Identification Of Differentially Expressed Proteins In High And Low Virulent Strains Of Mycoplasma Bovis And Their Roles In The Virulence Strains

Mycoplasma bovis is a wall-less bacterium and belongs to the class of the Mollicutes.It is one of the important pathogens causing bovine respiratory diseases,which leads a variety of clinical manifestations,including bovine bronchial pneumonia,otitis,mastitis,and genital disorders,tenosynovitis,arthritis,meningitis,keratoconjunctivitis,abortion and infertility.Due to its unclear virulence factors and limited understanding of the pathogenesis,so far,there is still no effective vaccine to prevent M.bovis infections.Antibiotic treatment has almost no effect,and the resistance of pathogens to antibiotics is increasing,resulting in lack of effective prevention and control measures.It has brought huge economic losses to the beef cattle and dairy industries.The virulent strain of M.bovis HB0801 P1 and its 150 passages of attenuated strain P150 obtained in our previous studies,were selected to investigate.To reveal the molecular basis of the attenuation and potential virulence factors,we used isobaric tags for relative and absolute Quantitation(iTRAQ)coupled with tandem mass spectrometry for comparison of proteins abundances between two strains.The main results are as follows:1.A total of 592 proteins were quantified from two strains by iTRAQ,corresponding to the 77.7% of the predicted ORFs(592/762).Thirty-seven proteins showed differential expressions,including 25 down-regulated proteins and 12 up-regulated in its attenuated strain.2.In order to explore the relationship between differential proteins and virulence,combining the difference in protein expression and the operability of codon mutations,a total of 16 proteins down-regulated in attenuated strains was investigated,including Mbov＿135,158,282,283,299,312,338,364,418,461,468,469,654,656,720,721 and two proteins Mbov＿086 and Mbov＿699 with the equal amount of expression were studied.After mutating UGG into UGG by overlap extension PCR,17 genes were successfully cloned(Mbov＿135 unsuccessfully)and transformed into E.coli.expression system,and 15 purified recombinant proteins were finally obtained(Mbov＿086,158,282,283,299,312,338,364,461,468,469,656,699,720,721),two recombinant bacteria(Mbov＿418 and Mbov＿654)did not express recombinant proteins.Then,a monoclonal antibody against the purified recombinant protein rMbov＿469 and the rabbit polyclonal antibodies against the purified recombinant proteins rMbov＿086,338 and rMbov＿699 were prepared for subsequent experiments.3.Subsequently,the reliability of proteomic results were confirmed by quantitative RT-PCR and Western Blot,at the mRNA expression level and protein expression level.Then,we analyzed these differentially expressed proteins roles in pathogenicity of M.bovis,coupling with their molecular characteristics and published papers.The results showed that among the down-regulated proteins in the attenuated strain,a few were linked to virulence,including NADH oxidase,Alcohol dehydrogenase(ADH),MethylenetetrahydrofolatetRNA-(uracil-5-)-methyltransferase,lipoprotein,ABC transporter,adhesion-related proteins,etc..Whereas in the attenuated strain,up-regulated proteins affected by culture conditions,might be stress-related proteins.4.The six purified proteins(Mbov＿282,Mbov＿299,Mbov＿338,Mbov＿461,Mbov＿469 and Mbov＿721)were analyzed by Western Blot.The results showed that they were recognized by M.bovis positive serum and identified as novel immunogenic proteins.5.To further explore the relationship between the differential proteins and the virulence,we selected a down-regulated protein in attenuated strain,M.bovis ADH and explored its functions of pathogenicity.The purified recombinant ADH(rADH)protein showed enzymatic activity.Western Blot combined with indirect immunofluorescence assay showed that ADH was distributed in the cytoplasm and membrane of M.bovis.Moreover,ADH was an immunogenic protein of mycoplasma,which was conserved among different strains.Indirect immunofluorescence combined with laser confocal assay showed that rADH can bind to the embryonic bovine lung cells(EBL).Furthermore,antirADH serum significantly reduced the abilities of adhesion to EBL cells,comparing to its pre-immune serum.Furthermore,Dot-blot and enzyme-linked immunosorbent assay showed that rADH bind to fibronectin in a dose-dependent manner.These results indicate that ADH acts as a cell adhesion factor in M.bovis infection by interacting with host fibronectin.In conclusion,the ADH protein seem to be involved in the M.bovis adhesion to host cells and play an important role in the pathogenesis of M.bovis.Differential protein expression profiles of M.bovis were obtained by the iTRAQ.Among of down-regulated proteins in attenuated strains,ADH was found to be an adhesionrelated protein,suggesting that it may be a new potential virulence factor.This study laid a foundation for further research the molecular mechanisms of M.bovis pathogenesis.At the same time,the novel immunogenic proteins may become vaccine candidate antigens and diagnostic markers in the future.