In Silico Analysis Of Differential Proteins Critical To Virulence Between Mycoplasma Bovis HB0801 And Its Attenuated Strains

Mycoplasma bovis(M.bovis)is a member of the Mycoplasmataceae family in the class of Mollicutes.It is very significant since it leads to massive economic losses for cattle industry by causing different diseases including bovine respiratory disease(BRD)complex,bovine pneumonia,mastitis and arthritis etc.To get a novel vaccine against M.bovis related diseases,M.bovis HB0801 was continuously passaged previously in the lab and the attenuated phenotypes of HB0801 derived strains were identified.Among them,the strains at passage number 115,150 and 180 referred as M.bovis HB0801-P115,HB0801-P150 and HB0801-P180 and further abbreviated as P115,P150 and P180 respectively in the present study.These passaged strains were further characterized by sequencing the genomes and revealing the proteome profiles.Hence,we aimed to understand the pathogenesis and mechanism of attenuation by analyzing the genomics and proteomics data with various tools of bioinformatics.Following results were mainly obtained.Comparative genomics analysisThe bioinformatics analysis was performed for genes related to deleted region of attenuated strains and genes having single nucleotide polymorphisms(SNPs).Different bioinformatics tools including metabolic pathways,SNPs types and location,whole genome paralogues analysis,protein-protein interactions,whole genome secretory proteins analysis,KEGG enrichment analysis,genomics and pathogenic island analysis,functional conservative domains,protein modeling and whole genome virulent factor database analysis.The SNP data was categorized into three categories.In one category,SNPs were present only in one passage.In second category,SNPs were present in two passages while in third category,SNPs were present in all three passages.The whole genome paralogues analysis highlighted four genes in deleted region while 14 genes in SNPs data having paralogues in genome of M.bovis HB0801.The secretory proteins analysis revealed that two proteins in deleted region while 10 proteins in SNPs data were found secretory.The functional conservative domains analysis for these secretory proteins was performed and domains were highlighted using structural bioinformatics tools.Moreover,protein-protein interactions highlighted five proteins interacting together.Interestingly,KEGG enrichment analysis highlighted the genes enriched in one pathway were overlapped withfindings of protein-protein interaction.Furthermore,whole genome virulent factor database highlighted some significant virulence factors including genes in genomics data.The analysis results highlighted and prioritized 11 genes in genomics data which might mostly contribute to the attenuation.The most significant 11 genes in genomics data encode Ascorbate-specific PTS system enzyme IIB and IIA components,Enolase,Llactate dehydrogenase,Pyruvate kinase,Glycerol and Multiple sugar ABC transporter ATP binding proteins,NADH dehydrogenase,Phosphate acetyltransferase,transketolase and Vsp.Comparative proteomics analysisThe proteomics data was handled in the same way where the most significant proteins were down-regulated during the process of attenuation.Interestingly,paralogues and secretory proteins were not detected in down-regulated proteins.Protein-protein interactions highlighted two proteins from proteomics data were interacting with genomics data proteins.Moreover,KEGG enrichment analysis highlighted glyceraldehyde 3-phosphate dehydrogenase enriched in pathways with genomics data and overlapped with findings of protein-protein interaction.Furthermore,whole genome virulent factor database also highlighted some significant virulence factors in proteomics data.The analysis of proteomics data highlighted 8 proteins most significant regarding virulence of bacteria including glyceraldehyde 3-phosphate dehydrogenase,dihydrolipoamide dehydrogenase,molecular chaperone Dna K,elongation factor,alcohol dehydrogenase,deoxyribose-phosphate aldolase,L-ribulose-5-phosphate 3-epimerase and3-dehydro-L-gulonate-6-phosphate decarboxylase.H2O2 production in M.bovis virulent and passaged strainsIn order to confirm H2O2 as a virulent factor,production of H2O2 in M.bovis strains representing eight passages from P1 to P180 was progressively decreased with increase of passages and attenuation.Hence,this decreasing tendency with increasing passage number confirmed H2O2 as a virulent factor.However,eight mutants specific to eight individual genes within the deleted region in attenuated strains did not alter H2O2 production which is similar to P1.These mutants were transposon disrupted mutants.Confirmation of specific disrupted proteinWhole cell proteins(WCPs)of the transposon disrupted mutants of deleted region genes were separated by SDS-PAGE to highlight the specific disrupted protein in the mutant.The specific disrupted protein was located and highlighted on the basis of molecular weight of the specific disrupted protein.Taken altogether,these results enriched M.bovis genomics and proteomics database and pointed out direction for exploring mechanism of M.bovis virulence.