The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

The obligate intracellular protozoan parasite Toxoplasma gondii is able to establish persistent infections within human and animal hosts. We have shown recently that T. gondii down‐regulates IFN‐γ‐induced MHC class II expression in murine bone marrow‐derived macrophages (BMMΦ). As shown in this study, the capacity of IFN‐γ‐activated murine BMMΦ to present ovalbumin to CD4⁺ T cell hybridomas was dose‐dependently inhibited by T. gondii. IFN‐γ‐induced up‐regulation of H2‐Aa, H2‐Ab, H2‐Eb, H2‐Ma, H2‐Mb, H2‐Oa and invariant chain transcripts was prominently down‐regulated by T. gondii. Furthermore, mRNA levels of class II transactivator and interferon‐regulatory factor‐1 were significantly diminished. Electromobilityshift assays demonstrated a decrease in the binding activity of nuclear extracts to the IFN‐γ‐activated site after infection with T. gondii, indicating parasitic interference with IFN‐γ‐induced signaling. However, neither the expression of the IFN‐γR nor the IFN‐γ‐induced tyrosine phosphorylation of IFN‐γR α chain and signal transducer and activator of transcription (STAT) 1α was diminished by T. gondii. IFN‐γ‐induced nuclear translocation of STAT1α was nevertheless inhibited after infection as demonstrated by immunofluorescence microscopy and subcellular fractionation analyses. In conclusion, this novel mechanism of microbial interference with MHC class II gene expression may contribute to intracellular survival and establishment of persistent infection with T. gondii.

The obligate intracellular protozoan parasite Toxoplasma gondii has been shown to protect different cell types from apoptosis induced by a variety of pro-apoptotic treatments. However, the precise cell biological mechanisms of this inhibition remained unknown. As shown in this study, apoptosis in human-derived HL-60 and U937 cells induced by treatment with actinomycin D or TNF-α in combination with cycloheximide, respectively, was indeed dose-dependently downregulated by prior infection with T. gondii, as determined by DNA fragmentation assays. Cleavage of caspase 3 and caspase 9 after treatment with pro-apoptotic stimuli was considerably diminished by T. gondii. Furthermore, release of mitochondrial cytochrome c during apoptosis in HL-60 cells was prevented by intracellular parasites and this was correlated with the absence of DNA strand breaks on the single cell level. Inhibition of cytochrome c release coincided with a twofold upregulation of Mcl-1 protein levels in HL-60 and U937 cells, while Bcl-2 expression did not increase after infection. Parasitic interference with the caspase cascade led to a reduced proteolytic cleavage of the nuclear target molecule protein kinase Cδ. In parallel, poly(ADP-ribose) polymerase protein levels were prominently downregulated by T. gondii, irrespective of whether HL-60 and U937 cells had been treated with pro-apototic stimuli or left untreated. However, poly(ADP-ribose) polymerase mRNA levels remained unchanged after infection as determined by RT-PCR analyses. These observations suggest that T. gondii has evolved different mechanisms that may contribute to downregulation of host cell apoptosis, namely inhibition of cytochrome c release and subsequent caspase activation as well as downregulation of poly(ADP-ribose) polymerase protein levels.