Quinine localizes to a non-acidic compartment within the food vacuole of the malaria parasite Plasmodium falciparum

Plasmodium falciparum cultures of two clonal parasite strains (3D7 and Dd2) with different pfmdr1 copy numbers and quinine (QN) sensitivity were used in this study. The 3D7 strain has one pfmdr1 copy and is sensitive to QN. The Dd2 strain has four pfmdr1 copies [22] and is resistant to QN. Cultures were maintained in candle jars at 37°C under the Trager and Jensen method for malaria parasite culturing [23]. Media was changed every 24 hours. Type O+ serum at 10% and red blood cells (Research Blood Components, LLC: Boston, MA, USA) at 2% haematocrit in malaria culture media (MCM) [24] were used in culturing and experimental conditions.

Live asynchronous cultures at 5-8% parasitaemia with 1μM of QN were imaged over an 11-hour period using fluorescence microscopy. Parasitized RBC preparations were plated onto 35mm glass bottom dishes (MatTek, Corp.: Ashland, MA, USA) coated with Poly-L-lysine. A coverslip was carefully applied, and the excess fluid gently squeezed out. The coverslip was sealed with warmed Vaseline to prevent the sample from drying out. Time-lapse imaging of multiple parasites was carried out with a Prior Scientific (Prior Scientific: Rockland, MA, USA) motorized x-y stage. An incubator box maintained the microscope work area including the objective at 37°C. Because P. falciparum grows well at 3% oxygen but will tolerate levels as low as 0.5% [25], air was in the incubator box, and the samples were sealed during imaging as stated above. LysoTracker Red DND-99 (Invitrogen: Carlsbad, CA, USA) (50nM) was used to stain the food vacuole and DRAQ5 (Biostatus Limited: Leicestershire, UK) (1μM) was used to stain DNA [26]. Fluorescence images were acquired using an Olympus FV1000 inverted IX81 microscope with a 63x 1.42 NA oil immersion objective at the UNC-Chapel Hill Michael Hooker Microscopy Core Facility. Serial sections with step sizes of 0.20 or 0.25μm were gathered using sequential scanning at 405nm (QN), 559nm (LysoTracker Red), and 635nm (DRAQ5) excitation, and 435±25nm, 595±25nm and 705±50nm emission filters for QN, LysoTracker Red, and DRAQ5 respectively. Confocal pinhole was set to maximum (800μm, ~8 Airy units). Laser transmitted light differential interference contrast (DIC) images were collected at an optimum z-level separately. Image stacks were deconvolved using the iterative blind deconvolution method with Volocity Image Analysis software, version 5.52 (Perkin Elmer: Waltham, MA, USA). Occasionally, Tetraspec 0.1μm beads (Molecular Probes, OR) were added to the sample and used for deconvolution with measured point spread functions. Three-dimensional (3-D) opacity volume rendering and 3-D co-localization analysis was carried out with Volocity.

Two-dimensional (2-D) fluorescence and DIC images of both 3D7 (one pfmdr1 copy) and Dd2 (four pfmdr1 copies) parasites showed that quinine (QN) co-localizes with haemozoin, which suggests localization in the parasite food vacuole (Figure 1). Similar results were found using a Leica SP2 microscope with a 351nm excitation laser (data not shown). The co-localization was highly repeatable and seen in four fields in at least 10 experiments. Parasites were also imaged at earlier time points following the addition of QN (beginning at one hour of exposure through 11 hours). Quinine fluorescence was found in the food vacuole as early as one hour of exposure and remained there for the duration of the experiment (Figure 2). This effect appears to be independent of pfmdr1 copy number as experiments in both strains rendered similar results. The same pattern was seen regardless of whether the infected red cells were maintained on the heated microscope stage or in a candle jar, indicating that photobleaching was not a factor. The parasites did not significantly progress through the intraerythrocytic cell cycle during this 11-hour period consistent with a previously observed maturation delay in the intraerythrocytic cell cycle following QN exposure at IC50 and IC99 concentrations for 12 hours [27]. Thus, the apparent halt in cell cycle progression observed in the current study is likely due to the previously reported QN-induced maturation delay phenomenon.

To further characterize subcellular localization of QN, co-localization of QN with either LysoTracker Red, which localizes to acidic compartments, or DRAQ5, which localizes to the nucleus, was investigated using 3-D reconstruction of z-stack fluorescence images. As expected, no co-localization with DRAQ5 was seen. However, QN was found to localize in a separate but adjacent compartment from that in which LysoTracker Red was localized (Figure 3). This 3-D imaging method does not allow for haemozoin to be visualized. The pattern of co-localization was the same for 3D7 and Dd2 parasites, despite their differences in pfmdr1 copy number. These observations suggest that QN localizes to a distinct, non-acidic compartment within the parasite food vacuole, possibly overlapping with haemozoin in parasites with both low and high pfmdr1 copy number.