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Hemmung der Cytochrom c-induzierten Caspase-Aktivierung durch Toxoplasma gondii in vitro und in vivo:molekulare Mechanismen und parasitäre Effektormoleküle

dc.contributor.advisorLüder, Carsten Prof.
dc.contributor.authorGraumann, Kristinde
dc.titleHemmung der Cytochrom c-induzierten Caspase-Aktivierung durch Toxoplasma gondii in vitro und in vivo:molekulare Mechanismen und parasitäre Effektormolekülede
dc.contributor.refereeGroß, Uwe Prof.
dc.subject.dnb000 Allgemeines, Wissenschaftde
dc.subject.gokWU 000de
dc.description.abstractengToxoplasma gondii is an obligate intracellular parasite and is able to infect almost all nucleated cells of humans and warm-blooded animals. Infection of immunocompetent individuals is mostly asymptomatic and leads to a lifelong persistence of the parasite particularly in the central nervous system and muscle cells of the host. T. gondii has evolved different strategies to evade the immune system of the host and to facilitate its own intracellular survival. Apoptosis is a common form of programmed cell death which eliminates damaged or unnecessary cells in a multicellular organism. Apoptosis also represents an important defense mechanism of both innate and adaptive immune responses to combat intracellular pathogens. Some pathogens including T. gondii are able to inhibit apoptosis of its host cell and thereby facilitate its own survival and development in the host. T. gondii inhibits cytochrome c-induced activation of the caspase 9-caspase 3/7-cascade in a cell-free system in a dose-dependent manner. In the present study it could be shown for the first time that inhibition of cytochrome c-induced caspase 3/7-activity by the parasite also plays an important role in infected cells. Studies in caspase 9-deficient Jurkat cells and caspase 9-retransfected cells showed a 30% stronger inhibition of apoptosis in caspase 9-expressing cells than in caspase 9-deficient cells thus indicating that this level of inhibition was due to parasite interference with caspase 3/7-activation. After electroporation of Jurkat cells in medium with and without cytochrome c, it was shown that the cytochrome c-induced caspase 3/7-activity was completely inhibited by the parasite without affecting cytosolic levels of cytochrome c in the host cell. In addition, we observed a dose-dependent inhibition of cytochrome c-induced caspase 3/7-activity in cytosolic lysates from cells infected with increasing numbers of the parasite. Efforts to unravel the molecular mechanism of the reduced activation of cytochrome c-induced caspase-activation in the presence of T. gondii showed that the recruitment of caspase 9 to the apoptosis activating factor-1 (APAF-1) and therefore activation of the initiator caspase 9 was inhibited by the parasite. In a reconstituted test system consisting of recombinant APAF-1, caspase 9 and cytochrome c, complete lysate of T. gondii inhibited cytochrome c-induced activation of caspase 9 dose-dependently. Thus, in a test system with defined components, our result displayed for the first time that T. gondii inhibits activation of caspase 9 and even blocks this activation after the apoptosome was already formed. The cell-free system was also used to characterize effector proteins of the T. gondii-mediated inhibition of the caspase cascade. In previous studies, NTPase and HSP70 of T. gondii were identified as possible effectors for the anti-apoptotic effect of the parasite. This study revealed that the NTPase of T. gondii indeed represents an anti-apoptotic protein of the parasite. Immunodepletion of NTPase from T. gondii lysate showed a dose-dependent abrogation of the inhibitory effect of complete parasite lysate on cytochrome c-induced caspase activity. Investigations with a non-hydrolyzable ATP analogon, App(NH)p, as well as use of high concentration of ATP indicated that the hydrolase-activity of T. gondii NTPase was not required for the inhibition of cytochrome c-induced caspase activation. Clonal lineages of T. gondii express different isoforms of NTPase with different hydrolase activities. However, investigations on a possible strain specificity of the parasite-mediated inhibition of the caspase cascade showed comparable inhibition by members of the three different clonal lineages on cytochrome c-induced caspase activation. This inhibition thus occurred independent of the expression of a specific isoform of NTPase. In this study, recombinant NTPase and HSP70 of T. gondii were expressed in E. coli. Bacterial expressed T. gondii HSP70 had no effect on cytochrome c-induced caspase 3/7 activity. In contrast, after purification under denaturing conditions, recombinant T. gondii NTPase showed an inhibition of cytochrome c-induced caspase activity. Furthermore, a functionally active NTPase inhibited cytochrome c-induced activation of caspase 3/7 dose-dependently and at a concentration of 0,5de
dc.contributor.coRefereeBraus, Gerhard Prof.
dc.subject.topicBiology (incl. Psychology)de
dc.subject.gerToxoplasma gondiide
dc.subject.gerCytochrom c-induzierte Caspase-Aktivitätde
dc.subject.engToxoplasma gondiide
dc.subject.engcytochrome c-induced caspase-activityde
dc.affiliation.instituteBiologische Fakultätde

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