title: 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.
authors.affiliation: Department of Chemistry, Program in Tumor Biology, Lombardi Cancer Center, Georgetown University, 37 and O Streets, Washington, DC 20057, USA.
authors.forename: Bojana
authors.initials: B
authors.lastname: Gligorijevic
authors.forename: Tyler
authors.initials: T
authors.lastname: Bennett
authors.forename: Ryan
authors.initials: R
authors.lastname: McAllister
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Urbach
authors.forename: Paul D
authors.initials: PD
authors.lastname: Roepe
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D002738:Chloroquine / Q000494:pharmacology; D004351:Drug Resistance; D004912:Erythrocytes / Q000469:parasitology*; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology*; D018613:Microscopy, Confocal / Q000379:methods*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D000962:Antimalarials; D005456:Fluorescent Dyes; D002738:Chloroquine