DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

Background: Toxoplasma gondiiis an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host's DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages inT. gondii-infected cellsin vitro; (ii) to ascertain causes of DNA damage inT. gondii-infected cells; and (iii) to investigate activation of DNA damage responses duringT. gondiiinfection. Methods: HeLa, Vero and HEK293 cells were infected withT. gondiiat a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) gamma H2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2 ',7 '-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results: gamma H2AX levels in the infected HeLa cells were significantly increased over time duringT. gondiiinfection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished gamma H2AX in host cells. The phosphorylated ATM/CHK2 were elevated inT. gondii-infected cells. Conclusions: Toxoplasma gondiiinfection triggered DNA DSBs with ROS as a major player in host cellsin vitro. It also activated DNA damage response pathway ATM/CHK2.Toxoplasma gondiimanages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.