Peroxisomal catalase in extrusion apparatus posterior vacuole of microsporidian spores.

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A remarkable survival adaptation of microsporidians is the spore extrusion apparatus (EXA), which is equipped to explosively discharge a tube through which the infective sporoplasm is sent into a host cell. The EXA consists of a membrane-associated aperture, polaroplast, polar filament protein, and a posterior vacuole. When the spore activates, the EXA discharges a long, fine tube by an apparent eversion process. When the tube is fully formed, the sporoplasm moves down the tube and ends up in an enveloped compartment thought to be derived from the everted extrusion apparatus membrane. The energy source for spore discharge is believed to reside in the EXA posterior vacuole. In activated spores, the posterior vacuole swells before and during spore extrusion. We report here the presence of catalase in the posterior vacuole of the spores of a microsporidian, Spraguea lophii. This was first made evident when significant levels of molecular oxygen were detected in a medium containing the discharging spores. Clarkstyle oxygen microelectrodes (Model 7376C, Diamond General, Ann Arbor, Michigan) were used to measure dissolved oxygen in the medium. The oxygen appeared only when low levels of hydrogen peroxide were added to the medium, either before or during spore discharge. In the absence of hydrogen peroxide, no oxygen formed in the medium containing the firing spores. The medium was recovered from the fired spores and was analyzed for catalase enzyme using western blot analysis and using the protocol described earlier (1). The results indicate that catalase was present in the medium, and that the molecular weight corresponded to control catalase (Fig. 1A). This finding supports the old hypothesis that the EXA contents may be released directly into the surrounding medium during tube eversion. Catalase crystals were successfully isolated using the protocol described by Reinlein (2). The catalase-rich medium was placed in 10% NaCl, containing 2 mM KH2PO4 at pH 5.5, and after 1 h, this was dialyzed against 1 mM ammonium chloride (pH 5.5). To recrystalize the catalase, there were two cycles of 10-h dialysis. For clean crystals, the entire sample was recycled through the procedure a second time. The crystals tested positive for catalase on the basis of western blots with rabbit anti-bovine catalase (Rockland Immunochemicals, Gilbertsville, Pennsylvania). To identify the specific location of the catalase, spores of S. lophii were subjected to the alkaline-diaminobenzidine (DAB) procedure as reported by Novikoff and Goldfischer (3). The DAB reaction localized to the posterior vacuole before and during spore activation (Fig. 1B). After spore discharge, no detectable DAB activity was found in either the spore ghosts or the extruded sporoplasms. Although the mechanism by which catalase works in the posterior vacuole is unclear, it may be associated with the oxidation of long chain fatty acids. Docosahexaenoic acid, commonly associated with peroxisomes, is significantly represented in S. lophii spores (4). A function of peroxisomes is to manage the betaoxidation of long chain fatty acids. Acyl-coA oxidase, an essential enzyme in this process, produces hydrogen peroxide. The catalase in the area can convert the hydrogen peroxide to water and oxygen. The rapid oxidation of the long chain fatty acids may effect the swelling of the posterior vacuole and induce spore discharge.