On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization

Abstract

Leishmania major is a protozoan parasite from one of the most ancient phylogenic branches of unicellular eukaryotes, and containing only one giant mitochondrion. Here we report that staurosporine, that induces apoptosis in all mammalian nucleated cells, also induces in L. major a death process with several cytoplasmic and nuclear features of apoptosis, including cell shrinkage, phosphatidyl serine exposure, maintenance of plasma membrane integrity, mitochondrial transmembrane potential (DCm) loss and cytochrome c release, nuclear chromatin condensation and fragmentation, and DNA degradation. Nuclear apoptosis-like features were prevented by cysteine proteinase inhibitors, and cell free assays using dying L. major cytoplasmic extracts indicated that the cysteine proteinases involved (i) also induced nuclear apoptosis-like features in isolated mammalian nuclei, and (ii) shared at least two nuclear substrates, but no cleavage site preference, with human effector caspases. Finally, isolated L. major mitochondria released cytochrome c and cysteine proteinases with nuclear pro-apoptotic activity when incubated with human recombinant Bax, even (although much less efficiently) when Bax was deleted of its transmembrane domain required for insertion in mitochondrial outermembranes, implying that L. major mitochondrion may express proteins able to interact with Bax. The recruitment of cysteine proteinases and mitochondria to the cell death machinery may be of very ancient evolutionary origin. Alternately, host/ parasite interactions may have exerted selective pressures on the cell death phenotype of kinetoplastid parasites, resulting in the more recent emergence of an apoptotic machinery through a process of convergent evolution.

Antimonial-induced Increase in Intracellular Ca2+ through Non-selective Cation Channels in the Host and the Parasite Is Responsible for Apoptosis of Intracellular Leishmania donovani Amastigotes

“…This observation differs from previous studies with free-swimming kinetoplastid parasites, where DNA fragmentation in multiples of oligonucleosomal fragments were reported during apoptotic death induced by various stimuli (1, 5, 4, 30). Our observations are similar to DNA fragmentation in staurosporine-induced apoptotic death of L. major, where no DNA breakage into oligonucleosomal fragments was detected (11). PAT is known to induce DNA fragmentation in axenically grown amastigotes of L. infantum (8), but its effect on intracellular amastigote DNA is not known.…”

“…Hydrogen peroxide (H 2 O 2 ) induces nuclear condensation, DNA ladder formation, and activation of caspase-like activity in L. donovani promastigotes that occur through a Ca 2ϩ -dependent mechanism (4,9). The protein kinase inhibitor staurosporine, known to induce apoptosis in all mammalian nucleated cells (10), stimulates apoptosis-like death in the promastigotes of L. major (11). Not only the Leishmania spp.…”

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

“…26 Although no adaptor domains such as Death domains, DED, CARD or Pyrin have been found in any lower eukaryote, homologues of proteins implicated in the process of cell death, may exist in unicellular organisms. 31 ± 33 In two recent studies, 34,35 cell death has been studied in more detail. Several cytoplasmic and nuclear features of apoptosis, such as mitochondrial transmembrane potential loss, cytochrome c release, nuclear condensation and fragmentation, were observed when staurosporine, a drug known to induce apoptosis in mammalian cells was used to stimulate cell death.…”

“…Several cytoplasmic and nuclear features of apoptosis, such as mitochondrial transmembrane potential loss, cytochrome c release, nuclear condensation and fragmentation, were observed when staurosporine, a drug known to induce apoptosis in mammalian cells was used to stimulate cell death. 34 Using human isolated nuclei as targets, nuclear DNA degradation was observed when a staurosporine treated cytoplasmic extract of promastigotes was used. The DNA fragmentation was prevented in the presence of the inhibitor E64 suggesting that cytoplasmic extracts of staurosporine treated parasites contained cysteine proteases activity involved in the DNA fragmentation process.…”

Looking for death at the core of life in the light of evolution

“…47 PCD induction involves intercellular signaling in response to environmental changes, and may participate in various important functions 16,40 including enforcement of cell differentiation, selection of the fittest cells in a given environment, 43 or the building of transient multicellular bodies made up of dead cell corpses favoring the persistence of long-lived, resistant, resting spores. 45 The involvement of ancestral, evolutionary conserved molecular mechanisms of PCD has been suggested by the identification of a potential role for cysteine proteases in kinetoplastids 48 and dinoflagellates, 46 for an AIF homologue in slime molds, 49 and for a metacaspase in yeast. 50 But it is the bacteria that may provide today the most fascinating model for addressing the question of the possible emergence and selection of PCD in a broad evolutionary perspective.…”