title: A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases.
abstract: Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: J-H
authors.initials: JH
authors.lastname: Ch'ng
authors.forename: S R
authors.initials: SR
authors.lastname: Kotturi
authors.forename: A G-L
authors.initials: AG
authors.lastname: Chong
authors.forename: M J
authors.initials: MJ
authors.lastname: Lear
authors.forename: K S-W
authors.initials: KS
authors.lastname: Tan
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D057056:Cysteine Proteases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008288:Malaria / Q000469:parasitology*; D008322:Mammals / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008954:Models, Biological; D009336:Necrosis; D010271:Parasites / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010963:Plasmodium falciparum / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D015800:Protozoan Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D015800:Protozoan Proteins; D002738:Chloroquine; D004247:DNA; D057056:Cysteine Proteases; D002118:Calcium