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Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum.

ABSTRACT: Malaria continues to impose a significant health burden in the continent of Africa with 213 million cases in 2018 alone, representing 93% of cases worldwide. Because of high transmission of malaria within the continent, the selection pressures to develop drug resistance in African parasites are distinct compared to the rest of the world. In light of the spread of resistance to artemisinin conferred by the C580Y mutation in the PfKelch13 propeller domain in Southeast Asia, and its independent emergence in South America, it is important to study genetic determinants of resistance in the African context using African parasites. Through in vitro evolution of Senegalese parasites, we had previously generated the artemisinin-resistant parasites Pikine_R and Thiès_R and established pfcoronin mutations to be sufficient to confer artemisinin resistance in the standard ring-stage survival assay (RSA). In the current study, we used genetic analysis of revertants to demonstrate pfcoronin to be the major driver of elevated RSA in the artemisinin-resistant parasites Pikine_R and Thiès_R evolved in vitro. We interrogated the role of a second gene PF3D7_1433800, which also had mutations in both the Pikine_R and Thiès_R selected lines, but found no evidence of a contribution to reduced susceptibility in the RSA survival assay. Nevertheless, our genetic analysis demonstrates that parasite genetic background is important in the level of pfcoronin mediated RSA survival, and therefore we cannot rule out a role for PF3D7_1433800 in other genetic backgrounds. Finally, we tested the potential synergy between the mutations of pfcoronin and pfkelch13 through the generation of single and double mutants in the Pikine genetic background and found that the contribution of pfcoronin to reduced susceptibility is masked by the presence of pfkelch13. This phenomenon was also observed in the 3D7 background, suggesting that pfcoronin may mediate its effects via the same pathway as pfkelch13. Investigating the biology of proteins containing the beta-propeller domain could further elucidate the different pathways that the parasite could use to attain resistance.

SUBMITTER: Sharma AI

PROVIDER: S-EPMC7793257 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum.

Sharma Aabha I AI Shin Sara H SH Bopp Selina S Volkman Sarah K SK Hartl Daniel L DL Wirth Dyann F DF

PLoS genetics 20201228 12

Malaria continues to impose a significant health burden in the continent of Africa with 213 million cases in 2018 alone, representing 93% of cases worldwide. Because of high transmission of malaria within the continent, the selection pressures to develop drug resistance in African parasites are distinct compared to the rest of the world. In light of the spread of resistance to artemisinin conferred by the C580Y mutation in the PfKelch13 propeller domain in Southeast Asia, and its independent eme ...[more]

PMID: 33370279

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Project description:BackgroundArtemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance.MethodsSixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates.ResultsAmong these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0-3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance.ConclusionsThis study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

Project description:Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance.In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance.We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups.P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)

Dataset Information

Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum.

Publications

Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum.

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