Abstract
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.
Highlights
Toxoplasma gondii is a pervasive obligate intracellular protozoan parasite known to infect human and all other warm-blooded animals
Since the complemented strain was generated using the MRC5 mutant strain that had been in culture for approximately 3 months it is possible that those mutations existed prior to the re-introduction of TgMSH1
It is important to note that the accumulation of single nucleotide variations (SNV) in the MRC5 mutant and derived clones appears to be limited to the mitochondrial DNA (mtDNA) as no variations were detected in either a 417-base pair fragment of nuclear DNA or a 1128 base pairs fragment of apicoplast DNA in any of the clones and strains sequenced
Summary
Toxoplasma gondii is a pervasive obligate intracellular protozoan parasite known to infect human and all other warm-blooded animals. Infection with Toxoplasma is usually asymptomatic in most healthy individuals as a robust immune response is able to exert pressure on the rapidly dividing parasites and forcing them into a dormant form, thereby limiting the tissue destruction caused by the parasite. First line therapy against acute toxoplasmosis consists of the antifolate pyrimethamine, in combination with sulphadiazine. These compounds have no effect on the dormant encysted form of the parasite but there are significant side effects associated with their use as they can affect the host’s folate synthesis. Considering that approximately a third of the world’s population is infected with this pathogen and given the significant patient population with compromised immune systems, infection with Toxoplasma is a serious public health concern—the discovery and study of alternative therapies is a priority [2]
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