| Bovine Tuberculosis(bTB)is a chronic wasting zoonotic disease caused by Mycobacterium tuberculosis complex(MTBC).The main pathogens are Mycobacterium bovis(M.bovis)and Mycobacterium tuberculosis(M.tuberculosis).At present,the disease has caused huge economic losses to the cattle industry in many countries around the world,and it has also caused a huge threat to human public health security.It has been 100 years since the first application of Bacillus Calmette-Guerin(BCG),but there are still no effective prevention and control measures for adult tuberculosis and bovine tuberculosis.The main reason is that the clinical infection mechanism of MTBC is so complicated that the pathogenic mechanism has not been fully resolved.One of the original intentions may be that different types of M.tuberculosis have different innate immune responses to the host alveolar macrophages.In this study,M.bovis clinical strains and M.tuberculosis clinical strains were used to isolate and establish primary bovine alveolar macrophages(bAMs)infection model.The differences between M.bovis and M.tb clinical strains and the innate immune response mechanism of bovine primary BAMS were analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS)and bioinformatics.At the same time,combined with bacterial proteomics and the latest research,we constructed a prokaryotic expression vector with M.tuberculosis secreted protein MTB3 9a(PPE18)as the research object,purified the protein and analyzed its activity.And we detected the serum reactivity of healthy and clinical tuberculosis patients by Western blot to reveal whether it can be used as a clinical diagnostic marker of tuberculosis.This study provides a theoretical reference for exploring the pathogenic mechanism of bovine tuberculosis caused by M.bovis and M.tb infection.The following results have been obtained through research:1.Nine differentially expressed proteins were identified by M.bovis and M.tb proteomics analysis,including three up-regulated proteins(o07412,o53873,p9wki3),and six down regulated proteins(p9wgi5,p9wh33,p9wh39,p9wh87,p9whc5,p9wqd1).Go functional annotation showed that the differentially up-regulated proteins of M.bovis and M.tb were annotated in plasma membrane,peripheral and intracellular cell composition.It is mainly involved in the biological process of M.bovis.Subcellular localization showed that one protein was located on the cell membrane and two proteins were located in the cytoplasm.CoG analysis showed that differentially up-regulated proteins were related to nucleotide transport,metabolism,replication,recombination,repair and secondary metabolite biosynthesis.KEGG pathway analysis showed that the differentially up-regulated proteins of M.bovis and M.tb were located in drug metabolism and nucleotide excision repair pathways.These two proteins are mainly concentrated in a series of cellular components related to the structure of ribosomes,participate in the composition of M.bovis ribosomes,and are closely related to the synthesis of intracellular proteins.In addition,these six down-regulated proteins activate molecular functions related to the structural composition of ribosomes,are related to the ribosome biogenesis of M.bovis and M.tb,and are enriched in the pathways of drug and other enzyme metabolism.The protein interaction network showed that only down-regulated proteins P9WH39,P9WH87,P9WH33,and P9WHC5 interacted in the M.bovis_CvsM.tb_C group.2.A total of 21 differentially phosphorylated proteins were identified by M.bovis and M.tb proteomics analysis.Go enrichment analysis showed that the phosphorylation of M.bovis and M.tb proteins was significantly enriched in the biological processes related to macromolecular metabolism,and the molecular function was related to molecular binding.In cell composition,differential protein phosphorylation exists in almost all parts of the cell.3.After 6 hours of infection with M.bovis and M.tb,the proteome of BAM was analyzed.The results showed that 17 proteins were up-regulated and 1 protein was down-regulated by M.tb infection;60 proteins were up-regulated and 3 proteins were down-regulated after M bovis infection.Attacking BAM by M.bovis and M.tb activates the autophagy-related processes of macrophages,the inflammatory-related processes and the defense process against pathogenic bacteria.At the same time,after M.tb is infected with BAM,the energy metabolism in BAM is activated sharply.M.bovis infection of BAM can cause changes in the expression of proteins involved in energy metabolism,autophagy,apoptosis,lysosomes and inflammation in cells.Differential proteins are mainly involved in various energy metabolism,autophagy and immune-related pathways,including tuberculosis,lysosome,phagosome,Th17 cell differentiation and oxidative phosphorylation.4.Based on the results of the first chapter and the latest research,we screened Mtb39a(ppe18)as a potential diagnostic marker.The prokaryotic expression of Mtb39a recombinant protein was realized by constructing the prokaryotic expression vector of Mtb39a.Western blot showed that the purified Mtb39a recombinant protein was reactive to the serum of healthy and clinical tuberculosis patients.Conclusion:1.Through proteomic analysis of M.bovis and M.tb,it is determined that their differential proteins are mainly located in the cytoplasm and participate in the regulation and assembly of ribosomes.It is mainly concentrated in the metabolic pathways of drugs and other enzymes,and is related to the replication and synthesis of M.bovis and drug resistance.2.Through the analysis of the protein phosphorylation of M.bovis and M.tb,it is found that the differentially phosphorylated proteins are mainly located in the cytoplasm,cell periphery and plasma membrane area,participate in the metabolic process of mycobacteria,and have a high degree of binding function.The pathogenicity of M.bovis is related to the adaptability of the host cell.3.Experiments have confirmed that BAM can respond differently when defending against M.bovis and M.tb infections,including the activation of various autophagy and inflammation-related processes and signaling pathways.In addition,M.bovis can inhibit BAM’s autophagy and inflammation-related proteins and their signaling pathways,avoiding being cleared by BAM and colonizing the host.When M.tb is infected with BAM,the energy metabolism in the cell is rapidly activated,which is the defense activity of BAM against M.tb attack.4.Mtb3 9a recombinant protein can be used as a potential diagnostic marker for M.tb.These results analyzed the difference between M.bovis and M.tb clinical strains at protein level,revealed the difference of immune response of BAM infected by M.bovis and M.tb,and verified that Mtb39a can be used as a clinical diagnosis marker for tuberculosis.It is of great significance to study the mechanism of Mycobacterium infection,lay a foundation for the study of the difference of M.bovis and M.tb virulence,and it is of great significance to make a new strategy for TB prevention and treatment. |
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