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.

Acidocalcisome is required for autophagy inTrypanosoma brucei

“…25,27,28 Upon starvation, the size and number of acidocalcisomes were not perceptibly changed as compared with control cells according to anti-TbVP1 staining (Fig. S1A).…”

A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

“…Given the association of DV permeabilization with PCD features (4,5) and the centrality of the DV to the erythrocytic stage of P. falciparum, it was a natural progression to the search for novel compounds that compromise the DV. Despite the popularity of molecular-target-based high-throughput screening methods, these assays tend to result in few leads; this is presumably due to the ease with which mutations conferring resistance are obtained and physical barriers to the drug targets, among other factors (16).…”

“…Our laboratory has in recent years uncovered a mode of cell death associated with permeabilization of the digestive vacuole (DV). Treatment with low micromolar concentrations of chloroquine (CQ) induced phenotypes reminiscent of programmed cell death (PCD) in mammalian cells, such as DNA fragmentation, mitochondrial dysfunction, and activation of clan CA cysteine proteases (4,5). However, CQ was long ago shelved as malaria chemotherapy because of the emergence of resistant mutants.…”

Cell death offers exceptional cellular and molecular windows for pharmacological interventions in protozoan parasites

“…The hallmark features of programmed cell death in P. falciparum and mammals have revealed fascinating analogies between them across both subkingdoms. The diagram below exemplifies programmed cell death-mediated by cys protease (s) in CQ-sensitive laboratory strain of P. falciparum, 3D7 (MRA-102, MR4, ATCC, Manassas, Va, USA) and caspase-3 in animals ( Figure 6) [70].…”