GABARAPL2 Is Critical For Growth Restriction Of Toxoplasma Gondii In HeLa Cells Treated With Gamma Interferon

IFN-y-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection.A core set of autophagy proteins is required for IFN-y-mediated clearance of T.gondii in the mouse because of their control of several downstream effectors,including IRGs and GBPs.However,these effectors are absent(i.e.,IRGs)from or nonessential(i.e.,GBPs)in IFN-y-activated human cells,raising the question of how these cells control parasite replication.It has been reported that IFN-? stimulates non-acidification-dependent growth restriction of T.gondii in HeLa cells.Studies have defined a novel role for ubiquitination,recruitment of autophagy adaptors and LC3 in the strain-specific control of T.gondii replication in IFN-y-activated human cells.Mice that lacked GABARAPL2 but not LC3 were defective in the IFN-y-induced clearance of T.gondii.However,there are differences between IFN-y stimulated human and mouse cells in inhibition of T.gondii.Therefore,it is important to verify the role of GABARAPL2 in HeLa cells in the inhibition of T.gondii.In this study,we first generated the GABARAPL2 knockout(KO)HeLa cells by the clustered regularly interspersed short palindromic repeats(CRISPR)/Cas9 method,and found that GABARAPL2 could be recruited to the T.gondii PVs in HeLa cells induced by IFN-y,and GABARAPL2 played a key role in inhibiting the growth of T.gondii.At the same time,we also found that in IFN-y-activated cells,large accumulations of GABARAPL2 were recruited to membranes surrounding PVs of T.gondii,but not directly bound to the PVs.Then GAB ARAPL2 or GABARAPL2(G116A)protein were re-expressed in GABARAPL2KO cells.Indirect immunofluorescence assay showed that GABARAPL2(G116A)protein were not recruited to membranes surrounding PVs of T.gondii,which indicated that GABARAPL2 were recruited to the PVs of T.gondii by lipidation.In addition,Ubiquitin,autophagy adaptors(p62,NDP52,OPTN)and autophagy related protein ATG5 are required for the localization and function of GABARAPL2 in the IFN-?-induced immune response.The deletion of autophagy adaptors p62,NDP52,OPTN and ATG5 seriously affected the inhibitory of IFN-? on T.gondii growth.These provide further understanding of a noncanonical autophagy responsible for IFN-?-dependent inhibition of T.gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.