Screening And Functional Identification Of Proteins Interacting With P1 Protein Of Mycoplasma Pneumoniae

Background: Mycoplasma pneumoniae is the most common pathogen causing human respiratory infections.In addition to causing community-acquired pneumonia in children and adults,it can lead to a variety of extra-pulmonary conditions.M.pneumoniae has a small genome and a simple structure.The P1 protein in its tip structure dominates the adhesion and sliding of M.pneumoniae on the host cell membrane.The absence of P1 makes M.pneumoniae lose its pathogenicity,indicating that it plays an vital role in the pathogenesis.However,the receptor of P1 protein and adhesion mechanism of M.pneumoniae to host cells remain controversial.This study aimed to screen the proteins that can interact with P1 protein on the membrane of human bronchial epithelial(BEAS-2B)cells and verify whether they can play the corresponding role as an adhesion receptor.The identification of receptor proteins can facilitate us better understand the pathogenic mechanism exploited by M.pneumoniae,and provide new ideas for the prevention and treatment of M.pneumoniae.Object: To screen the receptors of recombinant P1 protein on BEAS-2B cell membrane by using the modified virus overlay protein binding assay,and verify its function.To provide evidence for clarifying the adhesion mechanism of M.pneumoniae and the function of P1 protein.Methods:1.RP1-C/p ET-30a(+)vector was constructed,the 1,160-1,498 amino acid residues at the carboxyl end of P1 protein were expressed and purified.After the eluent was subjected to ultrafiltration and concentration,the concentration of recombinant P1 protein(rP1-C)was detected by BCA method.2.Preparation of rP1-C antibody:RP1-C was mixed with Freund’s adjuvant,emulsified and injected subcutaneously to immunize New Zealand rabbits.Blood was taken from the heart and the serum was preliminarily purified by saturated ammonium sulfate method,and then the antibody was further purified by CNBr-activated Sephaose 4B combined with rP1-C.3.The membrane proteins were extracted,and the modified VOPBA and LC-MS mass spectrometry were used to screen the proteins in BEAS-2B membrane that can specifically bind to rP1-C.The screened proteins were verified by Western blotting.Then,indirect immunofluorescence was used to observe the distribution of receptor protein in cells.4.The interaction between rP1-C and receptor protein was confirmed by Far-western blotting,indirect enzyme-linked immunosorbent assay(ELISA),Co-immunoprecipitation and double immunofluorescence assays.5.M.pneumoniae and rP1-C were preincubated with the receptor protein and the BEAS-2B cells were preincubated with anti-receptor protein antibodies to reduce the available binding sites of M.pneumoniae and rP1-C to BEAS-2B.Then,indirect immunofluorescence was used to observe whether the adhesion of M.pneumoniae and rP1-C to cells was affected by the receptor protein and its antibodies.6.The cells were transfected with short interfering RNA(siRNA)to interfere with the expression of receptor protein in the cells.Western blotting and Real Time Quantitative PCR were used to detect the expression of the protein.Indirect immunofluorescence was then performed to observe the adherence of M.pneumoniae and rP1-C to BEAS-2B cells.7.Lentivirus vector CMV-GFP-puro-Vimentin was used to construct receptor protein overexpressing cell lines.Western blotting and Real Time Quantitative PCR were used to detect protein expression levels.indirect immunofluorescence was subsequently performed to observe the adherence of M.pneumoniae and rP1-C to BEAS-2B cells.Results:1.The protein was successfully expressed and the expressed proteins were analyzed by SDS-PAGE and Western blotting,there was an obvious band at a molecular weight of about 43 k Da,indicating that the protein was successfully expressed.The protein concentration was detected to be about 1.5 mg/m L.2.The results of SDS-PAGE and Western blotting showed that the molecular weight of the heavy chain and light chain of the polyclonal antibody was about 50 k Da and 25 k Da,respectively,indicating that the rP1-C polyclonal antibody was successfully prepared and purified.3.The results of Modified VOPBA and LC-MS showed that Vimentin and beta-4 Tubulin have the highest score,which are most likely receptor proteins of rP1-C.And the results of indirect immunofluorescence demonstrated that Vimentin and beta-4 Tubulin are mainly distributed in the cytoplasm and membrane of cells.4.RP1-C specifically binds to Vimentin,but does not bind significantly to beta-4 Tubulin.5.The adhesion of M.pneumoniae and rP1-C to cells was significantly reduced in the Vimentin pre-incubation group and the anti-Vimentin antibody pre-incubation group,indicating that Vimentin was indeed related to the adhesion of M.pneumoniae and rP1-C to cells.6.The expression of vimentin in siRNA-vimentin transfected cells was significantly reduced,the amount of adhesion of rP1-C and M.pneumoniae to BEAS-2B cells was significantly lower than that of the control siRNA-transfected cells,indicating that vimentin played an indispensable role in the adhesion process of M.pneumoniae and rP1-C to cells.7.The Vimentin-overexpressing cell line was successfully constructed,and the expression of Vimentin was increased,as well as the adhesion of M.pneumoniae and rP1-C to cells,indicating that vimentin could indeed act as an adhesion receptor for M.pneumoniae.Conclusion:Vimentin may serve as an adhesion receptor forP1 protein of M.pneumoniae,playing a vital role in the adhesion of M.pneumoniae to BEAS-2B cells.