| Background:Toxoplasma gondii(T.gondii)is an opportunistic pathogenic parasite that can infect most warm blooded animals,including humans.T.gondii is one of the most successful pathogens in the world in terms of its geographic distribution or the diversity of its host range.T.gondii has a variety of secretory organelles,including rhoptry,dense granule,and micronene.During T.gondii infection,many effector molecules are released into the cytoplasm of the host cells and the parasitophorous vacuole(PV)to mediate its invasion,multiplication and cell rupture,and participate in the regulation of host immune function for the survival of T.gondii in the host cells.Among these effectors,TgROP18? is considered to be the key virulence factor of T.gondii.As a member of kinase ROP2 subfamily,TgROP18? is secreted into the host cells by the rhoptires in the apical organelles,and locates on the parasite vacuolar membranes(PVMs)of T.gondii in the process of invasion.As a Serine/Threonine protein kinase,TgROP18?exerts its fucntions by targeting and phosphorylating host proteins.BioID2 method is a biotin ligase based on Aquifex aeolicus(thermophilic bacteria).BirA*is fused with bait proteins and biotinylated with neighboring proteins.These biotinylated proteins can be selectively separated by conventional biotin capture methods and identified by mass spectrometry(MS).BioID2 method was used to screen the protein interacting with TgROP18? in living cells to reveal the mechanism of TgROP18? virulence.Method:1.The ROP18-BirA*-FLAG recombinant strain was constructed by CRISP-Cas9 technology.2.The invasion and proliferation of RH-ROP18-BirA*-FLAG in HFF cells were detected with indirect immunofluorescence assay(IFA).3.The transcription and expression of TRIM21 in HFF cells infected by T.gondii was detected by real time q-PCR and western blotting.4.The effect of IFN-y-induced TRIM21 on the proliferation and the ubiquitin labeling on PVM of T.gondii was detected by IFA.5.The regulation of NF-?B by TRIM21 was detected through Co-IP and western blotting.6.The interaction between TgROP18? and TRIM21 were detected by FRET,Co-IP,and western blotting.Results:1.RH-ROP18-BirA*-FLAG strain was successfully constructed,and nine ubiquitin related proteins interacting with TgROP18? were identified by mass spectrometry.2.The invasion efficiency and mice virulence of RH-ROP18-BirA*-FLAG strain remained unchanged.3.TRIM21 knocking down reduced the inhibitory effect of IFN-y on the proliferation of T.gondii,and the ubiquitin labeling of PVM in host cells.4.TRIM21 overexpression inhibited the proliferation of CEP strain,but not RH strain in host cells.5.TRIM21 interacted with I?B-? to inhibit the interaction of p65 and I?B-?,and then activate NF-?B pathway to inhibit the proliferation of T.gondii.6.TgROP18? interacted with the PRY-SPRY domain of TRIM21 to promote the phosphorylation level of TRIM21,and induced the degradation of TRIM21 through lysosomal pathway.The degradation of TRIM21 was closely related to the kinase activity of TgROP18?.TgROP18? promoted the proliferation of T.gondii in TRIM21 overexpressed cells.Conclusion:TRIM21 plays an important role in the inhibition of T.gondii proliferation induced by IFN-y.In the TRIM21 knockdown cells,the effect of IFN-y on the ubiquitin labeling of PVM and the inhibition of proliferation were significantly reduced.In this study,BioID2 method was used for the first time to screen the host ubiquitin related proteins interacting with type I ROP18;TRIM21 as a substrate of type I ROP18 was also reported for the first time.TRIM21 was firstly found to interacting with I?B-? to inhibit the binding of p65 and I?B-?,and then activated NF-?B pathway which inhibited the proliferation of T gondii.TgROP18? was firstly found to interact with TRIM21,promote the phosphorylation level of TRIM21 and induce degradation of TRIM21 through the lysosomal pathway,thus block the inhibitory effect of IFN-?-TRIM21-NF-?B on the proliferation of T.gondii and realizing the immune escape of T.gondii. |
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