We report on the development of a new SYBR Green I-based plate assay for analyzing the activities of antimalarial drugs against intraerythrocytic Plasmodium falciparum. This assay is considerably faster, less labor-intensive, and less expensive than conventional radiotracer (e.g., [3 H]hypoxanthine and [ 3 H]ethanolamine)-based assays or P. falciparum lactate dehydrogenase activity-based assays. The assay significantly improves the pace at which antimalarial drug discovery efforts may proceed.The continued emergence and spread of multidrug-resistant strains of Plasmodium falciparum and P. vivax are arguably the most pressing problems in the area of infectious diseases today. Also, although the recent deciphering of the P. falciparum genome reveals many promising new drug targets, the financial cost of bringing drugs to the clinic is a major obstacle in the development of new antimalarials (6). A faster, less expensive, high-throughput means of screening the activities of drugs against a variety of malarial parasite strains would greatly assist preclinical drug development.Quantitative assessment of the effects of drugs on parasite growth and development can be achieved by direct (but extremely tedious) microscopic examination of blood smears. An alternative assay is measurement of the effect of drug exposure by determination of the level of incorporation of radiolabeled hypoxanthine. While the latter method can be automated, it requires radioactive materials and is not convenient for detection of parasite stage-specific effects. Another assay measures parasite lactate dehydrogenase activity by methods that do not require radioisotopes. However, this assay requires multiple processing steps and expensive reagents and is not particularly cost-effective for large-scale drug screening efforts.We have thus endeavored to develop more rapid and convenient cell-based assays for quantifying antimalarial drug activities. We have strived to enhance simplicity and reduce cost. In this paper, we report on the development of one such assay that relies on the fluorophore SYBR Green I. MATERIALS AND METHODSCell culture. Asexual culture is routinely performed. Parasite cultures are initiated from stabilates preserved in liquid nitrogen (the level of parasitemia during storage is Ն10%). Following the initiation of a fresh culture, at least two full life cycles (96 h) are completed before parasites are used for assays. In general, cultures are synchronized in the laboratory, and assays are initiated when the parasites are at the ring stage. However, we find that this assay is equally applicable to asynchronous culture and that similar 50% inhibitory concentrations (IC 50 s) are calculated from data with asynchronous and synchronous cultures (data not shown). Prior to assay initiation, the level of parasitemia of an aliquot of a stock culture is measured by light microscopy following Giemsa staining or by fluorescence-activated cell sorter analysis after staining with propidium iodide. In general, stock cultures with 5 to 10% paras...

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In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.

Mutations in the Plasmodium falciparum pfcrt gene cause resistance to the 4 -amino quinoline chloroquine (CQ) and other antimalarial drugs. Mutations and/or overexpression of a P. falciparum multidrug resistance gene homologue (pfmdr1) may further modify or tailor the degree of quinoline drug resistance. Recently (M.T. Ferdig et al., Molecular Microbiology 52: 985-997 [2004]) QTL analysis further implicated a region of P. falciparum chromosome 13 as a partner (with pfcrt) in conferring resistance to the first quinoline -based antimalarial drug, quinine (QN). Since QN resistance (QNR) and CQR are often (but not always) observed together in parasite strains, since elevated cytosolic pH is frequently (but not always) found in CQR parasites, and since the chr 13 segment linked to QNR prominently harbors a gene encoding what appears to be a P. falciparum Na + /H + exchanger (PfNHE), we have systematically measured cytosolic pH and PfNHE activity for an extended series of parasite strains used in the QTL analysis. Altered PfNHE activity does not correlate with CQR as previously proposed, but significantly elevated PfNHE activity is found for strains with high levels of QNR, regardless their CQR status. We propose that either an elevated pH cyt or a higher vacuolar pH -to -cytosolic pH gradient contributes to one common route to malarial QNR that is also characterized by recently defined chr 13 -chr 9 pairwise interactions. Based on sequence analysis we propose a model whereby observed polymorphisms in PfNHE may lead to altered Na + /H + set point regulation in QNR parasites.