Title: Dissecting the molecular basis of PfCRT-mediated antimalarial drug resistance
Description: The protozoan parasite Plasmodium falciparum is responsible for the deadliest form of malaria, which causes 584,000 fatalities annually and whose complications include coma, anemia, respiratory distress, and renal failure. Although malaria eradication efforts were hindered by the rise of chloroquine (CQ) resistance (CQR), CQ continues to be clinically deployed in resistance-free regions. CQR is primarily mediated by mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene, which also modulates parasite susceptibility to first-line artemisinin-based combination therapies (ACTs). In certain geographical regions (e.g. Africa), mutant pfcrt alleles display considerable fitness costs and have undergone attrition in the absence of CQ pressure. Surveillance of resistant field isolates presently centers on the PfCRT mutation K76T, ubiquitous among CQ-resistant parasites and always accompanied by ≥3 additional mutations. Despite the global adoption of K76T as a molecular marker of CQR, the contributions of this and other mutations to P. falciparum drug resistance versus fitness had not been previously defined.

AIMS: We aimed to address the following: (1) Do PfCRT mutations beyond PfCRT K76T directly contribute to CQR? (2) Do PfCRT mutations contribute to parasite fitness during the pathogenic asexual blood stage? (3) Are there predictable mutational paths in the evolution of pfcrt-mediated drug resistance? (4) How do PfCRT mutations impact current antimalarials, including the first-line ACTs? APPROACH: Using zinc finger nucleases, we generated isogenic, pfcrt-modified blood-stage P. falciparum parasites encoding wild-type (CQ-sensitive) or variant PfCRT haplotypes. Variants included a combinatorial library of alleles harboring 1-4 mutations comprising the simplest CQ-resistant haplotype (Ecu1110). Additional genetic dissections of full-length or partial pfcrt alleles encompassed the most common variants found in Africa and Asia, including a unique fitness-neutral mutant allele (Cam734) that has undergone expansion in Southeast Asia. Parasite antimalarial drug susceptibility was determined using IC50-based (cytostatic) assays or parasite survival-based (cytocidal) assays and was combined with data from flow cytometric parasite growth competition assays to computationally model mutant pfcrt evolution.

To further define the biochemical impacts of PfCRT mutations, our studies leveraged metabolomic, heme fractionation, and drug transport studies. RESULTS: Key findings emerging from our studies included the following: (1) PfCRT K76T is insufficient for CQR and an inaccessible first mutational step in pfcrt evolution; (2) Alongside proliferation rates, parasite resistance gains dictate a constrained pfcrt mutational landscape and predict important roles for the active metabolites of CQ and amodiaquine in guiding pfcrt evolution; (3) To various degrees, PfCRT polymorphisms beyond K76T increase the potency of both the artemisinin and partner drug components of first-line ACT regimens; (4) Emerging PfCRT mutations (e.g. A144F) directly contribute to the enhanced fitness of pfcrt alleles and are necessary for multidrug resistance, independent of K76T. CONCLUSIONS: Our studies uncovered multiple pleiotropic contributions of PfCRT mutations to antimalarial drug resistance, countering earlier dogma that non-K76T mutations are merely compensatory.

Evolutionary modeling revealed parasites’ ability to navigate constrained mutational landscapes and evolve drug resistance via rare mutational bursts. These results collectively highlight the capacity of PfCRT to acquire novel mutations that successfully balance parasite multidrug resistance with the essential role of PfCRT in maintaining digestive vacuole physiology. Our studies are of direct relevance to the regional recommendations of antimalarials, whose activity is influenced by, and in certain cases enhanced against, pfcrt-mutant parasites.