Pulcini S , Staines HM , Lee AH , Shafik SH , Bouyer G , Moore CM , Daley DA , Hoke MJ , Altenhofen LM , Painter HJ , Mu J , Ferguson DJ , Llinás M , Martin RE , Fidock DA , Cooper RA , Krishna S .

R01 AI050234 NIAID NIH HHS , R01 AI071121 NIAID NIH HHS , T32 AI106711 NIAID NIH HHS , R01 AI50234 NIAID NIH HHS , R37 AI050234 NIAID NIH HHS

	Figure 1. PfCRT mutations.(a) Schematic representation of PfCRT and positions of previously identified polymorphisms871 from field isolates (green circles) and from drug-pressured laboratory lines (purple circles). The critical CQ resistance mutation site (K76) is shaded red, and the two residues at which mutations are described in this study are shaded in orange (C101) and blue (L272). (b) PfCRT haplotypes included this study.
	Figure 2. Representative morphology of parasite lines FCBC101F and 3D7L272F.(a) Appearance of enlarged FVs in fixed FCBC101F parasites (left panel), when compared with parental FCB parasites of similar developmental stages (right panel). (b) Images of live FCBC101F and FCB trophozoite-stage parasites, using bright-field and dark-field microscopy (left and right panels, respectively). (c) Appearance of enlarged FVs in fixed 3D7L272F parasites (left panel), when compared with parental 3D7 parasites of similar developmental stages (right panel). The diameter of a RBC is ~7 μm.
	Figure 3. Morphological comparisons of parasites with mutations in pfcrt compared with their parental controls.(a) Parasites were synchronized by sorbitol lysis and areas of FVs and parasites (approximately 38 h post-invasion) were measured and expressed as ratios (AFV/AParasite). 3D7, FCB and Dd2Dd2 (open bars; n = 72, 31 and 42, respectively) and 3D7L272F, FCBC101F and Dd2Dd2 L272F (closed bars; n = 84, 58 and 88, respectively) parasites were analyzed and significant enlargement of the FV was confirmed in 3D7L272F, FCBC101F and Dd2Dd2 L272F parasites relative to 3D7, FCB and Dd2Dd2, respectively (*p < 0.0001: two-tailed, unpaired, Student’s t-test). (b,c) Transmission electron micrographs of 3D7 and 3D7L272F parasites, respectively, showing the food vacuole (FV) and nucleus (N). Note the enlarged electron lucent FV in 3D7L272F (suggesting changes in the process of hemoglobin degradation and formation of hemozoin crystals). RBCs infected with 3D7L272F displayed approximately 7.5-fold more knobs (arrowheads) on the host surface than 3D7-infected RBCs, although this is likely due to sub-population selection rather than a direct link to the mutation in pfcrt. (insert) Detail of a knob. Bars represent 1 μm (b,c) and 100 nm (insert).
	Figure 4. Introduction of PfCRT L272F into Dd2.(a) Schematic of zinc-finger nuclease (ZFN)-mediated generation of vacuole-enlarged parasites. Dd2 parasites were first enriched for the episomal pcrtDd2 L272F-hdhfr or pcrtDd2-hdhfr donor plasmids (latter not shown). The donor plasmids encoded a cDNA copy of the Dd2 pfcrt allele (dark blue, plasmid), either L272F-mutated (dark blue bump) or wild-type (not shown), followed by a dhfr selection cassette (light grey). Each donor-enriched parasite was then transfected with the pZFNpfcrt-bsd plasmid, expressing the genomic (light blue) pfcrt intron 1-targeting ZFN pair (ZFN L and ZFN R, orange) and the bsd selection cassette (dark grey). ZFN-induced recombination in pfcrt yielded either control Dd2Dd2 (not shown) or Dd2Dd2 L272F parasites (dark blue, locus). (b) PCR verification of parental, recombinant control, and Dd2Dd2 L272F parasite clones. Primer (p) positions are shown in panel a. (c) Light microscope analysis of representative examples of parental, recombinant control, and experimental parasite clones. Ring morphology for each parasite Dd2, Dd2Dd2, Dd2Dd2 L272F, and GC03 was normal. Progression through the trophozoite and schizont stages showed normal morphological development except for the Dd2Dd2 L272F clone, which exhibited the characteristic enlarged vacuole and diffuse hemozoin phenotypes seen in 3D7L272F and FCBC101F Giemsa stained parasites. (d) Transmission electron micrographs of Dd2Dd2 (i) and Dd2Dd2 L272F parasites (ii) showing similar cytoplasmic appearances except for the enlarged food vacuole (FV) in Dd2Dd2 L272F. Note neither parasite exhibits knobs. This confirms similar morphological appearances to those of 3D7 and 3D7L272F, respectively but without knob formation. N—Nucleus. Bars represent 1 μm.
