Assessing the function of the alternative electron transport chain in the Cryptosporidium parvum mitosome

Cryptosporidium parvum and C. hominis possess a remanent mitochondrion called the mitosome, which lacks DNA, the tricarboxylic acid cycle, a conventional electron transport chain, and ATP synthesis. The mitosome retains ubiquinone and iron-sulfur cluster biosynthesis pathways, which require protein import that relies on the membrane potential. It was previously proposed that the membrane potential may be generated by a transhydrogenase (TH) that pumps protons out of the mitosome. This pathway was also proposed to rely on an alternative oxidase (AOX) and type II NADH dehydrogenase (NDH2), which also exists in plants, some fungi, and several protozoan parasites. To examine this model, we determined the location and function of AOX and NDH2 in C. parvum. Surprisingly, we observed that NDH2 was localized to parasite surface membranes instead of the mitosome. Furthermore, a ∆ndh2 knockout (KO) strain was readily obtained, indicating that this protein is not essential for parasite growth. Although AOX exhibited a mitosome-like staining pattern, we readily obtained a ∆aox KO strain, which did not exhibit a difference in MitoTracker, indicating that AOX is likely not involved in membrane potential and is dispensable for parasite growth. The growth of the ∆aox strain was inhibited by the AOX inhibitors, salicylhydroxamic acid and 8-hydroxyquinoline, to the same extent as wild type, indicating that AOX is not the target of these inhibitors in C. parvum. Collectively, our studies indicate that NDH2 and AOX are non-essential genes in C. parvum, supporting an alternative mechanism for maintaining the mitosome membrane potential.

Cryptosporidiosis is a leading cause of diarrhea in young children and immunocompromised individuals, particularly AIDS/HIV patients. The only FDA-approved drug against cryptosporidiosis, nitazoxanide, has limited effectiveness in immunocompromised patients and is not approved for use in children under 1 year. Genomic analysis and previous studies proposed an alternative respiration pathway involving alternative oxidase (AOX) and type II NAD(P)H dehydrogenase (NDH2), which are thought to generate the mitosome membrane potential in Cryptosporidium parvum. Additionally, AOX was nominated as potential drug targets, based on its absence in mammalian hosts and sensitivity of parasite growth to known inhibitors of AOX. However, our study demonstrated that NDH2 is not localized in the mitosome, AOX is non-essential for parasite growth, and knockout lines lacking this enzyme are equally sensitive to AOX inhibitors. These findings indicate that AOX and NDH2 are not ideal candidates for future drug development against cryptosporidiosis and force a re-evaluation of models of how the mitosome generates its membrane potential.