Die Rolle der Apoptose und des humanen Transkriptionsfaktors STAT1 bei der intrazellulären Abwehr von Trypanosoma cruzi

The protozoan hemoflagellate Trypanosoma cruzi (T. cruzi) is the etiologic agent of the zoonotic Chagas’ disease that affects approximately eight million people in Central and South America causing dilated cardiomyopathy or megavisceral disease with debilitating morbidity or death. Nowadays, Chagas’ disease is classified as a neglected tropical disease (NTD) and is the leading cause of heart disease in Latin America among people living in poverty with an immense socio-economic burden. The parasite is traditionally transmitted by reduviid bugs, by blood transfusion, organ transplantation, congenitally or orally by contaminated food and is able to persist for decades finally causing chronic Chagas’ disease. To elucidate the etiopathogenesis of chronic chagasic cardiomyopathy, this doctoral thesis demonstrates that infection with the extracellular form of T. cruzi, termed trypomastigotes, induces apoptosis in primary rat cardiomyocytes ex vivo. Furthermore, a new purification method for the intracellular life-cycle stage of amastigotes was established and, additionally, it was demonstrated that cardiomyocytes also showed apoptosis after inoculation with purified amastigotes. Up-regulation of the apoptotic gene bax by trypomastigotes and down-regulation of the anti-apoptotic gene bcl-2 by amastigotes was associated with the increase of apoptosis in cardiomyocytes as well as an activation of the human transcription factor signal transducer and activator of transcription 3 (STAT3). There was evidence that Toll-like receptors are involved in the intracellular recognition of T. cruzi after host cell invasion by this protozoan, since gene expression of tlr7 was found to be up-regulated. Purified glycosylphosphatidylinositols (GPIs) from trypomastigotes, however, showed neither an effect on cardiomyocyte apoptosis nor on STAT activation but down-regulated tlr7 expression. To conclude this first part of the thesis, cardiomyopathy in chronic Chagas’ disease might be in part due to apoptosis of cardiomyocytes directly induced by T. cruzi. In the second part of the thesis it is reported that stimulation of host cells with interferon-γ (IFNγ) impaired multiplication of intracellular T.cruzi-amastigotes in infected human cells via the transcription factor STAT1, which was ineffective in STAT1-negative cells. Furthermore, a solid activation of STAT1 through phosphorylation of both tyrosine residue 701 and serine residue 727 in T. cruzi-infected cells were observed, even in the absence of pre-treatment of host cells with IFNγ. STAT1 phosphorylation was accompanied by an increase in binding to STAT-specific DNA elements and concomitant up-regulation of STAT1-regulated target genes. The total amount of cytoplasmic STAT1-protein was increased in response to T.cruzi invasion in an infectious-dose dependent manner both at mRNA and protein level, indicating a positive feedback loop for STAT1 expression. Nevertheless, amastigotes successfully replicating intracellularly inhibited STAT1 signaling by specifically impairing the phosphorylation status of STAT1 serine 727, but not of tyrosine 701, indicating a strategy of circumventing the protective effect of fully-activated STAT1. Summarizing these findings, it has been clearly shown that the IFNγ/STAT1-signalling pathway plays a key role in the combat between T. cruzi parasites and their host cells.