Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America.

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Antimalarial drugs influence the pH dependent solubility of heme via apparent nucleation phenomena.

Recently, we measured a more acid digestive vacuolar pH for drug resistant Plasmodium falciparum [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press; Ursos LMB, Dzekunov S, Roepe PD. Mol Biochem Parasitol 2000;in press]. We suggested this acidification contributes to drug resistance via the profound effects that pH has on the solubility of unpolymerized heme found in the vacuole (ferriprotoporphyrin IX mu oxo dimers). In this report, we measure how FPIX concentration, time, NaCl concentration, and several antimalarial drugs affect FPIX pH dependent solubility. Aggregation is essentially instantaneous below pH 5.3, but at vacuolar pH previously measured for HB3 parasites [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press] can increase to several minutes as NaCl is lowered. As FPIX is decreased, the midpoint of the pH dependent solubility curve shifts to higher values. Addition of antimalarial drugs also increases the midpoint of the pH dependent FPIX solubility curve, with the net shift proportional to the relative affinity of the drug for FPIX. Surprisingly, however, for all drugs tested shifts of essentially identical magnitude are found at all drug: FPIX molar ratios inspected, spanning eight orders of magnitude (to as low as 0.0000001:1). This suggests that changes in pH dependent FPIX solubility by addition of antimalarial drugs is via previously unrecognized drug/FPIX nucleation phenomena. These data could have important implications for understanding the role of previously observed changes in pH(vac) [Dzekunov S, Ursos LMB, Roepe PD. Mol Biochem Parasitol 2000;in press; Ursos LMB, Dzekunov S, Roepe PD. Mol Biochem Parasitol 2000;in press] upon development of antimalarial drug resistance.

Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance.

The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

Digestive vacuolar pH of intact intraerythrocytic P. falciparum either sensitive or resistant to chloroquine.

We present the first single cell-level analysis of digestive vacuolar pH for representative chloroquine resistant (strain Dd2) versus sensitive (strain HB3) malarial parasites. Human red blood cells harboring intact intraerythrocytic parasites were attached to glass substrate, continuously perfused with appropriate buffer, and pH was analyzed via single cell imaging and photometry techniques. We find that digestive vacuolar pH (pH(vac)) is near 5.6 for HB3 parasites. Surprisingly, we also find that pH(vac) of Dd2 is more acidic relative to HB3. Notably, in vitro pH titration of hematin confirms a very steep transition between soluble heme (capable of binding chloroquine) and insoluble heme (not capable of binding chloroquine, but still capable of polymerization to hemozoin) with a distinct midpoint at pH 5.6. We suggest the similarity between the hematin pH titration midpoint and the measured value of HB3 pH(vac) is not coincidental, and that decreased pH(vac) for Dd2 titrates limited initial drug target (i.e. soluble heme) to lower concentration. That is, changes in pH(vac) for drug resistant Dd2 relative to drug sensitive HB3 are consistent with lowering drug target levels, but not directly lowering vacuolar concentrations of drug via the predictions of weak base partitioning theory. Regardless, lowering either would of course decrease the efficiency of drug/target interaction and hence the net cellular accumulation of drug over time, as is typically observed for resistant parasites. These observations contrast sharply with the common expectation that decreased chloroquine accumulation in drug resistant malarial parasites is likely linked to elevated pH(vac,) but nonetheless illustrate important differences in vacuolar ion transport for drug resistant malarial parasites. In the accompanying paper (Ursos, L. et al., following paper this issue) we describe how pH(vac) is affected by exposure to chloroquine and verapamil for HB3 versus Dd2.

The effects of chloroquine and verapamil on digestive vacuolar pH of P. falciparum either sensitive or resistant to chloroquine.

In the preceding paper, we present a novel method for measuring the digestive vacuolar pH (pH(vac)) of the malarial parasite Plasmodium falciparum, and show that, surprisingly, pH(vac) is lower for chloroquine resistant (CQR) Dd2 parasites relative to chloroquine sensitive (CQS) HB3. These data may have important consequences for elucidating mechanisms of antimalarial drug resistance and for developing new antimalarial therapy. Additional issues central to a better understanding of antimalarial pharmacology and antimalarial drug resistance require detailed comparative data on the effects of key drugs and other compounds on parasite biophysical parameters such as pH(vac), measured under close-to-physiologic conditions. Since the methods we develop in the previous paper allow us to record fluorescence signals from spatially well-defined regions of the living parasite while they are under continuous perfusion, it is relatively straightforward for us to test how antimalarial drugs (e. g. chloroquine, CQ) and other compounds (e.g. the chemoreversal agent verapamil [VPL]) affect pH(vac). In this paper, we measure both short term (i.e. initial perfusion conditions) and longer-term effects of CQ and VPL for living, intraerythrocytic CQS (HB3) and CQR (Dd2) malarial parasites under constant perfusion with physiologically relevant buffers. We find that VPL normalizes pH(vac) for Dd2 to a value near that measured for HB3, but has no effect on pH(vac) for HB3. Longer term CQ exposure is found to alter pH(vac) for HB3 but not Dd2, and short-term exposure to the drug has no significant effect in either strain. The results may help resolve longstanding debate regarding the effects of CQ and VPL on parasite physiology, and further support our evolving hypothesis for the mechanism of CQ resistance.