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Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility.

ABSTRACT: Drug resistance is an obstacle to global malaria control, as evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of the PfKelch13 protein in Southeast Asia. Identifying genetic determinants of ART resistance in African-derived parasites is important for surveillance and for understanding the mechanism of resistance. In this study, we carried out long-term in vitro selection of two recently isolated West African parasites (from Pikine and Thiès, Senegal) with increasing concentrations of dihydroartemisinin (DHA), the biologically active form of ART, over a 4-y period. We isolated two parasite clones, one from each original isolate, that exhibited enhanced survival to DHA in the ring-stage survival assay. Whole-genome sequence analysis identified 10 mutations in seven different genes. We chose to focus on the gene encoding PfCoronin, a member of the WD40-propeller domain protein family, because mutations in this gene occurred in both independent selections, and the protein shares the ?-propeller motif with PfKelch13 protein. For functional validation, when pfcoronin mutations were introduced into the parental parasites by CRISPR/Cas9-mediated gene editing, these mutations were sufficient to reduce ART susceptibility in the parental lines. The discovery of a second gene for ART resistance may yield insights into the molecular mechanisms of resistance. It also suggests that pfcoronin mutants could emerge as a nonkelch13 type of resistance to ART in natural settings.

SUBMITTER: Demas AR

PROVIDER: S-EPMC6294886 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Mutations in <i>Plasmodium falciparum</i> actin-binding protein coronin confer reduced artemisinin susceptibility.

Demas Allison R AR Sharma Aabha I AI Wong Wesley W Early Angela M AM Redmond Seth S Bopp Selina S Neafsey Daniel E DE Volkman Sarah K SK Hartl Daniel L DL Wirth Dyann F DF

Proceedings of the National Academy of Sciences of the United States of America 20181112 50

Drug resistance is an obstacle to global malaria control, as evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of the <i>Pf</i>Kelch13 protein in Southeast Asia. Identifying genetic determinants of ART resistance in African-derived parasites is important for surveillance and for understanding the mechanism of resistance. In this study, we carried out long-term in vitro selection of two recently isolated West African parasites (from Pikine a ...[more]

PMID: 30420498

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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:BackgroundGliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin.MethodsHere the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion.ResultsA combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane.ConclusionsThese data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites.

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Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility.

Publications

Mutations in <i>Plasmodium falciparum</i> actin-binding protein coronin confer reduced artemisinin susceptibility.

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