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

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• 作者：Bojana Gligorijevic ; Tyler Bennett ; Ryan McAllister ; Jeffrey S. Urbach ; Paul D. Roepe
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：October 17, 2006
• 年：2006
• 卷：45
• 期：41
• 页码：12411 - 12423
• 全文大小：320K
• 年卷期：v.45,no.41(October 17, 2006)
• ISSN：1520-4995

文摘

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], wereported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initialapplication to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparummalarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of theparasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previousreports 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 oftenlinked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisitionof SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterativedeconvolution 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 viatransfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. Wefind that relative to both CQS strains, both CQR strains show significantly increased DV volume as theorganelle forms upon entry into the trophozoite stage of development and that this persists until thetrophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelleforms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATPdepletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, thesedata suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.