Toxoplasma gondii infection-driven DNA damage and chromosome instability in human host cells

Abstract text

Introduction: The obligate intracellular apicomplexan parasite Toxoplasma gondii is a globally spread zoonotic protozoa causing severe health problems in humans and animals. Prenatal infections may cause abortion or severely affect progeny welfare, and acute T. gondii infections may become life-threatening in immunocompromised patients. Recently, we reported that T. gondii infection induces an increased percentage of binucleated cells, host cell chromosome segregation defects and cytokinesis failure in bovine endothelial cells, which also showed cell cycle arrest in S-phase, suggesting that parasite infection may directly interfere with host DNA synthesis or impair DNA quality. Objectives: Our aim here was to study if T. gondii infection induces host cell DNA damage. Materials & methods: DNA was quantified via FACS-based approaches. DNA damage was studied via comet assays and immunofluorescence-based detection of typical markers for DNA damage foci (γH2AX), S- (PCNA) and G2-phase (Geminin). Protein expression of ataxia-telangiectasia mutated (ATM)- and ATM-and Rad3 (ATR)-related key markers was assessed by Western blotting. Intracellular and extracellular ROS was quantified via DCHF-DA- and AmplexRed assay. Results: T. gondii infection led to host cell S-phase arrest, binucleated cell phenotype and micronuclei formation already 15 min after the invasion. Both, comet assays and γH2AX detection revealed that T. gondii triggered DNA double-strand breaks shortly after infection and at a higher percentage in S-phase. However, ROS levels were not changed between 15-180 min p. i. The results showed that the DNA damage response (DDR)-, ATM-dependent pathway was activated in T. gondii-infected cells. Interestingly, effector molecules of the overall DDR pathway like phospho-p21 and cyclin E1 were also found enhanced after infection. To address the role of cyclin E in this process, a T. gondii mutant (RHΔhce1, RHΔmyr1), which fails to induce host cell cyclin E1 expression, was here used for infection. Overall, infections with mutated tachyzoites induced host cell DNA damage equal to control parasites, thus denying cyclin E-dependent reactions. Conclusion: Current data suggest that T. gondii triggers genome instability and DNA strand breaks early after infection. Both processes are neither dependent on host cellular ROS or cyclin E1 upregulation.