Study On Secretome Of Mycoplasma Bovis With Genomic Bioinformatics Prediction And Proteome Approach And Confirmation Of Secreted Antigenic MbovP0581 Candidate:Muhammad Zubair

Mycoplasma bovis is one of the most vital pathogens in cattle that causes several forms of infection including calf pneumonia,arthritis,otitis media and mastitis,keratoconjunctivitis,genital diseases and various clinical related illnesses in cattle leading to the significant economic losses.Poor understanding about pathogenesis has put forward the challenges regarding its early diagnosis and potent vaccine.Since secreted proteins have a tricky role in M.bovis pathogenesis,this study was designed to systematically reveal M.bovis secretome and potential role in virulence of the pathogen.We followed Genomic bioinformatics prediction and Proteomics as combined approach for the investigation of secreted proteins.By using bioinformatics tools,a total of 246 secreted proteins were predicted based on M.bovis genome.Among them,14 are classical,154 nonclassical and 78 both pathways.Then by using 2-dimensional gel electrophoresis(2-DE)and Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),169 proteins were revealed.Of them,60 were previously predicted to be secreted including 3 classical,43 non-classical,and 14 both classical and non-classical.Subcellular localization revealed 8.3%(5/60)cell membrane,5%(3/60)extracellular region,31.6%(19/60)in cytoplasm,and 55%(33/60)unknown.KEGG Enrichment,Gene Ontology(GO)and protein-protein interaction analyses were performed for these sixty secreted proteins to know about the functions and mutually interacting proteins.Further 8 proteins(Mbov P0038,Mbov P0338,Mbov P0341,Mbov P0520,Mbov P0581,Mbov P0674,Mbov P0693,Mbov P0845)were predicted to be virulence-related factors with VFDB.Complete genes of Mbov P0581(ABC transporter protein)and Mbov P0674(putative lipoprotein)were expressed in E.coli,cloned and purified.Western blot analysis showed that both proteins are highly antigenic proteins which reacted with sera of cattle experimentally infected with M.bovis.However,only Mbov P0581 is a secreted protein,while Mbov P0674 is under detectable level.In addition,Mbov P0581 was investigated for its adhesion to EBL cells and found it to be an adhesin of M.bovis to EBL.This finding intensifies its role in pathogenesis and can be the potent source diagnostic marker of vaccine candidate.In conclusion,the present study was the first one that has investigated the secretome profile of Mycoplasma bovis by using combined approach of whole genome and proteome(2-dimensional gel electrophoresis)analyses.We determined that Mycoplasma bovis has at least 60 secreted proteins and 8 top secreted proteins related to virulence were proposed.Among these,Mbov P0581 was confirmed as a secreted and antigenic protein as well as an adhesin to EBL cells.Our work might contribute to the complete understanding of pathogenesis and discovery of the biomarkers for early diagnosis and to develop an effective vaccine against Mycoplasma bovis.