Mitochondrial heteroplasmy is responsible for Atovaquone drug resistance in Plasmodium falciparum

Abstract

24Malaria is the most significant parasitic disease affecting humans, with 212 25 million cases and 429,000 deaths in 2015 1 , and resistance to existing drugs endangers 26 the global malaria elimination campaign. Atovaquone (ATO) is a safe and potent 27 antimalarial drug that acts on cytochrome b (cyt. b) of the mitochondrial electron 28 transport chain (mtETC) in Plasmodium falciparum, yet treatment failures result in 29 resistance-conferring SNPs in cyt. b. Herein we report that rather than the expected de 30 novo selection of resistance, previously unknown mitochondrial diversity is the genetic 31 mechanism responsible for resistance to ATO, and potentially other cyt. b targeted 32 drugs. We found that P. falciparum harbors cryptic cyt. b. Y268S alleles in the multi-33 copy (~22 copies) mitochondrial genome prior to drug treatment, a phenomenon known 34 as mitochondrial heteroplasmy. Parasites with cryptic Y268S alleles readily evolve into 35 highly resistant parasites with >95% Y268S copies under in vitro ATO selection. Further 36 we uncovered high mitochondrial diversity in a global collection of 1279 genomes in 37 which heteroplasmic polymorphisms were >3-fold more prevalent than homoplasmic 38 SNPs. Moreover, significantly higher mitochondrial genome copy number was found in 39Asia (e.g., Cambodia) versus Africa (e.g., Ghana). Similarly, ATO drug selections in 40 vitro induced >3-fold mitochondrial copy number increases in ATO resistant lines. 41Hidden mitochondrial diversity is a previously unknown mechanism of antimalarial drug 42 resistance and characterization of mitochondrial heteroplasmy will be of paramount P275T, K272R, G280D, L283I, V284K, L144S and F267V 18-20 (Extended Data Table 1). 62Second, drug susceptibility studies with ATO resistant Y268S mutants demonstrate a 63 broad range of potency rather than a dichotomous response that would be expected 64 from a single SNP. By using paired parasites collected upon patient admission and 65 subsequent treatment failure from Phase II trials of ATO (Extended Data Table 2), we 66 discovered three distinct in vitro ATO resistance phenotypes (Table 1). A recrudescent 67 isolate (TM90-C6B) initially typed as Y268 (WT) exhibited low level ATO resistance, 68 other isolates with Y268S from recrudescent isolates (e.g., TM90-C2B) demonstrated 69 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/232033 doi: bioRxiv preprint first posted online Dec. 10, 2017; moderate resistance to ATO and myxothiazol, and two isolates from ATO-70 pyrimethamine treatment failures showed extreme resistance (TM92-C1086 and TM92-71 C1088) (Table 1). Surprisingly, the extreme ATO resistance phenotype produced 72 greatly reduced susceptibility to a broad range of mtETC inhibitors (Table 1), even 73 though the parasites expressed the common Y268S/N SNPs and no apparent 74 resistance-associated SNPs in candidate mtETC encoding gene...

Selection of Plasmodium falciparum cytochrome B mutants by putative PfNDH2 inhibitors

“…In the ATQ pressure experiments, four independent populations were obtained from Dd2: a PfCytB K272R mutant and three M133I mutants with an ATG → ATT or ATG → ATA codon change (Table 1). However, ATQ pressure on 106/1 yielded one population carrying PfCytB Y268S, a mutation frequently detected in clinical failures of atovaquone-proguanil (26,27) and recently obtained from human isolates by in vitro selection (28).…”

“…The nuclear pfdhod gene is normally single-copy in P. falciparum but amplification has been found with increases in ATQ EC 50 levels upon DSM1 selection (28,35,38). We tested for pfdhod CNV in the clones listed in Table 2.…”

“…In this work, no evidence for amplification of pfcoxI, pfcoxIII, pfcytB, or pfdhod was found in ETC inhibitor-resistant mutants. Mitochondrial heteroplasmy and mutations that arise with replication of mtDNA during mitosis may cause clinical failures after ETC inhibitor treatment (28,46). Reduced susceptibility of the V259L mutants to ATQ, CK-2-68, and RYL-552 suggests a general effect of this mutation on the binding ability of the PfCytB Q o site.…”

Elucidating Mechanisms of Drug-Resistant Plasmodium falciparum

“…In Plasmodium, ARTs have been found to trigger rapid ROS-dependent depolarization of mitochondrial and plasma membrane potentials (Antoine et al, 2014). Of note, Southeast Asian Cambodian parasites have been observed to generally have more mitochondrial genomes than West African Ghanaian parasites, allowing for more genetic diversity through heteroplasmy and perhaps also cushioning ART-caused loss of mitochondrial function (Siegel et al, 2017). DHA-treated ARTsensitive W2 parasites were reported to have low levels of survival in a modified in vitro assay, attributed to dormant parasites whose subsequent recovery has been associated with the presence of transcriptionally active mitochondria (Peatey et al, 2015).…”

Template copy number and the sensitivity of quantitative PCR for Plasmodium falciparum in asymptomatic individuals

“…Each candidate assay was screened using NCBI Primer-BLAST [ 11 ] to: (1) estimate its template copy number (number of DNA sequences yielding a positive fluorescent signal) based on the P. falciparum 3D7 genome (ASM2762), (2) examine the AT content of DNA sequences amplified and (3) test for cross-reactivity with host or pathogen DNA. In contrast, because the template copy numbers for mitochondrial DNA sequences cannot be estimated in silico, representative median estimates of mitochondrial genome copy numbers were used for the cytb and coxI assays [ 12 ].…”