Molecular and Functional Characterization of Programmed Cell Death (PCD) in Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite which leads to widespread infections in humans and animals worldwide. Infection is typically asymptomatic in immunocompetent hosts, but may lead to severe symptoms in immunocompromized individuals. Apoptosis, a form of programmed cell death (PCD), is a highly regulated molecular process of cell suicide that is crucial to the development, homeostasis and integrity of multicellular organisms. During recent years, there has been increasing evidence for the occurrence of an apoptotic cell death in unicellular organisms including protozoan parasites. The mechanisms of how T. gondii dies are largely unknown but knowledge of putative regulated cell death pathways are of major scientific interest and may lead to the development of novel antiparasitic therapies. Here, we demonstrated the occurrence of an apoptosis-like cell death in T. gondii. Staurosporine and miltefosine treatment resulted in the presence of apoptotic markers in T. gondii, including DNA strand breaks, reduction in cell size, and phosphatidylserine translocation. We found that apoptosis-like cell death obviously did not contribute to the regulation of tachyzoites density during acute infection in the peritoneal cavity of infected mice. However, TUNEL-positive parasites were detected in bradyzoite-containing tissue cysts from the brains of chronically infected mice, suggesting the parasites employed apoptosis-like cell death during chronic infection. Furthermore, number of extracellular TUNEL-positive parasites increased after treatment with drugs commonly used to treat toxoplasmosis. Intriguingly, treatment with clindamycin led to the emergence of intracellular TUNEL-positive parasites that partially showed abnormal morphology. Data mining of the Toxoplasma genome resource identified 17 putative cell death-associated genes encoding proteases, a nuclease and several apoptosis regulators. Here, we focused on three putative T. gondii Bax inhibitors: TgBI-1, TgBI-2 and TgBI-3. To unravel possible functions of TgBIs in the regulation of apoptosis, we stably transfected HeLa cells with the genes encoding TgBI-1, TgBI-2 and TgBI-3 and this was confirmed by immunofluorescence microscopy. Measurement of caspase 3/7 activity demonstrated an inhibitory effect of TgBI-1 and TgBI-2 in HeLa cells after treatment with staurosporine and brefeldin A. Moreover, TgBI-1, TgBI-2- and TgBI-3-expressing HeLa cells showed a lower induction of hypoploid DNA (subpeak G0/G1) after treatment with pro-apoptotic stimuli as above. These results suggested that T. gondii Bax inhibitors might fulfill conserved functions in regulating an apoptosis-like cell death in T. gondii. Furthermore, mitochondrial membrane potential (ΔΨm) in the majority of parasites remained intact and caspase-like activity increased only slightly during staurosporine-triggered cell death. In contrast, T. gondii lost their ΔΨm and rapidly presented strong caspase-like activity during miltefosine treatment. Consequently, protease inhibitors abrogated miltefosine-induced but not staurosporine-induced Toxoplasma cell death. Together, the results indicate the existence of an apoptosis-like cell death mechanism in T. gondii which might play a role during chronic infection. Additionally, we proof for the first time that treatment of intracellular parasites with the chemotherapeutic drug clindamycin induces an apoptosis-like cell death phenotype in a small proportion of parasites. Our results on TgBIs indicate evolutionary conserved protective effects of BI proteins from T. gondii to mammals and suggest a role of TgBI proteins in regulation of T. gondii apoptosis-like cell death. Furthermore, distinct apoptosis-like cell death pathways appear to exist in T. gondii; one involves both a loss of ΔΨm and an activation of caspase-like proteases, which is specifically triggered by miltefosine. Further experiments to unravel precise mechanisms of PCD in Toxoplasma are required. A better understanding on Toxoplasma PCD might provide new possibilities to combat the parasite particularly during its persistent bradyzoite stage