Mitochondrial Heteroplasmy Contributes to the Dynamic Atovaquone Resistance Response in Plasmodium falciparum

Of the considerable challenges researchers face in the control and elimination of malaria, the development of antimalarial drug resistance in parasite populations remains a significant hurdle to progress worldwide. Atovaquone is used in combination with proguanil (Malarone) as an antimalarial treatment in uncomplicated malaria, but is rendered ineffective by the rapid development of atovaquone resistance during treatment. Previous studies have established that de novo mutant parasites confer resistance to atovaquone with a substitution in amino acid 268 in the cytochrome b gene encoded by the parasite mitochondrial genome, yet much is still unknown about how this resistance develops, and whether parasites are inherently predisposed to resistance development. Here we report phenotypic characterization of isolates from patients that failed treatment in the original atovaquone Phase II studies in Thailand by using a diverse series of chemotypes that target mitochondrial functions. We defined their structure-activity relationships and observed broad resistance (5-30,000 fold in atovaquone), suggesting that cytochrome b mutations alone are not sufficient to explain this spectrum of resistance. We also report the first known in vitro selection that recapitulates the clinical Y268S mutation using the TM90-C2A genetic background, the pre-treatment parent for TM90-C2B. Selection of the Y268S mutation in TM90-C2A and others indicates that the parasite genetic background is critical in the selection of clinical atovaquone resistance, since selection attempts in multiple other genetic backgrounds results in mutations at positions other than amino acid 268. We implicate mitochondrial heteroplasmy in the development of sporadic, rapid resistance to atovaquone, where pre-existing low-level mutations in the multi-copy mitochondrial DNA can be quickly selected for in parasite populations. High-coverage mitochondrial deep-sequencing data showed that low-level Y268S mutants were present in admission parasites from the atovaquone Phase II clinical trials in Thailand, and recrudescent parasites either maintained high level Y268S mutation frequencies or gradually returned to cryptic Y268S levels. The phenomenon of gradual heteroplasmic conversion back to wild-type was noted in some ex vivo patient isolated parasites as well as some in vitro selected lines, which suggests that other factors are at play that influence heteroplasmy stability. In addition to mitochondrial heteroplasmy, the total mtDNA copy number is likely influencing phenotypes in a gene dose-dependent fashion. Further, pressure on the DHODH enzyme that results in DHODH copy number amplifications/mutations has been shown to influence mitochondrial heteroplasmy directly. Last, mitochondrial diversity was shown to be vastly underestimated without heteroplasmic loci being taken into account, as seen in the re-analysis of the Pf3K MalariaGEN genome dataset we performed. The complex interactions between these drug resistance mechanisms reveal the phenotypic and genotypic plasticity that the Plasmodium falciparum parasite utilizes are a clear fitness advantage in the face of mitochondrial stress, and further studies are required to determine the impact of this wide-ranging phenotype on the development of new mitochondria-targeted drugs