Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria

Abstract

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.

Mechanisms of hematin crystallization and inhibition by the antimalarial drug chloroquine

“…The parasite DV presents a complex environment. The vacuole comprises membrane interfaces (20), an acidic aqueous solution with pH 4.8−5.5 (30), and lipids, mostly mono-and diglycerides, resulting from the degradation of the transport vesicle membranes that carry hemoglobin into the DV (9,16,(18)(19)(20). The location and structure of the lipid subphase in the parasite DV is a subject of debate.…”

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

“…Spinning Disk Confocal Microscopy (SDCM) and data acquisition-We have previously described our customized SDCM apparatus in detail [1,28]. We used a Merzhauser motorized MS-2000 XY translation stage and an additional piezo table for Z -movement.…”

“…For fluorescence SDCM, excitation was with a Coherent Innova 300 I Ar/Kr laser (300 mW at 488 nm), and a NEOS acousto optic tunable filter was used for fast illumination control. Exposure time was typically 100 ms with laser power 100 mW and z-spacing was 200 nm (appropriate for iterative deconvolution as in [28]). Additional details of the system and data acquisition are described elsewhere [1,28].…”

“…Exposure time was typically 100 ms with laser power 100 mW and z-spacing was 200 nm (appropriate for iterative deconvolution as in [28]). Additional details of the system and data acquisition are described elsewhere [1,28]. After iterative deconvolution using experimentally derived point spread functions, the x, y resolution of fluorescence SDCM data is only slightly lower than that of classical LSCM confocal data (measured on our instrument to be 213 nm using 520 nm light [28]).…”

“…In this study we investigate the stage specificity of CQ in detail using recently developed imaging methods [28,29] to measure parameters that are central to various models for toxicity, but that have not previously been measurable for live parasites. Our results are in agreement with previous observations that the effect of CQ is both schedule and dose-dependent, but also clearly show that ring stage parasites are affected by CQ long before the mature DV is formed, and that schizont stage parasites are similarly sensitive to CQ long after Hz formation has plateaued.…”