Inhibition Of DNA Damage Repair In Host Cell By Toxoplasma Gondii Dense Granule GRA16

Toxoplasma gondii is a worldwide obligate parasite that infects humans and almost all warm-blooded animals.Its wide spread not only causes economic losses to animal husbandry,but also challenges the public health and safety in endemic areas.Generally,hosts infected by T.gondii suffer a chronic infection with T.gondii bradyzoites remaining dormant in the cysts inhabiting in the tissues of the host.While the immune system of the host fails or compromises,bradyzoites are activated and transform into proliferative tachyzoites,which may cause acute infection leading to death if untreated.To date,although several drugs are used to control acute infection by tachyzoites,there is no feasible way to eradicate the dormant cysts of T.gondii in the host.The interaction between T.gondii and host is among one of the most interesting research topics for scientists.T.gondii infection leads to disorders in a couple of host biological processes including cell cycle arrest,apoptosis and autophagy,however,studies on host DNA damage were rarely reported.DNA integrity is crucial for genetic stability and vital for cell proliferation,because RNAs and proteins as essential components in various biological processes are both derived from nuclear DNA.The aim of this study is to confirm the existence of DNA damage in T.gondii-infected host cells and to explore how T.gondii regulates host's DNA damage by its secretory effectors.Our data showed that T.gondii infection caused DNA damage in the host cells.This process was not related to apoptosis and depended on the invasion of T.gondii.ROS(Reactive oxygen species)was a critical factor that determined the degree of DNA damage as downregulation of ROS by ROS inhibitor NAC(N-acetylcysteine)significantly alleviated the DNA damage in host cells.T.gondii dense granule protein GRA16 was selected to evaluate its regulation on host DNA damage due to its binding ability with phosphatase PP2A,which was reported involving in DNA damage by dephosphorylation of DNA damage biomarker yH2AX.GRA16 was verified not to induce host DNA damage but to hinder DNA damage repair by reducing the expression of ?H2AX.yH2AX is under a dynamic balance during DNA damage and repair,Histone H2AX is phosphorylated into yH2AX when DNA damage begins,then yH2AX is dephosphorylated into H2AX after DNA damage is repaired.Regulation of reduced yH2AX by GRA16 was validated by accelerating the dephosphorylation of yH2AX rather than blocking the phosphorylation of H2AX.Furthermore,phosphatase PPM1D was confirmed involving in the downregulation of yH2AX by GRA16.1.Toxoplasma gondii infection causes DNA damage in host cells.293T,HeLa and Vero cells were infected with T.gondii(Multiplicity of infection,MOI=10),harvested at 0 h(uninfected),10 h,20 h and 30 h and then subjected to Western blot and immunofluorescence assay.DNA double strand break biomarker ?H2AX was used to indicate the DNA damage.Data showed that T.gondii infection induced large amount of yH2AX in different host cells during infection over time.This result clearly demonstrated that T.gondii infection can induce DNA damage in host cells.How DNA damage was induced was then investigated.Data showed that apoptosis was not related to the host DNA damage as no signal of apoptosis biomarker Caspase 3 was detected in the infected cells.Host DNA damage was reliant on the invasion of T.gondii.Block of invasion by positive serum significantly lessen the expression of yH2AX in host cells.Reactive oxygen species(ROS)was defined as a main factor that induced the host DNA damage,as elevated ROS level was observed in host cells and ROS inhibitor(N-acetylcysteine,NAC)treatment caused considerable decrease of yH2AX in infected cells.ATM/CHK2 signal pathway is closely related to DNA double strand break and regulates a couple of biological processes,such as DNA repair,cell cycle,and apoptosis.Western blot assay showed that this pathway was activated in the T.gondii-infected cells,indicating that host DNA damage may associated with many biological disorders in host cells during T.gondii infection.2.Toxoplasma gondii dense granule GRA16 attenuates the expression of yH2AX and hinders the DNA repair in host cells.It is well accepted that toxins and virulence factors secreted by pathogens are both capable to induce DNA damage in host cells.Does T.gondii own secretory effectors that regulate the DNA damage in host cell?Dense granule protein GRA16 is a potential candidate,due to its relocation from T.gohdii to host nucleus where DNA damage takes place and its binding to PP2A which is a key phosphatase acting on DNA damage biomarker yH2AX.Overexpression of GRA16 in 293T cells showed that GRA16 was unable to induce DNA damage in mammalian cells,however,GRA16 largely reduced the expression of yH2AX during camptothecin-induced DNA damage repair process,suggesting GRA16 may participate in host's DNA damage repair.Genome-editing CRISPR-CAS9 system was applied to knock out GRA16 in T.gondii RH?ku80 strain.Compared with wildtype RH?ku80 strain,RH?ku80?gral6 strain was unable to reduce the ?H2AX expression at early stage of infection,thus supporting the speculation that GRA16 attenuates the expression of yH2AX.?H2AX expression is closely related to DNA damage and DNA damage repair,since GRA16 downrcgulates the expression of yH2AX,the comet assay was then carried out to evaluate the function of GRA16 on DNA damage repair.Data shown that camptothecin-pretreated 293T cells that overexpressing GRA16 retained obvious DNA migration through the electrophoresis gel,suggesting that the downregulation of yH2AX by GRA16 may hinder the DNA damage repair process.3.GRA16 accelerates the dephosphorylation of ?H2AX through phosphatase PPMID.The amount of yH2AX is in a dynamic balance during DNA damage and repair,H2AX is phosphorylated and convert into ?H2AX once DNA damage occurs,while ?H2AX is dephosphorylated and return into H2AX when DNA repair is fixed.Consequently,there are two possible ways for GRA16 to lessen the expression of yH2AX:1)GRA16 inhibits the phosphorylation of H2AX;2)GRA16 accelerates the dephosphorylation of ?H2AX.Immunofluorescence result shown that GRA16 accelerates the dephosphorylation of ?H2AX rather than inhibiting the phosphorylation of yH2AX.Considering that GRA16 binds to phosphatase PP2A which is known participating in the dephosphorylation of yH2AX,this study constructed the full-length GRA16(1-505 aa)and truncated GRA16(1-237 aa)without binding ability to PP2A to test whether the decrease of the yH2AX amount by GRA16 is related to its binding with PP2A.Data shown that compared with full-length GRA16,truncated GRA16 remained the function that lessened the expression of yH2AX,implying that PP2A is not the key phosphatase involving in the decrease of ?H2AX.To find out which phosphatase works on the dephosphorylation of yH2AX,real-time PCR was performed to screen three phosphatase candidates:PPM1D,PPM1G and PP2A,which were reported acting on the dephosphorylation of yH2AX.The result showed that the expression pattern of PPM1D was in accordance with the dynamic change of ?H2AX in GRA 16-overexpressing HeLa cells during DNA damage repair.CRISPR-CAS9 system was then carried out to knockout PPM1D from HeLa cells,and the immunofluorescence result showed that,compared with wildtype cells,?ppmld cells revealed a notable less decrease of yH2AX by GRA16,indicating that GRA16 accelerates the dephosphorylation of ?H2AX through phosphatase PPM1D.In conclusion,this study confirmed the occurrence of ROS-related DNA damage in host cells during T.gondii infection.T.gondii GRA16 was tested to evaluate its function on host DNA damage,it reveals a capacity of accelerating the dephosphorylation of yH2AX and hindering the host DAN damage repair.The phosphatase PPM1D was verified taking part in the dephosphorylation of ?H2AX by GRA16.Our findings on host DNA damage during T.gondii infection contribute to a better understanding on interaction between T.gondii and its hosts.