Research On Toxoplasma Gondii ROP18 Interactome In Human Cells And Mechanism Of TgROP18_Ⅰ’s Disruption Of NMI’s Function

Background:Toxoplasma gondii rhoptry protein ROP18(TgROP18)is a key virulence factor secreted into the host cell during invasion,where it modulates the host cell response by interacting with its host targets.However,only a few TgROP18 targets have been identified.TgROP18 is known to mediate its effects in mice by targeting the immunity-related GTPases(IRGs).Given that IRGs are poorly conserved in humans,the role of TgROP18 during infection of human cells is unknown.Additionally,whether the sequence differences between the TgROPl 81 and TgROP18Ⅱ alleles result in any differences in functionality is unknown.Methods:In this study,we applied a high-throughput protein-protein interaction(PPI)screening in human cells using bimolecular fluorescence complementation(BiFC)to identify the targets of TgROP18Ⅰ and TgROP18Ⅱ.Fluorescence resonance energy transfer(FRET)and co-immunoprecipitation(Co-IP)were used to verify the PPI between TgROP18Ⅰ and NMI,IL20RB,P2RX1,IL21,or UBC.Bioinformatic analysis,including gene ontology(GO)analysis,search tool for the retrieval of interacting genes/proteins PPI network(STRING),and ingenuity pathway analysis were performed to investigate TgROP18’s interactome.Co-IP and immunofluorescence assay(IFA)were conducted to confirm the interaction between Tg-ROP18Ⅰ and NMI during Toxoplasma infection.Transgenetic parasites were constructed using homology modeling,phylogenetics and site-directed mutagenesis,in order to identify the residue of TgROP18Ⅰ required for NMI association.Protein level of NMI in subcellular fractions and IFN-y-activated sequence(GAS)were quantified by western blotting and ChIP in the presence and absence of TgROP18-expression,to evaluate the impact of TgROP18Ⅰ on NMI nuclear translocation and GAS binding.Parasite replication assay was applied to test if TgROP18Ⅰ facilitated parasite resistance to IFN-γ treatment via interference with NMI.Results:From a protein pool of more than 18,000 human cDNA expression library,492 and 141 proteins were identified as the targets of TgROP18Ⅰ and TgROP18Ⅱ,respectively.GO analysis,STRING,and ingenuity pathway analysis revealed that the majority of these proteins were associated with immune response and apoptosis.Among the proteins identified,NMI,IL20RB,P2RX1,IL21,and UBC were further verified as TgROP181 targets by FRET and Co-IP.Specifically,NMI was confirmed as a unique target of TgROP18Ⅰ,but not TgROP18Ⅱ.In transgenic parasites expressing TgROP18Ⅰ,TgROP18Ⅱ or HhROP18,only those parasites expressing TgROP18Ⅰ are capable of targeting NMI on the parasitophorous vacuolar membrane(PVM).Consistent with this distinct pattern,we also found that TgROP18Ⅰ interferes with the function of NMI by inhibiting its nuclear translocation and its association with a GAS in the IRF1 gene promoter.Finally,using homology modeling,phylogenetics and site-directed mutagenesis we have determined that the catalytic activity of TgROP18Ⅰ and a key amino acid in the substrate-binding loop of TgROP18Ⅰ(Arg-439,which is a proline in non-type Ⅰ-like alleles)is required for NMI association,and that this residue is one of the many that appear to be under diversifying selection between TgROP18Ⅰ and TgROP18Ⅱ alleles.Conclusion:The interactome of TgROP18 in human cells indicates a key role of TgROP18 in manipulating host’s immunity and cell apoptosis,which might contribute to the immune escape and successful parasitism of the parasite.TgROP18Ⅰtargets NMI via its catalytic activity and a key residue Arg-439 in its kinase domain,inhibiting NMI nuclear translocation and GAS binding,thus interfering with NMI’s function in human cells.