Research Article: Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

Date Published: November 10, 2016

Publisher: Public Library of Science

Author(s): Stanislaw J. Gabryszewski, Satish K. Dhingra, Jill M. Combrinck, Ian A. Lewis, Paul S. Callaghan, Matthew R. Hassett, Amila Siriwardana, Philipp P. Henrich, Andrew H. Lee, Nina F. Gnädig, Lise Musset, Manuel Llinás, Timothy J. Egan, Paul D. Roepe, David A. Fidock, Xin-zhuan Su.

http://doi.org/10.1371/journal.ppat.1005976

Abstract

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.

Partial Text

Human malaria remains a leading global health scourge in part due to multidrug resistance mechanisms evolved by Plasmodium falciparum, the protozoan species responsible for the most severe forms of disease [1]. Artemisinin-based combination therapies (ACTs) are the current first-line means of controlling pathogenic asexual blood-stage infections, including ones dominated with drug-resistant strains that arose during previous selective sweeps resulting from the global use of chloroquine (CQ) and sulfadoxine-pyrimethamine [2–4]. The 4-aminoquinoline compound CQ was especially pivotal earlier in reducing mortality rates [5]. However, the multi-focal emergence and spread of CQ resistance (CQR) contributed to stalled control measures and substantial increases in malaria-associated hospitalizations and deaths [6]. Nevertheless, owing to its safety, affordability, and established efficacy against non-resistant parasites, CQ continues to be deployed in regions that are free of CQR or that harbor CQ-sensitive P. vivax [7]. Interestingly, studies of infections with CQ-resistant P. falciparum strains in Guinea-Bissau recently revealed a ~5–fold increase in CQ efficacy upon doubling the standard dose in children aged <5 years, the age demographic at highest risk for malaria mortality [8]. These findings coincide with renewed efforts to delineate the molecular basis of resistance to antimalarials bearing the hallmark CQ-type quinoline moiety [9]. To ensure successful progression through their life cycle, drug-resistant P. falciparum parasites must balance the acquisition of resistance properties with the maintenance of required and often interrelated physiological processes. Focusing on the pathogenic intraerythrocytic stages of parasite growth, we explored herein how novel mutations comprising the unusually polymorphic Cam734 PfCRT variant contribute to this complex relationship. Our analysis of isogenic, pfcrt-modified lines reveals that multiple PfCRT mutations possess dual roles, contributing to both quinoline resistance and parasite proliferation. This was most notable for the A144F mutation that is unique to Cam734 PfCRT, which in addition to affecting growth rates proved to be indispensable for parasite resistance to multiple quinoline-type compounds, including CQ, QN, and the first-line ACT partner drug AQ. While these drug IC50 shifts are often relatively small, studies have shown that these translate into clear patterns of selection in field parasite populations [7,12]. The pleotropic requirement for the A144F mutation in Cam734 PfCRT-mediated drug resistance is reminiscent of earlier work, in which back-mutation of K76T ablated CQR and nearly halved the degree of parasite resistance to QN [52]. GC03Cam734 F144A parasites appeared CQ-sensitive, but nonetheless showed a 3–fold higher IC90 value for md-CQ, believed to be the major driver of selection for mutant pfcrt [16], relative to the fully sensitive GC03GC03 line (S2 Table). By comparison, GC03Cam734 parasites showed a 27–fold md-CQ IC90 increase. Similar findings were earlier observed with a PfCRT variant of 7G8 that carries the C350R mutation (the H209 isolate found in French Guiana) [50,60]. This variant was shown to mediate a phenotype of CQ tolerance, which manifested as low CQ IC50 values but elevated md-CQ IC90 values as well as parasite recrudescence after exposure to CQ concentrations lethal to CQ-sensitive parasites expressing wild-type pfcrt [50,60]. We note that parasites encoding Cam734 F144A PfCRT retained K76T as well as 7 other mutations [7]. The clear importance of mutations other than K76T in contributing to CQR can help explain, in areas where novel PfCRT variants have arisen, why the K76T mutation predicts clinical CQR with good sensitivity but only moderate specificity [17,62]. Another important factor driving the reduced specificity of the K76T marker is patient immunity, which in higher-transmission settings of Africa is known to help resolve CQ-resistant infections in CQ-treated patients [78].   Source: http://doi.org/10.1371/journal.ppat.1005976

 

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