Shafik SH , Cobbold SA , Barkat K , Richards SN , Lancaster NS , Llinás M , Hogg SJ , Summers RL , McConville MJ , Martin RE .

DP2 OD001315 NIH HHS

             cellular response to drug
             cellular response to chloroquine

                Plasmodium falciparum malaria

	Fig. 1: Screening of a solute library for cis-inhibitory activity identified potential substrates of PfCRT. a Schematic showing the cis-inhibition of [3H]CQ uptake via PfCRTDd2 or PfCRTEcu1110 by an unlabelled solute in the Xenopus oocyte system. The library included solutes known to exist in the parasitised erythrocyte, such as vitamins, carbohydrates, metal ions, lipids, amino acids, peptides and other organic and inorganic molecules. b, c The effect on [3H]CQ transport via PfCRT by a subset of solutes (2 mM) (b) or host-derived peptides (2 mM) (c). Non-expressing oocytes and those expressing PfCRT3D7 were included as negative controls; these oocytes accumulate a low level of [3H]CQ via simple diffusion of the neutral species15,16, reflecting the background level of [3H]CQ transport. Note that aspartate and vitamins E and B6 caused modest reductions in PfCRTDd2-mediated transport, but only aspartate was found to inhibit PfCRTEcu1110. A diverse range of peptides cause potent inhibition of both PfCRTDd2 or PfCRTEcu1110. d Heatmap of the cis-inhibition of [3H]CQ transport via PfCRTDd2 and PfCRTEcu1110 by 173 different solutes. e VF-6 (top) and HM-5 (bottom) inhibit [3H]CQ transport via PfCRTDd2 in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC50) for VF-6 and HM-5 are 207 ± 12 and 167 ± 14 µM, respectively. The data are the mean of n = 4 independent experiments (each yielding similar results and overlaid as individual data points in b and c), and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from the relevant PfCRTDd2 (red asterisks) or PfCRTEcu1110 (orange asterisks) control; *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). The source datasets are provided as a Source Data file.
	Fig. 2: PfCRT is trans-stimulated by peptides containing 4–11 amino acid residues. a Schematic showing the trans-stimulation of [3H]CQ transport via PfCRT by an unlabelled substrate in Xenopus oocytes. b, cTrans-stimulation of [3H]CQ transport via PfCRTDd2 or PfCRTEcu1110 (b) or via PfCRT3D7 (c) by a subset of host-derived peptides and peptide mimics (45 mM). The test solute was injected into the oocyte immediately prior to the commencement of the assay, with the buffer-only and LH treatments serving as injection controls21. The negative controls were non-expressing oocytes and those expressing an unrelated P. falciparum transporter, the nucleoside transporter 1 (PfNT175,76). The latter demonstrates that the expression of a similarly sized transporter in Xenopus oocytes does not affect the ability of the oocyte membrane to reseal following injection of a test solute21. dTrans-stimulation of [3H]CQ transport via PfCRT3D7 by VF-6 (45 mM) is pH-dependent, with the largest increase observed when the pH of the injection buffer was 5.5 (equating to 79 fmoles H+). eTrans-stimulation of [3H]CQ transport via PfCRT is unaffected by the removal of Na+ from the injection buffer. f Concentration-dependence of the trans-stimulation of [3H]CQ transport via PfCRT by VF-6 (left) and HM-5 (right). The non-expressing oocyte data overlays the data obtained with oocytes expressing PfNT1. g Heatmap of the trans-stimulation of [3H]CQ transport via PfCRT3D7, PfCRTEcu1110 or PfCRTDd2 by 173 different solutes. Only peptides and peptide mimics trans-stimulate PfCRT; all other solutes, including aspartate and vitamins E and B6, failed to increase [3H]CQ transport via PfCRT are thus unlikely to be substrates of the transporter. The data are the mean of n = 4 independent experiments (each yielding similar results and overlaid as individual data points in b, c and e), and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from the relevant PfCRTDd2 (red asterisks), PfCRTEcu1110 (orange asterisks) or PfCRT3D7 (blue asterisks) buffer-injected control; *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant (one-way ANOVA). SQV: saquinavir. The source datasets are provided as a Source Data file.
