The Knockout Of Irgm4,5,6 Genes In Canine Mdck Cells And Its Resistance To The Growth Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite that can infect all warm-blooded animals,including humans,in a global distribution.Toxoplasmosis is a comorbidity of human beings mainly through the consumption of meat containing bio-envelopes,or the ingestion of food or water contaminated with oocysts.In recent years,Toxoplasmosis spread widely in the aquaculture industry,especially pigs,which can cause abortion or stillbirth in pregnant sows,which seriously endangers the development of public health and animal husbandry.At present,there are no effective measures to prevent toxoplasmosis.Therefore,it is urgent to find an ideal and effective method to inhibit T.gondii infection.Current studies have shown that interferon can induce the expression of a large number of endogenous innate immune proteins and play a role in the resistance to T.gondii infection in cellular immunity against T.gondii infection in mice.It contains immune-related GTPases?IRGs?and guanylate-binding proteins?GBPs?.IRGs are structurally and functionally divided into GMS proteins and GKS proteins.GMS-like IRGs?Irgm1,Irgm2,Irgm3?are "guided" to GKS-like IRGs?Irga6,Irgb6,Irgb10,etc.?to the PVM surface by the principle of missing-self.It also causes the aggregation of GBPs to form a "splitting membrane complex",which in turn causes vesicles of PVM to expose T.gondii,which allows T.gondii to be recognized by natural immune molecules in the host cells,which in turn kills T.gondii.In human cells,the IRGM protein can kill invading endogenous pathogens such as M.tuberculosis by an autophagy system.However,studies have shown that GBPs in human cells cannot be recruited to the surface of PVM to exert anti-parasitic effects.It is speculated that during the evolution process,the function of GBPs in the immune process against Toxoplasma infection is gradually weakened.From the perspective of evolution,it is analyzed that the kinship of dogs is closer to humans.Is the immune mechanism of toxoplasma infection in dogs more similar to humans than mice?Therefore,this study firstly used the method of lentiviral packaging to detect the localization detection of IRGs and GBPs protein in wild-type cells.this study identified canine-derived Irgm4,Irgm5,Irgm6 proteins by sequence alignment with mice,and designed specific forward and reverse g RNAs,using CRISPR/Cas9 knockout technology for Irgm4 in canine MDCK cells.Irgm5 and Irgm6 were knocked out one by one,and verified by PCRamplification and sequencing to establish an Irgmknockout cell line.Secondly,in the case of IFN-? induction and non-induction,the growth of T.gondii in wild-type MDCK cells,Irgm^4/5/6-/-knockout cell lines and Irgm^4/5/6 gene replenishing cells were compared.The results showed that the growth of T.gondii was not significantly different in the absence of IFN-?induction,whereas in the case of IFN-? induction,Irgm^4/5/6-/-knockout cells were compared with wild-type MDCK cells.The growth rate of T.gondii was significantly increased,indicating that Irgms is an IFN-?-inducible gene in canine cells and plays a role in the growth of T.gondii.In order to further explore the synergistic protein of Irgms inhibiting the growth of T.gondii in canine cells,the localization of related proteins and T.gondii PVM was detected by laser confocal method.The results showed that Irgb11 and Irgb12 were recruited to the surface of T.gondii PVM to resist arching.The role of worms,in which Irgb12 plays a leading role,and GBP1 and GBP5 are not recruited to the surface of PVM,indicating that GBPs in canine cells can not inhibit the growth of T.gondii by destroying PVM.This is similar to human GBPs.In this study,for the first time,Irgm4,Irgm5,and Irgm6 in canine MDCK cells were knocked out one by one,and the growth of T.gondii was confirmed by Irgm^4/5/6-/-knockout cells.The results showed that in MDCK cells,CRISPR-Cas9 gene knockout technique was used to verify that Irgm4,Irgm5 and Irgm6 were able to inhibit T.gondii induced by IFN-?.Irgms protein can induce the recruitment of Irgbs protein to the surface of T.gondii PVM to form a structure similar to the "splitting membrane complex" to destroy PVM and thereby inhibit the growth of T.gondii.This study lays the foundation for further research on the host's natural immune mechanism to inhibit T.gondii infection.