A broad distribution of the alternative oxidase in microsporidian parasites

Abstract

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

Figure 1. Alignment of A. locustae, T. hominis, S. guttatum and T. brucei AOX sequences.

The four-helix bundles are underlined with a solid line. The putative quinone binding site is underlined with a broken line. Conserved amino acid sites are marked with a star and semi conserved sites are marked with dots.

Figure 2. Transfection of S. cerevisiae cells with AOX-GFP constructs.

The left panel represents the signal from the GFP-AOX constructs, the centre image represents the mitotracker signal accumulated in the yeast mitochondria. The right panel shows the composite image of the GFP and the red mitotracker signal. The top set (ThAOX) represents the T. hominis AOX-GFP construct, the lower set (AlAOX) represents the A. locustae AOX-GFP construct. In each set of panels a single yeast cell is shown with the branched mitochondrial network around the periphery of the cell that has an approximate diameter of 6 µm.

Figure 3. Phylogenetic analyses of microsporidian AOX sequences.

A. Global MrBayes AOX phylogeny, posterior probabilities and PhyML bootstraps from an analysis of 500 bootstrapped datasets are shown above and below respectively, key and well supported clades (>70% Bootstrap). B. Short alignment PhyML phylogeny including the translated amplified sequences from all four microsporidia. Bootstrap support from 100 datasets is shown next to microporidian nodes. C. Microsporidian distribution of the alternative oxidase gene plotted onto a phylogeny of microsporidian SSU rDNA sequences. Scale bars in all trees indicate substitutions per site.

Figure 4. Western blots of the membrane preparations from C41 E. coli strains expressing microsporidian AOX proteins.

Lane 1 shows A. locustae rAOX and Lane 2 shows T. hominis rAOX. Lane 3 shows purified alternative oxidase protein from Sauromatum guttatum.

Figure 5. Hypothetical scheme of function of the alternative oxidase in the microsporidian cell.

Microsporidian cells are known to contain glycolytic enzymes, though no obvious mechanism exists for reoxidising NADH to NAD+. The glycerol-3-phosphate shuttle is encoded in many microsporidian genomes. If this shuttle is coupled to an alternative oxidase protein in the mitosome, it could potentially represent a mechanism for regenerating NAD+.