Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly.

Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome cin vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway.

Mammalian apoptotic signalling pathways: multiple targets of protozoan parasites to activate or deactivate host cell death.

Programmed cell death is an essential mechanism of the host to combat infectious agents and to regulate immunity during infection. Consequently, activation and deactivation of the hosts' cell death pathways by protozoan parasites play critical roles in parasite control, pathogenesis, immune evasion and parasite dissemination within the host. Here, we discuss advances in the understanding of these fascinating host-parasite interactions with special emphasis on how protozoa can modulate the cell death apparatus of its host.

Toxoplasma gondii infection confers resistance against BimS-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic Bax.

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein Bim(S) to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated Bim(S)-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of Bim(S) was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of Bim(S)-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.

Modulation of caspase activation by Toxoplasma gondii.

Apoptosis plays crucial roles for the outcome of infection with various infectious agents. The host's apoptotic program may be modulated after infection in order to combat the pathogen or to restrict the immune response. In addition, distinct microorganisms alter the apoptotic program of the host in order to meet the requirements for their further distribution. The activation of caspases (i.e., cysteine proteases with specificity for aspartic acid residues) preludes the disassembly of the cell in response to apoptosis-inducing stimuli. This depends on the proteolytic cleavage of inactive proforms into catalytically active subunits. Analyses of the proteolysis and the enzymatic activity of caspases therefore represent valuable tools to study apoptotic programs during infection. The apicomplexan parasite Toxoplasma gondii interferes with the caspase cascade of its host cell in order to facilitate intracellular survival. The modulation of caspase activation by T. gondii is determined by SDS-PAGE and immunoblotting with caspase-specific antibodies. Furthermore, the impact of the parasite on caspase activity is fluorimetrically determined by measuring the cleavage of caspase-specific substrate analogues.

Fas/CD95-mediated apoptosis of type II cells is blocked by Toxoplasma gondii primarily via interference with the mitochondrial amplification loop.

The intracellular protozoan Toxoplasma gondii induces persistent infections in various hosts and is an important opportunistic pathogen of humans with immature or deficient immune responses. The ability to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of its host cell. Fas/CD95 triggers an apoptotic cascade that is crucial for immunity and the outcome of infectious diseases. We have determined the mechanism by which T. gondii counteracts death receptor-mediated cell death in type II cells that transduce Fas/CD95 ligation via caspase 8-mediated activation of the mitochondrial amplification loop. The results showed that infection with T. gondii significantly reduced Fas/CD95-triggered apoptosis in HeLa cells by inhibiting the activities of initiator caspases 8 and 9 and effector caspase 3/7. Parasitic infection dose dependently diminished cleavage of caspase 8, the BH3-only protein Bid, and the downstream caspases 9 and 3. Importantly, interference with Fas/CD95-triggered caspase 8 and caspase 3/7 activities after parasitic infection was largely dependent on the presence of caspase 9. Within the mitochondrial amplification loop, T. gondii significantly inhibited the Fas/CD95-triggered release of cytochrome c into the host cell cytosol. These results indicate that T. gondii inhibits Fas/CD95-mediated apoptosis in type II cells primarily by decreasing the apoptogenic function of mitochondria.

Toxoplasma gondii inhibits Fas/CD95-triggered cell death by inducing aberrant processing and degradation of caspase 8.

Ligation of the death receptor Fas/CD95 activates an apoptotic cascade and plays critical roles during infectious diseases. Previous work has established that infection with the intracellular parasite Toxoplasma gondii renders cells resistant to multiple inducers of apoptosis. However, the effect of T. gondii on the death receptor pathway is poorly characterized. Here we have determined the impact of the parasite on apoptosis in type I cells that transduce Fas/CD95 engagement via the death receptor pathway without the need of a mitochondrial amplification loop. The results have shown that T. gondii significantly reduced Fas/CD95-triggered apoptosis by impairing activation of the initiator caspase 8. Parasitic infection diminished the cellular amount of procaspase 8, resulting in its decreased recruitment to the death-inducing signalling complex and the impaired activation of effector caspases. Remarkably, downregulation of caspase 8 protein in T. gondii-infected cells also occurred in the absence of Fas/CD95 engagement and was associated with the appearance of non-canonical caspase 8 cleavage fragments. Distinct parasite proteins were associated with caspase 8 and its proteolytic fragments. These findings indicate that T. gondii aberrantly processes and finally degrades the initiator caspase 8, thereby, blocking Fas/CD95-mediated apoptosis which signals independently of the apoptogenic function of host cell mitochondria.

Toxoplasma gondii glycosylphosphatidylinositols are not involved in T. gondii-induced host cell survival.

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.