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.
Highlights
A better understanding of the mechanisms involved in protozoan cell death could provide an opportunity to design parasitespecific pro-apoptotic drugs in the aim of controlling parasitic disease. protozoan parasites exhibit most of the cellular and molecular markers described in higher eukaryotes [1], information on molecular pathways involved in protozoan cell death (CD) is still limited [2]
PfMCA1-cd-Sc expression was induced in yeast metacaspase null mutants, producing the expected 35kDa polypeptide that was recognized by anti-M2 antibody (Figure 1, lane 8)
Galactose incubation allowed the expression of two other metacaspases in the Dyca1 cells, i.e the full length YCA1 and the catalytic domain of the L. major metacaspase (LmjMCA-cd)
Summary
Protozoan parasites exhibit most of the cellular and molecular markers described in higher eukaryotes [1], information on molecular pathways involved in protozoan cell death (CD) is still limited [2]. It is known, that the metazoan caspase genes encoding major CD effector proteases are absent in prokaryotes, plants, fungi and protozoan parasites such as Leishmania and Plasmodium [3,4]. The structural homology of the catalytic domains between metacaspase and caspase suggests that metacaspase could be involved in CD through an apoptotic-like pathway [7,8,9]. The importance of the CD effector function of metacaspase has been supported by experimental evidence in Arabidopsis thaliana (A. thaliana) and in Leishmania major (L. major) [14,15]
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