Mycoplasma Genitalium Protein Of Adhesion Suppresses T Cell Activation Through The CypA-CaN-NFAT Pathway

Background: Mycoplasma genitalium(Mg)is the smallest prokaryotic microorganism that lacks cell walls and can grow and reproduce in inanimate medium causing a variety of genitourinary diseases.M.genitalium protein of adhesion(MgPa)is vital to M.genitalium adhesion and host invasion.Cyclophilin A(CypA)is identificated as the MgPa receptor in our previous study.Studies have confirmed that Cyclosporin A(CsA)combined with CypA can down-regulate the expression of Calcineurin(CaN),inhibit the nuclear translocation of nuclear factor of activated T cells(NFAT),reduce the transcription of IL-2 and IFN-γ mRNA,and thus inhibit the activation of T cells.In this study,we investigate whether MgPa can suppresses T-cell activation after it interacts with CypA on the host T cell membrane to further elucidate the immunosuppressive mechanism of M.genitalium on host cells.Objiect: To explore whether rMgPa can block the calcineurin-NFAT signaling pathway and prevent T cell activation and thus play immunosuppressive function to host cell,which benefit to elucidate the biological function of MgPa and the immunosuppressive mechanism of M.genitalium.Methods:1.The distribution of CypA,Co-localization and intracellular interaction of MgPa-CypA in T cells transfected with pcDNA3.1(+)/MgPa were identified by indirect immunofluorescence assay.2.The adhesion of recombinant protein(rMgPa)and M.genitalium to Jurkat cells and whether CypA and the corresponding antibody could inhibit the adhesion of rMgPa to Jurkat cells were detected by indirect immunofluorescence assay.3.The expression of CypA was detected using Western blot(WB)after the CypA-siRNA was transfected into Jurkat cells.4.WB and indirect enzyme-linked immunosorbent assay(ELISA)were performed to detect the phosphorylation and activity of calcineurin(CaN)after rMgPa treated T cells activated by CD3-antibody and CD28-antibody.5.Indirect immunofluorescence was examined to detect the nuclear translocation of nuclear factor of activated T cell(NFAT)in Jurkat cells pretreated with rMgPa.6.Real-time fluorescent quantitative PCR(RT-qPCR)and ELISA were used to detect the transcription levels and protein levels of IL-2 and IFN-γ after activated T cells pretreated with rMgPa.7.Flow cytometry was used to detect the expression of T cell surface molecules CD25 and CD69 and intracellular cytokines IL-2 and IFN-γ in T cells pretreated with rMgPa.Results:1.CypA was mainly distributed in cytoplasmic matrix and nucleus,and could co-located with rMgPa in the cell membrane of Jurkat cells.2.M.genitalium adhered and invaded host cells via rMgPa.CypA and CypA antibodies could partially inhibit rMgPa adhesion to Jurkat cells.3.rMgPa can inhibit the phosphorylation and activity of CaN.The interference of CypA-siRNA weakened the ability of rMgPa to inhibit CaN phosphorylation and activity in Jurkat cells.4.PMA/Ionomycin could induce the NFAT translocation in T cells,which was influenced by rMgPa.The nuclear translocation of NFAT was recovered after CypA-siRNA was transfected Jurkat cells.5.rMgPa could inhibit mRNA and protein expression of IL-2 and INF-γ.Compared with the rMgPa experimental group,the mRNA and protein expression of IL-2 and INF-γ were upregulated in the CypA-siRNA transfected Jurkat cells.6.The IL-2,INF-γ,CD69,and CD25 expression were very low in unstimulated T cells.After stimulation with CD3/CD28 antibodies,the levels of IL-2,INF-γ,CD25 and CD69 were significantly increased.The expression of IL-2,INF-γ,CD25 and CD69 in T cells was suppressed after rMgPa and CaN inhibitors pretreated.The inhibition of rMgPa to T cell activation was improved in the CypA-siRNA transfected T cells.Conclusion:1.MgPa can bind with the CypA receptor on T cell membrane to mediate Mg adhesion and invasion into T cells;2.MgPa can suppress inhibit human T cell activation through the CypA-CaN-NFAT pathway.