Plasmodium falciparum Metacaspase PfMCA-1 Triggers a z-VAD-fmk Inhibitable Protease to Promote Cell Death

Abstract

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

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

“…High level of metacaspase results in the translocation of endonuclease G (LdEndoG) from mitochondria to the nucleus for DNA degradation [67]. Surprisingly, the initiator proteins, for example, MCA-Arginine protease from both protozoan parasite species, such as Plasmodium falciparum and Leishmania major can elicit apoptotic cell death through a Z-VAD-FMK inhibitable effector protease [68].…”

The first suicides: a legacy inherited by parasitic protozoans from prokaryote ancestors

“…Biochemical evidence for a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, for example in B. hominis and Plasmodium spp, although no ‘classical’ caspases have been found in unicellular organisms [42,69-71]. Contenders for the cysteine proteases involved in apoptosis include members of an ancient family of clan CD cysteine proteases - the metacaspases [72-74].…”

Autophagy in Apicomplexa: a life sustaining death mechanism?

“…Apoptotic death in higher eukaryotes is mediated by specific effectors that include the caspase family of proteases [15]. The presence of related molecules, the metacaspases, in yeast [12] and parasitic protozoa, including Plasmodium [16, 17], Toxoplasma (annotated in the genome), and the kinetoplastids [18, 19] suggest a primordial form of apoptosis probably exists in unicellular eukaryotes (reviewed in [2, 4, 14, 20]. This view is further supported by morphological and biochemical features consistent with progression of the apoptotic death pathway defined for higher eukaryotes (Box 1).…”