	Fig. 3: Characterisation of the transport of a host-derived hexapeptide via PfCRT. a Schematic showing the transport of [3H]VF-6 via PfCRT in Xenopus oocytes. b–e [3H]VF-6 transport via PfCRT is approximately linear with time for at least 2 h (b), is dependent on both the pH of the injection buffer (the highest level of transport occurred at pH 5.5, equating to 79 fmoles H+) (c) and the membrane potential (with the rate of VF-6 efflux steadily decreasing as the membrane potential became more positive) (d), and is saturable (PfCRT3D7 possesses a slightly lower affinity and higher Vmax relative to the mutant transporters) (e). [3H]CQ influx is also dependent on the membrane potential (Supplementary Fig. 2g and Supplementary Note 3). f, gCis-inhibition of [3H]VF-6 transport via PfCRT by verapamil (VP) (f) and Q2C (g). h The half-maximum inhibitory concentrations (IC50s) for the cis-inhibition of [3H]VF-6 transport via PfCRT by drugs known to interact with the transporter. iTrans-stimulation of [3H]VF-6 transport via PfCRT by saquinavir (SQV). jTrans-inhibition of [3H]VF-6 transport via PfCRT by haemoglobin-peptides (45 mM). k Left: various other radiolabelled solutes are not transported out of the oocyte by PfCRT. Right: in the same assay, [3H]CQ is effluxed via PfCRTEcu1110 and PfCRTDd2 and [3H]hypoxanthine is effluxed by PfNT1. The data are the mean of n = 5 independent experiments (each yielding similar results and overlaid as individual data points in j and k), and the error is the SEM. Where not visible, the error bars fall within the symbols. The non-expressing oocyte data overlays the data obtained with oocytes expressing PfNT1 in b, c, e–g and i. The asterisks denote a significant difference from the relevant PfCRTDd2 (red asterisks), PfCRTEcu1110 (orange asterisks), PfCRT3D7 (blue asterisks) or non-expressing (black asterisks) control; *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant (one-way ANOVA). The source datasets are provided as a Source Data file.
	Fig. 4: Diverse field and laboratory-derived isoforms of PfCRT transport the VF-6 peptide. a The VF-6 transport activities, including kinetic parameters, of various field and laboratory-derived PfCRT isoforms in Xenopus oocytes (Km, Michaelis–Menten constant; Vmax, maximum velocity). b The capacity of PfCRT to transport VF-6 decays exponentially as the protein’s CQ transport activity increases (R2 = 0.93). Exceptions to this trend include PfCRTCam734, L272F-PfCRTDd2, L272F-PfCRT3D7 and C101F-PfCRTDd2. c [3H]CQ transport (left) and [3H]VF-6 transport (right) via epitope-tagged versions of PfCRT3D7 and PfCRTDd2. The version of PfCRTDd2 carrying a C-terminal 3xmyc tag does not mediate [3H]CQ transport. The other four variants of PfCRTDd2 retain all or most of their CQ transport activity. By contrast, none of the tagged versions of PfCRT3D7 or PfCRTDd2 transport [3H]VF-6. The fusion of polypeptides to PfCRT can therefore abolish its ability to transport peptides, even when the protein remains able to transport CQ. The data are the mean of n = 5 independent experiments (each yielding similar results and overlaid as individual data points in a and c), and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from the relevant PfCRTDd2 (red asterisks) or PfCRT3D7 (blue asterisks) control; *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). The source datasets are provided as a Source Data file.
	Fig. 5: Peptide mimics are substrates of PfCRT in situ. a Left: schematic showing the H+-efflux assay. If a protonated solute is a substrate of PfCRT it will cause a H+ leak when effluxed from the DV. Inhibition of the DV’s H+-ATPase by concanamycin A enables this H+ leak to be detected (with a pH-sensitive probe) as an increase in the rate of DV alkalinisation. Right: several peptide mimics (10 μM) increase the rate of DV alkalinisation in C2GC03, C67G8 and C4Dd2 parasites. CQ (the positive control) causes the rate of DV alkalinisation to increase in the C67G8 and C4Dd2 lines and to decrease slightly in C2GC03 parasites. This is consistent with previous applications of the H+-efflux assay36,37 and with the abilities of PfCRTEcu1110, PfCRT7G8 and PfCRTDd2 (but not PfCRT3D7) to transport CQ when tested under normal conditions in the oocyte system15,16. Unless labelled ns, P < 0.05 relative to the absence of a test solute. b Left: schematic showing the inhibition of PfCRT by verapamil (VP) or the quinine dimer Q2C in the H+-efflux assay. Right: the increase in the rate of DV alkalinisation caused by saquinavir (SQV; 10 μM) and Ac-YF-5-NH2 (10 μM) is inhibited by VP (50 μM) and Q2C (1 μM). The statistical analyses were performed relative to the SQV or Ac-YF-5-NH2 controls. c SQV (left) and Ac-YF-5-NH2 (right) increase the rate of DV alkalinisation in a concentration-dependent manner. d Concentration-dependence of the trans-stimulation of [3H]CQ transport via PfCRT by SQV (left) and Ac-YF-5-NH2 (right) in Xenopus oocytes. The non-expressing oocyte data overlays the data obtained with oocytes expressing PfNT1. The data are the mean of n = 5 independent experiments (each yielding similar results and overlaid as individual data points in a and b), and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from the relevant C4Dd2 (red asterisks), C67G8 (orange asterisks) or C2GC03 (blue asterisks) control; *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant (one-way ANOVA). The source datasets are provided as a Source Data file.
