Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum

Abstract

Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

Figure 1. pfcrt allelic exchange strategy and molecular characterization of clones.

(A) Schematic representation of single-site crossover between the transfection plasmid pBSD-7G8 and the endogenous pfcrt allele, leading to expression of a recombinant allele containing the 7G8 polymorphisms (black circles), transcribed from the endogenous 3.0 kb full-length promoter. In some parasites the downstream plasmid sequence integrated as tandem linear copies (delineated as square brackets with a copy number n≥0). The distal truncated locus harbored the Δ5′ UTR, which was previously found in luciferase assays to have minimal activity. E, EcoRI; B, BglII. (B) Southern blot hybridization of EcoRI/BglII–digested genomic DNA samples hybridized with a pfcrt probe from the 5′ UTR and exon 1 region (depicted in panel A). (C) PCR analyses of the recombinant clones and parental lines. (D) Transcript levels from the functional and truncated pfcrt loci (terminated by Py3′ and Pf3′ UTRs respectively in the case of the recombinant clones). Data are presented as a percentage of total pfcrt transcript levels normalized against the respective WT control (3D7 or D10). (E) Western blot analysis of recombinant and parental lines, probed with antibodies to PfCRT or the ER-Golgi marker PfERD2 . (F) Signals were quantified, normalized against PfERD2, and expressed as a proportion of the signals obtained in the appropriate parental line. (B–F) Lanes: 1-3D7, 2-3D7^C, 3-3D7^7G8-1, 4-3D7^7G8-2, 5-D10, 6-D10^C, 7-D10^7G8-1, and 8-D10^7G8-2. GC03 clones were also confirmed by PCR, sequencing, and Southern hybridization, and were found to have similar levels of pfcrt RNA and protein expression as compared with the 3D7 and D10 clones (data not shown).

Figure 2. In vitro response of pfcrt-modified clones to chloroquine and its primary metabolite monodesethylchloroquine.

In vitro [³H]-hypoxanthine incorporation assays were performed with the WT, control, and mutant pfcrt clones. All lines were tested in duplicate against CQ±VP and mdCQ±VP an average of 7 times (range 4–11; see summary in Table S1). Mean±SEM IC₅₀ values are presented for (A) CQ and (B) its metabolite mdCQ. Statistical comparisons comparing mutant pfcrt-modified lines against recombinant control lines of the same genetic backgrounds were performed using one-way ANOVA with a Bonferroni post-hoc test. **P<0.01; ***P<0.001. (C–E) Percent inhibition of growth (shown as means±SEMs derived from all assays) across a range of CQ concentrations for (C) 3D7, (D) D10, and (E) GC03 lines.

Figure 3. CQ recrudescence data for pfcrt -modified and parental clones.

Lines were subjected to 50 nM or 80 nM CQ for 6 days and assayed for recrudescence every 2–3 days from days 7–30. Pooled data from two independent experiments were plotted as the percent of positive wells as a function of time post-CQ exposure. The panels show (A) 3D7 and (B) D10 clones and controls, including recombinant clones pretreated with 50 or 80 nM CQ. All no-treatment controls were positive on day 7 (as shown for 7G8+80 nM CQ), as was GC03^7G8-1 treated with both 50 nM and 80 nM CQ.

Figure 4. Characterization of the French Guiana isolates G224 and H209.

(A–B) In vitro [³H]-hypoxanthine incorporation assays against CQ±VP (A) and mdCQ±VP (B) were performed with a CQ-sensitive (3D7) and a CQ-resistant (Dd2) control line. All lines were tested in duplicate on average 6 times (range 3–8). Mean±SEM IC₅₀ values were derived by linear extrapolation. (C) Percent inhibition of growth (means±SEM derived from all assays) determined across a range of CQ concentrations. (D) Lines were exposed to 50 nM or 80 nM CQ for 6 days and assayed for recrudescence every 2–3 days from days 7–30. All no-treatment controls were positive on day 7 (as shown for 7G8+80 nM CQ).

Figure 5. In vitro response of pfcrt-modified clones and French Guiana isolates to clinically important antimalarials.

In vitro [³H]-hypoxanthine incorporation assays were performed in duplicate an average of 6 separate times (range 3–12 independent assays). Mean±SEM IC₅₀ values are presented for (A) quinine (QN), (B) artemisinin (ART), (C) monodesethyl-amodiaquine (mdADQ), (D) lumefantrine (LMF), and (E) piperaquine (PIP). Statistical comparisons comparing mutant pfcrt-modified lines against recombinant control lines of the same genetic backgrounds were performed using unpaired student t tests. *P<0.05; **P<0.01. For brevity, a single recombinant mutant clone is presented for each host strain. Results from additional recombinant mutant lines are included in Table S1.