Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy

We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400-10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as "delayed death". Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR.