of studied proteins, cleared spore homogenate was centrifuged in a series at 20,000 × g for 20 min and at 200,000 × g for 1 h, and the pellets were resuspended, with the volume of TS brought up to that of the final supernatant prior to Western blot assay. The pelleting of mitG3PDH, AOX, and most of mitHsp70 by high-speed centrifugation has suggested their localization in some membrane compartment.

Fig. 2.

Identification of mitosomes in A. locustae spores. Ultrathin cryosections of mature spores were stained with affinity-purified and depleted anti-mitHsp70 Abs. Immunoelectron microscopy shows specific labeling of mitosomes, small (50- to 200-nm) structures of round profiles bounded by a double membrane (A to G). About 3 or 4 mitosomes were found per spore section, and some of them were aggregated in pairs (B, G). Mitosomes from panel B are shown enlarged (C, D). As expected, most of the labeling was present over the inner area of the organelles (mitosomal matrix). Microsporidial mitosomes appeared to be in close contact with ER cisternae (arrows). Gold grain size, 10 nm. Scale bar, 0.1 μm.

Fig. 3.

Immunolocalization of A. locustae AOX and mitG3PDH. Double labeling of spore cryosections with anti-AOX and anti-mitHsp70 Abs shows specific decoration of mitosomes and colocalization of both proteins in these organelles (A to D). Immunoelectron microscopy of spore cryosections with anti-mitG3PDH and anti-mitHsp70 Abs has shown similar colocalization of both proteins in A. locustae mitosomes (E to G). AOX and mitG3PDH labeling was seen mostly over the mitosomal membranes around the more electron-dense matrix. Scale bar, 0.1 μm.