Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of ironsulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells. Author summary Microsporidia are increasingly appreciated as ubiquitous pathogens with ecological, veterinary and medical importance. Here we provide detailed protocol for establishing a synchronised infection of microsporidian parasites T. hominis and E. cuniculi inside host RK13 cell lines. We demonstrate how this protocol in combination with molecular and cell biology techniques can be utilised to test the effects of anti-microsporidian drugs, and specific protein inhibitors on the parasite's cell biology and infection cycle progression. Using these techniques with bespoke specific antibodies against T. hominis alternative respiration (AR) components-alternative oxidase (AOX) and glycerol-3-phosphate dehydrogenase (mtG3PDH)-we demonstrate their life cycle stage-dependent mitosomal localisation, and in combination with the specific AOX-inhibitor colletochlorin B we show an essential function of the mitosomal AOX during an early phase of the T. hominis infection cycle. Using bioinformatics and specific inhibitors we show that the role of the microsporidian minimal mitochondria in AR was subsequently lost in the course of further reductive evolution in some lineages including that of E. cuniculi. Additional RNAseq across T. hominis life cycle and experiments using antibodies against the mitosomal iron-sulphur clusters biosynthesis (ISC) protein NFS indicated that unlike the phylogenetically-restricted AR, the conserved ISC function of the mitosome is active during T. hominis proliferation.

Automated summary

This article investigates the role of highly reduced forms of mitochondria called mitosomes in parasites, revealing their importance in the progression of the parasite life cycle and the biosynthesis of essential proteins. Additionally, the authors demonstrate that T. hominis is a useful model for studying microsporidian parasites and testing therapeutic agents.