Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth

Abstract

SummaryIn many apicomplexan parasites the entry of electrons from NADH into the electron transport chain is governed by type II NADH dehydrogenases (NDH2s) instead of a canonical complex I. Toxoplasma gondii expresses two NDH2 isoforms, TgNDH2-I and TgNDH2-II with no indication for stage-specific regulation. We dissected the orientation of both isoforms by using a split GFP assay and a protease protection assay after selective membrane permeabilization. The two approaches revealed that both TgNDH2 isoforms are internal enzymes facing with their active sites to the mitochondrial matrix. Single knockout mutants displayed a decreased replication rate and a reduced mitochondrial membrane potential, which were both more severe in the Tgndh2-II-deleted than in the Tgndh2-I-deleted mutant. Complementation with a myc-tagged, ectopic copy of the deleted gene restored the growth rate and the mitochondrial membrane potential. However, an overexpression of the remaining intact isoform could not restore the phenotype, suggesting that the two TgNDH2 isoforms are nonredundant and possess functional differences. Together, our studies indicate that although TgNDH2-I and TgNDH2-II are individually non-essential, the expression of both internal isoforms is required to maintain the mitochondrial physiology in T. gondii tachyzoites.

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…However, flux from both sources is much greater in T. gondii, and therefore, unlike Plasmodium, T. gondii tachyzoite proliferation is sensitive to TCA cycle and ETC perturbations. Disruption of BCKDH attenuates parasite intracellular development and virulence [46], while inhibition of Aco [54] or NDH [68] arrests growth, suggesting that an active respiratory chain is essential for tachyzoite growth, as previously hypothesized [4,54,82]. However, as above, the lethal effects of Aco inhibition may be due to citrate accumulation and cytotoxicity, or inhibition of Aco activity in the T. gondii apicoplast (Figure 3 and Box 3).…”

“…One might speculate, then, that dormant P. falciparum maintains some degree of metabolic activity, albeit in an altered manner that is not currently understood. Similarly, effects have been seen in T. gondii, where sublethal type II NADH dehydrogenase (NDH) inhibition leads to the formation of bradyzoites [68]. The metabolic state of bradyzoites is still poorly understood, although NDH (1 and 2) have similar mRNA levels to the tachyzoites, suggesting that at least some form of metabolism is maintained.…”

“…The precise mitochondrial membrane location of the Plasmodium enzyme is unknown, but it is dispensable in P. berghei blood stages [44]. In T. gondii, two mitochondrial NDH isoforms are present on the matrix side of the inner leaflet and are required (but individually dispensable) for ATP regulation and normal tachyzoite proliferation [68][69][70][71].…”

HDQ, a Potent Inhibitor of Plasmodium falciparum Proliferation, Binds to the Quinone Reduction Site of the Cytochrome bc 1 Complex

“…It is worth noting, however, that, historically, anti-infectives displaying polypharmacology show greater efficacy over single-targeting inhibitors (27). In Toxoplasma gondii, the deletion of type II NADH:dehydrogenase genes is not lethal but is required for optimal tachyzoite growth (35). Interestingly, HDQ has been shown to be synergistic with atovaquone for growth inhibition (36), which may be attributed to the polypharmacological effect of HDQ against the type II NADH: dehydrogenases as well as the Q o and Q i sites of the bc 1 complex.…”

Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity

“…[11] Little is known though about their respective roles; in organisms with multiple copies of Ndh-2, their roles are often non-redundant. [12] To further examine target engagement, we utilized a temperature-sensitive (TS) Mycobacterium smegmatis mutant to probe the activity of each compound against Ndh and NdhA individually. When grown above the permissive temperature, this TS strain downregulates expression of ndh due to a base substitution of G250 to T of the ndh gene that alters amino acid Gly84 (GGC) to Cys (TGC) of Ndh (Yano and Rubin, unpublished data).…”