Acidification of the digestive vacuole in Plasmodium falciparum malarial parasites is linked to chloroquine resistance

This investigation presents a novel method for measuring the digestive vacuolar pH (pH_vac) of the malarial parasite Plasmodium falciparum, and shows that, surprisingly, pH_vac is likely lower for chloroquine resistant (CQR) parasites relative to chloroquine sensitive (CQS). 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. I suggest that the similarity between the hematin pH titration midpoint and the measured value of pH_vac for CQS parasite is not coincidental, and that decreased pH_vac for CQR parasites 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 parasites are consistent with lowering drug target levels, but not directly lowering vacuolar concentrations of drug via the predictions of weak base partitioning theory. I also find that verapamil (VPL) normalizes pH_vac for some CQR parasites to a value near that measured for CQS parasites but has less effect on pH _vac of CQS parasites. In addition, longer term CQ exposure is found to alter pH_vac for HB3 (CQS strain) but not Dd2 (CQR strain), and short-term exposure to the drug has no significant effect in either strain. Other studies using spectroscopic methods show that heme precipitation is instantaneous below pH 5.3 and that increase in [NaCl] promotes this phenomenon. Nuclear magnetic resonance (NMR) spectroscopy techniques have also been employed to examine the interaction between heme and several quinoline antimalarials. These studies greatly help in the resolution of the mechanism of drug resistance in malarial parasites.