Study On The Protective Effect And Mechanism Of Subcutaneous With GAPDH In Mice Against Streptococcus Pneumoniae

Objective: Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a classical enzyme involved in glycolytic pathway.Currently,researches have shown that GAPDH could locate at the surface and inside of bacteria,and it takes part in other physical functions including apoptosis,DNA repair and autophagy.It has been reported that GAPDH is closely associated with virulence of bacteria and is considered as a good vaccine candidate for protecting pathogenic microorganisms.Furthermore,subcutaneous immunization is widely used as a safe and quick way.Therefore,our study aims to explore protective effect of subcutaneous immunization with GAPDH and further investigate the molecular mechanism,which lay a foundation for preclinical study.Methods: The recombinant GAPDH(rGAPDH)protein was successfully expressed through prokaryotic expression and purified using a nickelnitrilotriacetic acid column according to the manufacture's instruction.Purified protein was incubated with polymyxin B(Pm B)-agarose to exclude LPS contamination.One week after last immunization in mice,we established the lethal-infection models and the focal infection model to evaluate the survival rates and bacterial loads in nasal washes and lung homogenates respectively.In vitro,to explore the molecular mechanism,Bone Marrow-Derived Dendritic Cells(BMDCs)were stimulated with rGAPDH.Enzyme-Linked Immunosorbent Assay(ELISA)was applied to detect the titers of antigen-specific antibody and the level of cytokines.The lung tissue sections were analyzed by hematoxylin-eosin(HE)staining to confirm their inflammatory changes.The costimulatory molecules on BMDCs and the apoptosis of BMDCs were tested by flow cytometry(FCM).Western blot was used to analyze the activated signaling pathways in BMDCs.Results:The recombinant protein of GAPDH was successfully prepared and analyzed by SDS-PAGE with purity of over 90%.The residual concentration of LPS was below 0.1 EU/ml,which meet the requirements of subsequent experiments.In vivo,high titers of rGAPDH specific antibody and elevated titers of Ig G subtype in mice were observed.In the lethal-infection model,rGAPDH-immunized mice showed higher survival rates than control,while in the focal infection model,immunization with rGAPDH could decline the colonization levels in both nasopharynx and lung.In vitro,rGAPDH induced phenotypic and functional maturation of BMDCs,because the high expression of CD40,CD86 and MHC II and the production of IL-12p70,IL-6 and TNF-? were observed after treatment with rGAPDH.However,the costimulatory molecules and cytokines declined significantly in TLR2-/-and TLR4-/-mice,indicating rGAPDH can be a potential ligand for both TLR2 and TLR4.Subsequent investigations suggested that rGAPDH could also activate the phosphorylation of MAPKs,PI3K-Akt and NF-?B.Meantime,upregulation of mir-146 a and downregulation of mir-27 a in BMDCs were observed.Conclusion: our findings confirm that rGAPDH,a housekeeping protein,is also qualified as a vaccine candidate protein and rGAPDH activates BMDCs in a TLR2 and TLR4 dependent manner.