Abstract 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.
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.
Abstract Bacillus thuringiensis (Bt) Cry toxins have been used widely in pest managements. However, Cry toxins are not effective against sap-sucking insects (Hemiptera), which limits the application of Bt for pest management. In order to extend the insecticidal spectrum of Bt toxins to the rice brown planthopper (BPH), Nilaparvata lugens , we modified Cry1Ab putative receptor binding domains with selected BPH gut-binding peptides (GBPs). Three surface exposed loops in the domain II of Cry1Ab were replaced with two GBPs (P2S and P1Z) respectively. Bioassay results showed that toxicity of modified toxin L2-P2S increased significantly (~9 folds) against BPH nymphs. In addition, damage of midgut cells was observed from the nymphs fed with L2-P2S. Our results indicate that modifying Cry toxins based on the toxin-gut interactions can broaden the insecticidal spectrum of Bt toxin. This method provides another approach for the development of transgenic crops with novel insecticidal activity against hemipteran insects and insect populations resistant to current Bt transgenic crops.
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