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. 2017 Apr 11;8(2):e00172-17.
doi: 10.1128/mBio.00172-17.

Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro

Affiliations

Plasmodium falciparum K13 Mutations Differentially Impact Ozonide Susceptibility and Parasite Fitness In Vitro

Judith Straimer et al. mBio. .

Abstract

The emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0-3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia.IMPORTANCE ACTs have helped halve the malaria disease burden in recent years; however, emerging resistance to ART derivatives threatens to reverse this substantial progress. Resistance is driven primarily by mutations in the P. falciparum K13 gene. These mutations pose a threat to ozonides, touted as promising alternatives to ARTs that share a similar mode of action. We report that DHA was considerably more potent than OZ439 and OZ277 against ART-sensitive asexual blood-stage parasites cultured in vitro We also document that mutant K13 significantly compromised the activity of the registered drug OZ277. In contrast, OZ439 remained effective against most parasite lines expressing mutant K13, with the exception of I543T that merits further monitoring in field-based OZ439 efficacy studies. K13 mutations differed considerably in their impact on parasite growth rates, in a strain-dependent context, with the most prevalent C580Y mutation being fitness neutral in recently culture-adapted strains from Cambodia, the epicenter of emerging ART resistance.

Keywords: K13/Kelch13; Plasmodium falciparum; artemisinin; drug resistance; fitness; gene editing; ozonides.

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Figures

FIG 1
FIG 1
DHA is a more potent inhibitor of parasite survival in vitro than OZ439 or OZ277, as defined in the ring-stage survival assay from 0 to 3 h (RSA0–3h). Results show the percentage of early ring-stage parasites expressing wild-type K13 (0 to 3 h postinvasion of human erythrocytes) that survive exposure to a 4-h pulse of DHA, OZ439, or OZ277 ranging in concentration from 700 nM to 0.6 nM, as measured by flow cytometry 68 h later. Data show mean ± standard error of the mean (SEM; error bars) percent survival from at least three independent experiments performed in duplicate compared with DMSO vehicle-treated parasites processed in parallel. Data show the combined results for percent survival in four parasite lines harboring the wild-type K13 allele: CamWT, Cam3.IIrev, V1/Sctrl, and Cam5rev. RSA0–3h dose-response curves are shown for DHA, OZ439, and OZ277.
FIG 2
FIG 2
K13 propeller mutations confer cross-resistance to OZ277 but not to OZ439 in clinical isolates and reference lines in vitro, as defined in the ring-stage survival assay from 0 to 3 h (RSA0–3h). Results show the percentage of early ring-stage parasites expressing wild-type or mutant K13 (0 to 3 h postinvasion of human erythrocytes) that survive exposure to a 4-h pulse of DHA, OZ439, or OZ277 ranging in concentration from 700 nM to 0.6 nM, as measured by flow cytometry 68 h later. Data show mean ± SEM percent survival for each line assayed in duplicate on at least three independent occasions with drug or DMSO as a control. (A, E, and I) RSA0–3h dose-response curves for Cam3.II parasites expressing K13 C580Y or R539T (mutation shown by the superscript) or the wild-type allele (indicated by the rev superscript). (B, F, and J) Dose-response curves for V1/S parasites expressing mutant K13 (mutation shown by the superscript) or wild-type K13 (ctrl superscript for control). (C, G, and K) Dose-response curves for Cam5 parasites expressing K13 I543T (shown in superscript) or the wild-type allele (indicated by the rev superscript). (D, H, and L) Dose-response curves for CamWT parasites expressing K13 C580Y (shown in superscript) or the wild-type allele. Student t tests compared percent survival values between each K13 mutant and its corresponding isogenic wild-type line, assayed for each of the four sets of parasite lines and for each drug individually when tested at 700 nM. These tests included calculations of the standard error of the difference between the means of samples being compared and the corresponding P values (results detailed in Table 1, with statistical outputs provided in Text S1 in the supplemental material). Cam5I543T and Cam5rev lines were also compared for percent survival when exposed to 175 nM OZ439. Values that are significantly different by Student t test are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.
FIG 3
FIG 3
K13 mutations confer an in vitro fitness cost in clinical isolates and reference lines. Results show differences in growth rates per 48-h generation of clinical isolates and reference lines harboring native or ZFN-edited K13 mutations (shown in superscript) relative to their isogenic parasite lines carrying the wild-type K13 allele (e.g., Cam3.IIC580Y versus Cam3.IIrev, showing a mean 0.06% reduction in the rate of growth of the C580Y mutant relative to its isogenic K13 wild-type control). Differences in growth rates were calculated as the percent change in K13 mutant allele frequency over a 60-day coculture period, as determined by pyrosequencing. Values are shown as means ± SEM (error bars) in two independent assays performed in duplicate.

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References

    1. World Health Organization 2016. Artemisinin and artemisinin-based combination therapy resistance. WHO reference number WHO/HTM/GMP/2016.11 Global Malaria Programme, World Health Organization, Geneva, Switzerland: http://www.who.int/malaria/publications/atoz/update-artemisinin-resistan....
    1. Woodrow CJ, White NJ. 2017. The clinical impact of artemisinin resistance in Southeast Asia and the potential for future spread. FEMS Microbiol Rev 41:34–48. doi:10.1093/femsre/fuw037. - DOI - PMC - PubMed
    1. White NJ. 2008. Qinghaosu (artemisinin): the price of success. Science 320:330–334. doi:10.1126/science.1155165. - DOI - PubMed
    1. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K, Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NP, Lindegardh N, Socheat D, White NJ. 2009. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361:455–467. doi:10.1056/NEJMoa0808859. - DOI - PMC - PubMed
    1. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, Artemisinin Resistance in Cambodia 1 (ARC1) Study Consortium . 2008. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med 359:2619–2620. doi:10.1056/NEJMc0805011. - DOI - PubMed