Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology.
Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens.
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America.
Stanislaw J
SJ
Gabryszewski
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America.
Satish K
SK
Dhingra
Division of Pharmacology, Department of Medicine, University of Cape Town, Cape Town, South Africa.
Jill M
http://orcid.org/0000-0003-0936-1761
JM
Combrinck
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Ian A
IA
Lewis
Departments of Chemistry and of Biochemistry and Cellular & Molecular Biology, Georgetown University, Washington, DC, United States of America.
Paul S
PS
Callaghan
Departments of Chemistry and of Biochemistry and Cellular & Molecular Biology, Georgetown University, Washington, DC, United States of America.
Matthew R
MR
Hassett
Departments of Chemistry and of Biochemistry and Cellular & Molecular Biology, Georgetown University, Washington, DC, United States of America.
Amila
A
Siriwardana
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America.
Philipp P
PP
Henrich
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America.
Andrew H
AH
Lee
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America.
Nina F
NF
Gnädig
Laboratoire de Parasitologie, WHO Collaborating Center for Surveillance of Anti-Malarial Drug Resistance, Institut Pasteur de la Guyane, Cayenne, French Guiana.
Lise
L
Musset
Departments of Biochemistry & Molecular Biology and Chemistry, Center for Malaria Research and Center for Infectious Diseases Dynamics, Pennsylvania State University, University Park, Pennsylvania, United States of America.
Manuel
M
Llinás
Department of Chemistry, University of Cape Town, Cape Town, South Africa.
Timothy J
TJ
Egan
Departments of Chemistry and of Biochemistry and Cellular & Molecular Biology, Georgetown University, Washington, DC, United States of America.
Paul D
PD
Roepe
Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, United States of America.|Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, New York, United States of America.
David A
http://orcid.org/0000-0001-6753-8938
DA
Fidock
D000634:Aminoquinolines / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics*; D056084:Genetic Fitness / Q000235:genetics*; D005838:Genotype; D006801:Humans; D016778:Malaria, Falciparum / Q000235:genetics*; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010963:Plasmodium falciparum / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics*; D014617:Vacuoles / Q000378:metabolism
D000634:Aminoquinolines; D000962:Antimalarials; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; C001920:4-aminoquinoline