DNA double-strand breaks in the Toxoplasma gondii -infected cells by the action of reactive oxygen species
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Parasites & Vectors Open Access
RESEARCH
DNA double‑strand breaks in the Toxoplasma gondii‑infected cells by the action of reactive oxygen species Haohan Zhuang1†, Chaoqun Yao2†, Xianfeng Zhao3, Xueqiu Chen1, Yimin Yang1, Siyang Huang4, Lingtao Pan1, Aifang Du1* and Yi Yang1*
Abstract 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. Keywords: Toxoplasma gondii, DNA damage, Reactive oxygen species, DNA damage response Background The protozoan parasite Toxoplasma gondii infects almost all warm-blooded animals including humans worldwide [1]. It modulates some biological processes of the infected cell, such as autophagy and apoptosis, to *Correspondence: [email protected]; [email protected] † Haohan Zhuang, Chaoqun Yao, equal contributors 1 Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, PR China Full list of author information is available at the end of the article
facilitate its survival and proliferation [2, 3]. DNA damage was also reported in the retina and the peripheral blood cells of T. gondii-infected mice [4, 5]. However, it remains to be elucidated how T. gondii causes host DNA damage and what the DNA damage responses are. DNA damage appears in different forms such as singlestrand breaks (SSBs), double-strand breaks (DSBs), missing bases and
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