A Research On The Mechanism Of Toxoplasma Gondii Host Dependancy Factor Cbl-b Inhibiting Host Innate Immune Signaling

Background:Toxoplasma gondii is intracellular parasitic protozoa.Due to the small genomes and a limited number of encoded proteins,T.gondii has to exploit host factors for entry,replication,and dissemination.Such host factors can be defined as host dependency factors(HDFs).Although HDFs partly affect host cell viability,they are essential for pathogen infection and are potential ideal targets for therapeutic intervention.However,information on these HDFs required for T.gondii infection is very scarce.The genome wide CRISPR/Cas9 library has been widely used in gene screening for drug target,tumor-acting,and viral infection.Compared with RNAi,genome-wide CRISPR lentiviral sgRNAs library screening has higher sensitivity and specificity in gene editing.A CRISPR/Cas9 sgRNAs lentiviral library targeting 19,050 human genes was used to edit the genes of HFF to screen for the potential HDFs of T.gondii parasitism.Methods:A library of cells with specific gene knockout was constructed using a CRISPR lentiviral sgRNAs library targeting 19,050 human genes and 1,864 miRNAs,and infected with T.gondii to screen for HDFs.Using RNAi to interfere with the HDF gene expression to verify the effect of the gene on the proliferation of T.gondii.The clustering and function of HDFs were analyzed by bioinformatics.Cbl-b KO cell lines and mice were constructed by CRISPR/Cas9 gene editing technology.The proliferation ability of T.gondii in Cbl-b KO cells and mice was determined to be the host dependency factor of T.gondii.The interaction between Cbl-b and MyD88 was verified by FRET and Co-IP,and the effect of Cbl-b on MyD88 ubiquitination was verified by Co-IP.The effects of Cbl-b gene knockout on different types of cytokines’transcription/secretion,and the propotion of the immune cell subsets induced by T.gondii infection were evaluated by real-time PCR/Cytometric bead arrays,and flow cytometry,respectively.Results:In this study,from a pool of 19050 human genes and 1864 human primiRNAs,1193 potential HDFs were identified,including 1183 genes and 10 primiRNAs(corresponding with 17 mature miRNAs).It was confirmed that the proliferation of T.gondii was significantly inhibited after 7 genes(USP19,Cbl-b,USP17L24,ENPP5,PIM1,HDAC7,ULK1)and 5 mature miRNAs(miR-642a-5p,miR-1270,miR-3065-5p,miR-22-5p,miR-656-5p)were knocked down or inhibited,respectively.Bioinformatic analysis revealed 53 HDFs were associated with regulation of host actin reorganization and 23 HDFs coded immune negative regulators.This result indicated that actin reorganization was essential for T.gondii infection,and some host immune negative regulators may be involved in disarming host defense.Cbl-b was screened to be one of the HDFs required by T.gondii.The proliferation of T.gondii was found to be significantly inhibited in Cbl-b knockout cell lines.The expression of Cbl-b increased with prolonged T.gondii infection,while the expression of MyD88 decreased in T.gondii infection group.Co-IP and FRET results showed that Cbl-b could interact with MyD88 and mediate the ubiquitination of MyD88.Cbl-b knockout(Cbl-b KO)mice were constructed in this study.Both the wild type C57BL/6J and Cbl-b KO mice were infected with T.gondii ME49 strain.On 13 days post infection,all the wild type mice died,while 80%of the Cbl-b KO mice survied till the end of the experiment.The parasitic copies in the lung and brain of the Cbl-b KO mice were significantly lower than that in the wild type mice.Compared with wild type mice,the percentages of B cells were increased,and macrophages were decreased in Cbl-b KO mice,the level of serum IFN-y and IL-6 were significantly increased in Cbl-b KO mice.Conclusion:T.gondii exploit host factors for successful infection and parasitism,through manipulating host cytoskeleton rearrangement and negatively regulating host’ s immunity,which might promote the intracellular proliferation and immune escape of T.gondii.T.gondii infection induces increased expression of Cbl-b.Cbl-b interacted with MyD88 when host cells were infected,and then ubiquitinated MyD88 for degradation,as a result,negatively regulated the host’s anti-T.gondii innate immunity.