title: Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum.
abstract: The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field.
authors.affiliation: Department of Microbiology and Immunology, Columbia University Medical Center, New York.
authors.forename: Stanislaw J
authors.initials: SJ
authors.lastname: Gabryszewski
authors.affiliation: Department of Physics, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Charin
authors.initials: C
authors.lastname: Modchang
authors.affiliation: Laboratoire de Parasitologie, WHO Collaborating Center for Surveillance of Anti-Malarial Drug Resistance, Institut Pasteur de la Guyane, Cayenne, French Guiana.
authors.forename: Lise
authors.initials: L
authors.lastname: Musset
authors.affiliation: Genomics and Evolutionary Medicine Unit, Center of Excellence in Malaria, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
authors.forename: Thanat
authors.initials: T
authors.lastname: Chookajorn
authors.affiliation: Department of Microbiology and Immunology, Columbia University Medical Center, New York Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, NY df2260@columbia.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
mesh_terms: D000483:Alleles; D000962:Antimalarials / Q000494:pharmacology; D002738:Chloroquine / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011804:Quinolines; D002738:Chloroquine; C037219:quinoline
keywords: Plasmodium falciparum malaria; chloroquine; drug resistance; evolutionary genetics; pfcrt chloroquine resistance transporter.; transfection