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

ABSTRACT: 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.

SUBMITTER: Graumann K

PROVIDER: S-EPMC5349237 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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

Graumann Kristin K Schaumburg Frieder F Reubold Thomas F TF Hippe Diana D Eschenburg Susanne S Lüder Carsten G K CG

Microbial cell (Graz, Austria) 20150504 5

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 cytoc ...[more]

PMID: 28357287

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Project description:UnlabelledInterleukin-1? (IL-1?) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1?; however, the host and parasite factors that mediate IL-1? production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1? transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1? release, suggesting a role for the inflammasome in T. gondii-induced IL-1? production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1? induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1?. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1? mRNA and protein release than did the type I and III strains. Since IL-1? transcript is known to be induced downstream of NF-?B signaling, we investigated a role for the GRA15 protein, which induces sustained NF-?B signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1? induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1? production.ImportanceMonocytes are immune cells that protect against infection by increasing inflammation and antimicrobial activities in the body. Upon infection with the parasitic pathogen Toxoplasma gondii, human monocytes release interleukin-1? (IL-1?), a "master regulator" of inflammation, which amplifies immune responses. Although inflammatory responses are critical for host defense against infection, excessive inflammation can result in tissue damage and pathology. This delicate balance underscores the importance of understanding the mechanisms that regulate IL-1? during infection. We have investigated the molecular pathway by which T. gondii induces the synthesis and release of IL-1? in human monocytes. We found that specific proteins in the parasite and the host cell coordinate to induce IL-1? production. This research is significant because it contributes to a greater understanding of human innate immunity to infection and IL-1? regulation, thereby enhancing our potential to modulate inflammation in the body.

Project description:BackgroundApoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed.MethodsApoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting.ResultsThe apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation.ConclusionsToxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

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

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

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