Molecular Mechanism Of Virulence Factor ROP18in Toxoplasmosis

Background:Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect allwarm-blooded animals. Toxoplasmosis is one of the more common parasitic zoonosesworld-wide. Infection in immunocompetent individuals usually is asymptomatic butinfection in immunocompromise would lead to serious complications. Nowtoxoplasmosis may be a major cause of morbidity and mortality in AIDS and organtransplant patients. Toxoplasma gondii hybridization experiments, genome analysis andtransgenic strain of Toxoplasma gondii prove that ROP18can increase the speed ofproliferation division of Toxoplasma gondii and cause lethal effect on infected animal, soROP18is an important virulence factors of Toxoplasma-gondii. In this study, wescreened the host cell proteins that can interact with Toxoplasma gondii ROP18by usingyeast two-hybrid system and constructed the expressing ROP18transgenic strain ofToxoplasma gondii. The interaction between p65and ROP18was confirmed throughcoimmunoprecipitation, GST pull-down, yeast-two-hybrid assays andimmunofluorescence. Our results provide the theory and experiment basis for delineatingthe molecular mechanism of toxoplasmosis. Objective:To screen the host cell proteins that can interact with Toxoplasma gondii ROP18by usingyeast two-hybrid system. The interaction between p65and ROP18was confirmedthrough coimmunoprecipitation, GST pull-down, yeast-two-hybrid assays andimmunofluorescence..Materials:The experiment consists of two parts:(1)The ROP18gene fragments were amplified byRT-PCR from mRNA of Toxoplasma gondii RH strain. The product of RT-PCR wasdigested with double restriction enzyme and was subcloned into the bait vector pGBKT7.The recombinant plasmid was transferred into yeast AH109strain. Its toxicity and theautonomous activating activity were tested. The human fetal brain cDNA library wasscreened with pGBKT7-ROP1825-251aaas the bait plasmid by yeast two-hybrid system.(2)The ROP18gene fragments were amplified by RT-PCR from RNA of Toxoplasmagondii RH strain. The product was subcloned into a PCR blunt II Topo vector to generatepROP18. The ROP18gene coding sequence was PCR-amplified from pROP18andsubcloned into pTUB8mycGFPPftailTY to generate pTUB8-ROP18-TY.pTUB8-ROP18-TY was electroporated into the parasites. Then Toxoplasma gondiisuspension respectively was transferred to bottles cultured adherent cells. Transfectantswere selected with25μg/ml mycophenolic acid and50μg/ml xanthine. The strains werecollected and injected into mice abdominal cavity. The expression of ROP18was alsodetected by immunofluorescence analysis. Giemsa assay was used to detect the rate ofproliferation between the RH strain and the expressing ROP18RH strain. The interactionbetween p65and ROP18was confirmed through coimmunoprecipitation, GST pull-downand yeast-two-hybrid assays. Immunofluorescence staining was performed to verify theco-localization of ROP18with p65in the cytosol of the infectious HFF cells.Results:1.Screening of host cell proteins that interact with Toxoplasma gondii ROP18(1) Endonuclease digestion analysis showed that the plasmid was constructedsuccessfully.(2) The yeast protein was extracted with glass beads (Sigma, USA), and WB analysis showed the expression of ROP18.(3) Using the selection procedures, eight novel host cell proteins were obtained: NF-κBp65, damage-specific DNA binding protein1(DDB1), torsin A interacting protein1(TOR1AIP1), integrin beta1, solute carrier family3(SLC3A2), tyrosylproteinsulfotransferase (TPST2), OCIA domain containing1(OCIAD1), Der1-like domainfamily member2(DERL2), in addition to Homo sapiens activating transcription factor6beta (ATF6).2. Construction of transgenic Toxoplasma gondii overexpressing ROP18(1) Endonuclease digestion analysis showed that the plasmid was constructedsuccessfully.(2) The cells infected with transgenic Toxoplasma gondii were extracted, fixed andstained with mouse anti-Ty-1antibody(green), DAPI (blue). Immunofluorescencestaining showed that ROP18was localized at the rhoptry of Toxoplasma gondii.(3) Giemsa assay showed that the proliferation of ROP18over-expressing RH strain ishigher than the RH strain. Therefore, ROP18promotes the proliferation of Toxoplasmagondii in cells.(4) Co-transformation Assay: AH109cells were co-transformed pGADT7-p65andpGBKT7-ROP18and then selected on supplemented minimal plates lacking tryptophan,leucine, histidine, and adenine. The interaction between p65and ROP18was revealedthrough staining for β-galactosidase activity with X-α-Gal. The co-transformation assayconfirmed that p65interacts with ROP18.(5) Immunofluorescence: HFF cells infected expressing ROP18transgenic strain wereextracted, fixed and stained with mouse anti-Ty1antibody (green), rabbit anti-P65antibody (red), DAPI (blue). The merged images show that p65was recruited around theparasites and co-distributed with ROP18.(6) Co-immunoprecipitation Assay: The293t cells were transfected with ROP18-Δ27-GFP and GFP. Immunoprecipitates were transferred to NC membranes and were hybridi-zed with anti-GFP antibody and anti-P65antibody. The results suggested that ROP18indeed interacts with p65in vivo. (7)GST-pull down: We carried out a pull-down assay in which histidine-taggedrecombinant p65-M was purified on Ni-NTA agarose beads and used as an affinitymatrix to absorb purified GST-ROP18in test tubes. The results showed that p65interactswith ROP18directly.Conclusions：(1) Eight ROP18-interacting host proteins have been obtained via yeast two-hybridsystem.(2) The transgenic strain of Toxoplasma gondii over-expressing ROP18was constructedsuccessfully.(3) NF-κB p65interacts with ROP18directly.