	Figure 5. CQ transport activity of the C101F and L272F variants of PfCRT in Xenopus oocytes.(a,b) The uptake of [3H]CQ into oocytes expressing PfCRT was measured in the absence (closed bars) or presence of 250 μM VP (light grey bars; a), 100 μM BSD (dark grey bars; b), or 500 μM BSD (open bars; b). Within each experiment, measurements were made from 10 oocytes per treatment and uptake was expressed relative to that measured in the PfCRTDd2-expressing oocytes under control conditions. The normalized data obtained from 4–5 separate experiments (each using oocytes from different frogs) were then averaged and are shown + SEM. Both panels show PfCRT-mediated CQ uptake, calculated by subtracting CQ uptake measured in PfCRT3D7-expressing oocytes (i.e. the component of CQ accumulation attributable to diffusion; see Supplementary Fig. S3) from that measured in oocytes expressing a variant of PfCRT. In the control treatments, the rates of CQ uptake (pmol/oocyte/h; n = 9 ± SEM) in oocytes expressing PfCRTDd2 and PfCRT3D7 were 23.6 ± 2.3 and 1.3 ± 0.2, respectively. ‘ns’ denotes no significant difference (p > 0.05) in CQ accumulation between oocytes expressing a PfCRT variant (in the presence or absence of VP or BSD) and that measured in the PfCRT3D7-expressing oocytes under control conditions.
	Figure 6. Hypothetical schematic model of the effects of PfCRT mutations.The FV is acidified by a vacuolar proton pump to create a suitable environment for hemoglobin digestion. The acidic nature of the FV also leads to near complete diprotonation of CQ, which diffuses across the FV membrane in an uncharged form (CQ) and accumulates as a charged form (either CQH+ or CQH22+, although predominantly CQH22+). CQH22+ interferes with the polymerization of toxic heme to non-toxic hemozoin, which leads to parasite death. In normal CQ-sensitive (CQS) parasites, PfCRT, which contains a positive charge in its pore (K76), exports its natural substrates but little, if any, CQH22+. Thus, CQH22+ accumulates in the FV and causes parasite death. In CQ-resistant (CQR) parasites, the positive change in the pore of PfCRT is lost (K76T) and both its natural substrates and CQH22+ are transported out of the FV. As CQH22+ cannot accumulate in the FV, the parasites become resistant to the drug. In 3D7L272F parasites (where the parent strain is already CQS), the mutation may reduce residual transport of CQH22+ out of the FV even further or completely, leading to a greater FV accumulation of CQH22+ and CQ-hypersensitivity or some other mechanism may be responsible for this phenomenon. The mutation also leads to a reduction in the export of natural substrates, resulting in a build-up of these substrates. This causes water to enter the FV by the process of osmosis, leading to swelling. In FCBC101F and Dd2Dd2 L272F parasites (where the parent strains are CQR), the mutations reduce the export of CQH22+ back towards levels measured in CQS lines and also reduce natural substrate export, leading to normal CQ sensitivity and FV swelling, respectively. Note mutations in PfCRT (orange graphic) are denoted by red transmembrane or loop regions, depending on the location of the amino acid change (see Fig. 1a).

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