Lack of mitochondrial MutS homolog 1 in Toxoplasma gondii disrupts maintenance and fidelity of mitochondrial DNA and reveals metabolic plasticity

The importance of maintaining the fidelity of the mitochondrial genome is underscored by the presence of various repair pathways within this organelle. Presumably, the repair of mitochondrial DNA would be of particular importance in organisms that possess only a single mitochondrion, like the human pathogens Plasmodium falciparum and Toxoplasma gondii. Understanding the machinery that maintains mitochondrial DNA in these parasites is of particular relevance, as mitochondrial function is a validated and effective target for anti-parasitic drugs. We previously determined that the Toxoplasma MutS homolog TgMSH1 localizes to the mitochondrion. MutS homologs are key components of the nuclear mismatch repair system in mammalian cells, and both yeast and plants possess MutS homologs that localize to the mitochondria where they regulate DNA stability. Here we show that the lack of TgMSH1 results in accumulation of single nucleotide variations in mitochondrial DNA and a reduction in mitochondrial DNA content. Additionally, parasites lacking TgMSH1 function can survive treatment with the cytochrome b inhibitor atovaquone. While the Tgmsh1 knockout strain has several missense mutations in cytochrome b, none affect amino acids known to be determinants of atovaquone sensitivity and atovaquone is still able to inhibit electron transport in the Tgmsh1 mutants. Furthermore, culture of Tgmsh1 mutant in the presence atovaquone leads to parasites with enhanced atovaquone resistance and complete shutdown of respiration. Thus, parasites lacking TgMSH1 overcome the disruption of mitochondrial DNA by adapting their physiology allowing them to forgo the need for oxidative phosphorylation. Consistent with this idea, the Tgmsh1 mutant is resistant to mitochondrial inhibitors with diverse targets and exhibits reduced ability to grow in the absence of glucose. This work shows TgMSH1 as critical for the maintenance and fidelity of the mitochondrial DNA in Toxoplasma, reveals a novel mechanism for atovaquone resistance, and exposes the physiological plasticity of this important human pathogen.

线粒体作为一类细胞器，其内部存在多种修复通路，凸显了维持线粒体基因组（mitochondrial genome）保真度的重要性。据推测，对于仅拥有单个线粒体的生物而言，线粒体DNA（mitochondrial DNA）的修复尤为关键，例如人类病原体恶性疟原虫（Plasmodium falciparum）与刚地弓形虫（Toxoplasma gondii）。鉴于线粒体功能已被证实为抗寄生虫药物的有效作用靶点，解析这类寄生虫体内维持线粒体DNA的分子机制具有重要的研究价值。我们此前已证实，刚地弓形虫的MutS同源蛋白TgMSH1定位于线粒体（mitochondrion）中。MutS同源蛋白是哺乳动物细胞核错配修复系统（nuclear mismatch repair system）的核心组分，酵母与植物均拥有定位于线粒体并调控DNA稳定性的MutS同源蛋白。本研究证实，TgMSH1缺失会导致线粒体DNA中出现单核苷酸变异（single nucleotide variations）的积累，并降低线粒体DNA的拷贝含量。此外，功能缺失TgMSH1的寄生虫可在细胞色素b（cytochrome b）抑制剂阿托伐醌（atovaquone）的处理下存活。尽管Tgmsh1敲除株在细胞色素b基因上存在多个错义突变（missense mutation），但这些突变均未影响已知的阿托伐醌敏感性决定氨基酸位点，且阿托伐醌仍可抑制Tgmsh1突变体的电子传递过程。此外，在含阿托伐醌的培养基中培养Tgmsh1突变体，可获得阿托伐醌抗性增强的寄生虫株，且这类寄生虫的呼吸作用完全停滞。由此可见，TgMSH1缺失的寄生虫可通过调整自身生理状态来应对线粒体DNA的损伤，从而无需依赖氧化磷酸化（oxidative phosphorylation）供能。与此结论相符，Tgmsh1突变体对多种靶向不同位点的线粒体抑制剂均具有抗性，且在无糖环境下的生长能力显著下降。本研究证实，TgMSH1对刚地弓形虫线粒体DNA的维持与保真度至关重要，揭示了一种全新的阿托伐醌抗性机制，并展现了这一重要人类病原体的生理可塑性。