The Function Of Host Cell Racl And DNA Methylation Of T.Gondii In T.Gondii Invasion And Stage-conversion

Background:Toxoplasma is an opportunistic pathogenic protozoan that infects all warm-blooded animals though various ways.The processes of T.gondii tachyzoites invading host cells include attachment to host cell membrane,formation of parasitophorous vacuole(PV),and so on.After infecting intermediate hosts including humans,T.gondii undergoes a two-stage conversion between tachyzoite which causes acute infection,and bradyzoite which accounts for chronic infection.So far,the mechanism for T.gondii invasion and stage-conversion is still unclear.Rac1 plays an important role in cytoskeleton reorganization.It was previously reported that host cell Racl was activated and concentrated on the parasitophorous vacuole membrane(PVM)during the invasion of T.gondii tachyzoites into a host cell.DNA methylation is a key epigenetic modification which confers phenotypic plasticity and adaptation.This study is aimed to reveal the function of host cell Racl and T.gondii DNA methylation in T.gondii invasion and stage-conversion.Methods:HFF cells were treated with Racl inhibitor(NSC23766)or left untreated,followed by infection with GFP-SAG1 RH strain T.gondii tachyzoites.The invasion efficiency of T.gondii was counted under a microscope.Immune fluorescence(IF)of host cell actin in the inhibitor treated or untreated cells were observed to show the different cytoskeleton reorganization.The co-localization of host cell Racl with T.gondii ROP2 was observed in the T.gondii infected cells under a fluorescence microscope.COS-7 cells over-expressed with CFP-Racl-WT were infected by T.gondii tachyzoites and incubated for different time.The Racl-positive PVs were counted under a microscope.The phosphorylation level of Raclin HFF cells infected with T.gondii for different time was detected by western blot.HFF cells infected with T.gondii tachyzoites were treated with Racl inhibitor(NSC23766)or left untreated for 24h,the replication rate of T.gondii were counted under a microscope.The T.gondii tachyzoite nuclear extract were prepared and used immediately for DNMT activity assay.The cDNA sequences of TgDNMTa and TgDNMTb were cloned into the pET32a(+)vector and expressed in E.coli.The purifications of the recombinant T.gondii DNMTs were used immediately for DNMT activity assay.RT-qPCR analysis was used to evaluate the DNMT transcription level in T.gondii tachyzoites and bradyzoites.DNA methylation was detected with genome-wide bisulfite sequencing.m5C locus were verified with qPCR following digestion with methylation sensitive or insensitive restriction digestion enzyme.Bioinformatics analysis were performed to analyze the functions of the the genes with different DNA methylation levels in tachyzoites and bradyzoites.T.gondii tachyzoites were treated with DNMT inhibitor(5-AzaC)or left untreated,the invasion efficiency and replication rate of T.gondii were counted under a microscope.The TgDNMTa konckout and TgDNMTb konckout ME49 strain T.gondii were constructed with CRISPR/CAS9 systerm.Results:T.gondii invasion efficiency into the normal cells was significantly higher than that into NSC23766 treated cells(p<0.05).The cytoskeleton reorganization was more active in the untreated cells comparing to the NSC23766 treated cells when the cells were infected with T.gondii.Host cell Racl co-localized with T.gondii ROP2 on the PVM.Racl was recruited to the PVM as soon as the invasion started and then disappeared from the PVM later.The phosphorylation level of host cell Racl increased with the infection time(0-6 hours)of T.gondii.Long term treatment with Racl inhibitor could reduce the replication rate of T.gondii in the PV(p<0.05).We identified two functional DNA methyltransferases,TgDNMTa and TgDNMTb,in T.gondii that may mediate DNA methylation.The E.coli expressed recombinant proteins showed intrinsic methyltransferase activity;both have higher transcription levels in bradyzoites than that in tachyzoites.We performed genome-wide analysis of DNA methylation in tachyzoites and bradyzoites.The results showed more methylation sites in bradyzoites than that in tachyzoites.The most significantly enriched GO-terms of the genes with different DNA methylation levels were associated with basal cellular processes such as energy metabolism and parasite resistance to host immunity.Tachyzoite proliferation in PV can be inhibited by the DNA methyltransferase inhibitor 5-AzaC(p<0.05).Conclusion:Rac1 regulated host cell cytoskeleton reorganization to facilitate T.gondii invasion.This study provided the first confirmation of DNA methylation in T.gondii.Bradyzoites have more methylated sites than that in the tachyzoites.DNA methylation of T.gondii may function in the tachyzoite-bradyzoite interconversion.