Artemisinin-based combination therapies (ACTs) have been pivotal in reducing the global burden of Plasmodium falciparum (Pf) malaria. Their clinical efficacy, however, is threatened by the recent emergence in Cambodia of artemisinin (ART) resistance, defined as reduced rates of parasite clearance. Cambodian parasites show a highly differentiated population structure with minimal chromosomal admixture, which, we hypothesize, allows resistant subpopulations to survive ART exposure by maintaining complex genetic traits in states of linkage disequilibrium.
In Aim 1 we test the hypothesis that the kelch gene (PF3D7_1343700), very recently reported to be associated with delayed parasite clearance in patient isolates, constitutes a central determinant of emerging ART resistance across Cambodian Pf subpopulations. To test this, we will use kelch-specific zinc finger nucleases (ZFNs) to revert kelch mutations to the wild-type allele in clinically defined resistant parasites, and to introduce the same mutations into sensitive parasites. Kelch-edited clones will be tested for ART resistance using ring-stage survival assays (RSA) that correlate closely with longer clearance half-lives in vivo and that identify the signature trait of ART-induced early ring stage parasite entry into quiescence.
In Aim 2 we address the hypothesis that ART resistance is multifactorial and are defined by subpopulation-specific complex genetic traits. To test this we will study patterns of inheritance in Pf genetic crosses between clinically defined ART-resistant isolates (representing each of the three subpopulations KH2-4) and the sensitive NF54 clone. These crosses take advantage of a new humanized mouse model that allows Pf sporozoites to develop in engrafted human hepatocytes and be recovered in infused human red blood cells. Recombinant progeny will be subjected to whole-genome sequence (WGS) analysis and their ART susceptibility will be quantified using RSA assays. Quantitative trait loci analysis will be used to localize the primary chromosomal regions associated with resistance, and candidate genes will be validated using ZFN-based gene editing. These studies are expected to quantify the role of kelch and define subpopulation-specific secondary determinants.
In Aim 3 we address the equally important topic of resistance to the ACT partner drugs, namely lumefantrine, amodiaquine, piperaquine and pyronaridine. Using the humanized mouse model, we will implement genetic crosses with field isolates resistant to amodiaquine or piperaquine, and perform in vitro selection studies for all drugs. WGS analysis will be followed by ZFN-based validation, and mechanistic studies will be implemented to test the hypothesis that Pf partner drug resistance is achieved via reduced drug accumulation and drug-heme binding. Our multidisciplinary approach to defining the genetic and molecular basis of resistance to ACT drugs will provide powerful new investigational tools, and be of direct translational impact in providing markers to readily track resistance and identify appropriate treatment and containment strategies.
The treatment of malaria is vitally dependent on artemisinin-based combination therapies, yet their continued efficacy is threatened by the emergence and spread of multidrug-resistant strains of the causative agent, the parasite Plasmodium falciparum. Here we present a comprehensive genetic approach to defining the P. falciparum genes that mediate resistance to artemisinin and its combination therapy partner drugs. The implementation of this project can be expected to yield molecular markers to detect the emergence and spread of resistance and to stimulate novel therapeutic approaches to effectively treat drug-resistant malaria.
|Sá, Juliana M; Kaslow, Sarah R; Krause, Michael A et al. (2018) Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model. Proc Natl Acad Sci U S A 115:12513-12518|
|Schuh, Anna Katharina; Rahbari, Mahsa; Heimsch, Kim C et al. (2018) Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum. ACS Infect Dis 4:1601-1612|
|Witkowski, Benoit; Duru, Valentine; Khim, Nimol et al. (2017) A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype-genotype association study. Lancet Infect Dis 17:174-183|
|Agrawal, Sonia; Moser, Kara A; Morton, Lindsay et al. (2017) Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity. J Infect Dis 216:468-476|
|Ng, Caroline L; Fidock, David A; Bogyo, Matthew (2017) Protein Degradation Systems as Antimalarial Therapeutic Targets. Trends Parasitol 33:731-743|
|Paquet, Tanya; Le Manach, Claire; Cabrera, Diego González et al. (2017) Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase. Sci Transl Med 9:|
|Dembele, L; Ang, X; Chavchich, M et al. (2017) The Plasmodium PI(4)K inhibitor KDU691 selectively inhibits dihydroartemisinin-pretreated Plasmodium falciparum ring-stage parasites. Sci Rep 7:2325|
|O'Neill, Paul M; Amewu, Richard K; Charman, Susan A et al. (2017) A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance. Nat Commun 8:15159|
|Rahbari, Mahsa; Rahlfs, Stefan; Przyborski, Jude M et al. (2017) Hydrogen peroxide dynamics in subcellular compartments of malaria parasites using genetically encoded redox probes. Sci Rep 7:10449|
|Blasco, Benjamin; Leroy, Didier; Fidock, David A (2017) Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic. Nat Med 23:917-928|
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