Background Toxoplasma gondii is 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 in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX 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 γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

IntroductionAutophagy has been recognized as a bona fide immunological process. Evidence has shown that this process in IFN-γ stimulated cells controls Toxoplasma gondii proliferation or eliminates its infection. However, little is known about the effect of T. gondii infection on the host cell autophagy in the absence of IFN-γ.MethodsMultiple autophagy detection methods and CRISPR/CAS9 technology were used to study T. gondii-induced autophagy in HeLa and several other mammalian cell lines.ResultsHere, we report increased LC3 II, autophagosome-like membrane structures, enhanced autophagic flux, and decreased lysosomes in a range of mammalian cell lines without IFN-γ treatment after T. gondii infection. Specifically, disruption of host atg5 (a necessary gene for autophagy) in HeLa cells promoted the intracellular replication of T. gondii, with the transcript level of rab11a increased, compared with that in wild-type cells. Further, after T. gondii infection, the abundance of Rab11A remained stable in wild-type HeLa cells but decreased in atg5−/− mutant. Disruption of rab11a in the HeLa cells compromised the proliferation of T. gondii, and increased the transcription of gra2 in the parasite. Compared to the T. gondii wild-type RH∆ku80 strain, the ∆gra2 mutant induces enhanced host autophagy in HeLa cells, and results in slower replication of the parasite.DiscussionCollectively, these results indicate that host cell autophagy can limit T. gondii proliferation in an IFN-γ independent manner, possibly by affecting the hijack of host Rab11A-positive vesicles by the parasite which involved TgGRA2. The findings provide novel insights into T. gondii infection in host cells and toxoplasmosis research.

Toxoplasma gondii switches back and forth between acute and chronic infections, mainly in response to host immunologic status, which is characterized by flexible but specific energy metabolism. Polysaccharide granules are accumulated in the acute infection stage of T. gondii that have been exposed to a chemical inhibitor of the PP2A subfamily.

Background: Toxoplasma gondii (T. gondii) is an obligate parasite of the warm-blooded animals with a worldwide distribution. Once having entered a host cell, it manipulates host’s DNA damage response that is yet to be investigated. The objectives of the present study were three-fold: 1) to assess DNA damages in T. gondii-infected cells in vitro; 2) to ascertain sources causing DNA damage in T. gondii-infected cells; 3) to investigate activation of DNA damage response during T. gondii infection.Methods: HeLa, Vero and HEK293 cells were infected with T. gondii at multiplicity of infection (MOI) of 10:1. Infected cells at 10 h, 20 h or 30 h post infection were analyzed for a DNA double strand breaks (DSBs) biomarker γH2AX using Western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were examined using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), and the impact of ROS on DNA damage was assessed by inhibition using a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage response in these T. gondii-infected cells was evaluated by detecting the expression of active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) with Western blot. Results: Compared to uninfected cells, γH2AX expression in the infected HeLa cells at 10 h, 20 h, and 30 h was increased over time during T. gondii infection. NAC treatment reduced ROS level in host cells and significantly decreased the expression of γH2AX. Expression of phosphorylated ATM/CHK2 was elevated in T. gondii-infected cells.Conclusion: T. gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also concomitantly activated DNA damage response pathway ATM/CHK2. T. gondii struggles a balance between survival and apoptosis of its host cells for the benefit of its own survival.