	Fig. 6: CQ-resistance-conferring isoforms of PfCRT increase parasite susceptibility to saquinavir. a The antiplasmodial activities of CQ and saquinavir (SQV) against CQ-sensitive (C2GC03 and 3D7) and CQ-resistant (C67G8, C4Dd2, 7G8 and Dd2) parasites in the presence or absence of verapamil (VP). The data are the mean of n = 5 independent experiments (each yielding similar results) and the error is the SEM. The asterisks denote a significant difference from the relevant control (red asterisks, C4Dd2 or Dd2 control; orange asterisks, C67G8 or 7G8 control; blue asterisks, C2GC03 or 3D7 control); **P < 0.01, ***P < 0.001, ns: non-significant (one-way ANOVA). The source datasets are provided as a Source Data file. b Mechanistic model for the increased susceptibility of CQ-resistant parasites to SQV. PfCRT3D7 (wild-type PfCRT) lacks detectable CQ transport activity, whereas the CQ-resistance-conferring isoforms of PfCRT (mutant PfCRT) efflux CQ from the DV. VP inhibits CQ transport via the mutant transporters, thereby re-sensitising the CQ-resistant parasites to this antimalarial drug. By contrast, PfCRT3D7 has a greater capacity for transporting SQV out of the DV than do the mutant isoforms. Moreover, it is the wild-type protein that confers reduced sensitivity to SQV. Given that VP (1) inhibits SQV transport via both the wild-type and mutant PfCRT transporters and (2) increases the antiplasmodial activity of SQV in all of the parasite types, our findings indicate that SQV acts on a target within the DV.
	Fig. 7: The peptide substrates of PfCRT accumulate in parasites expressing mutant isoforms of the transporter. Host-derived peptides in erythrocytes infected with C2GC03 or C4Dd2 parasites were quantified using tandem liquid-chromatography mass-spectrometry and the peptide levels within the C4Dd2 line were expressed relative to those measured in the C2GC03 parasites (Supplementary Data 3 and 5). For peptides containing 4–11 residues, a positive relationship exists between the ability to trans-stimulate CQ transport via PfCRT3D7 and accumulation within the CQ-resistant C4Dd2 parasites. The analysis used the PfCRT3D7trans-stimulation dataset, rather than that generated for PfCRTDd2, because the peptides most likely to accumulate in the C4Dd2 line will include those that are very poor substrates of (or no longer transported by) PfCRTDd2. An analysis of the data with a Bayesian Information Criteria model identified two distinct populations. The trans-stimulation data are the mean of four independent experiments (each yielding similar results) and the peptide accumulation data are the mean of 2–6 independent experiments (each yielding similar results). Error bars are shown for data points that are n ≥ 3; the Y error is the SD and the X error is the SEM. The source datasets are provided as a Source Data file.
	Fig. 8: Model for the natural function and physiological role of PfCRT. PfCRT transports host-derived peptides 4–11 residues in length, and of varying composition and charge, out of the DV and into the parasite’s cytosol. Wild-type PfCRT transports a broader range of peptides, and has a higher capacity for peptide transport, than most CQ-resistance-conferring isoforms of PfCRT. The diminished abilities of the mutant transporters to efflux peptides results in the accumulation of these peptides within CQ-resistant parasites. Moreover, the promiscuous degradation of the accumulated peptides leads to the build-up of small peptide fragments and amino acids, which exerts further osmotic pressure upon the DV and causes feedback inhibition of the digestion of host proteins. PfCRT therefore serves two roles: (1) it prevents osmotic stress of the DV by exporting peptides and (2) it delivers these peptides to the cytosol where they are degraded into amino acids to fuel parasite growth. AAT1: amino acid transporter 1.

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