Bio-marker Screening For Tuberculosis And Non-tuberculous Mycobacterial Diseases Differentiation

Part one.Screening of extracellular proteins as immunogenic markers for diagnostic and differential diagnosis of MTB and NTMObjective To screen and identify the specific immunogenic proteins for different mycobacterial species,including Mycobacterium tuberculosis(MTB)and the most isolated pathogenic Non-tuberculous Mycobacteria(NTM)species,so as to find new diagnostic markers for TB and NTM diseases.Methods MTB standard strain H37Rv and standard strains of five most isolated pathogenic NTM species[Including 3 slow-growing mycobacteria(SGM)species:M.kansasii,M.avium,intracellulare;2 rapid-growing mycobacteria(RGM)species:M fortuitum and M abscessus]were studied in the study.The protein in culture filtrate of the strains was collected and subjected to enzymatic disposition.After validation of the quality of the protein samples,the peptide fragments were analyzed by Label-free proteomics,and the signal peptides of the protein were predicted by the SignalP server based on the UniProt database for screening membrane-associated proteins or secreted proteins.A three-dimensional model was established in combination with SWISS-MODEL.T cell epitopes and B cell epitopes of these extracellular proteins were predicted in the IEDB database.At the same time,bioinformatics analysis was performed to enrich the GO function annotation and KEGG pathway annotation of extracellular proteins to compare the differences between nontuberculous mycobacteria and Mycobacterium tuberculosis.Results 1.The protein expression among MTB and the three SGM strains were very compatible,while the protein patterns of the two RGM strains were significantly different from the other SGM strains.20 specific antigenic proteins among the extracellular proteins were identified for Mycobacterium tuberculosis;while the counterpart numbers for M kansasii,M.avium,M intracellulare,M.fortuitum and M.abscessu were 27,32,52,26 and 2 respectively.2.The functions of the extracellular proteins with divergence between NTM species and MTB were mainly related with some important biological processes,such as transmembrane transport,transport enzyme and hydrolase activity,and membrane-anchored transporter.The two RGM,M fortuitum and.abscessus,demonstrarted evident difference with other SGM in copper ions transportation and the stability of copper ions,and the carboxypeptidase activity is also significantly different from that of MTB.3.The expression of the homologue proteins of LprA,LprG and PstS were elevated in MTB and M kansasii,but were absent in M aurantia and M abscessus,LprA and LprG were absent in M.avium and M.intracellulare,Conclusion 1.Specific immunogenic proteins were identified for MTB and 5 NTM species,which might be used as diagnosis markers for tuberculosis and NTM diseases.2.The difference in copper ion transport and carboxypeptidase activity may be related with strain virulence and strain survival.The difference in expressions of lprA and pstS-3 among different mycobacterial species might be related with pathogenicity.Some differential proteins could be used as candidate markers for tuberculosis and NTM disease diagnosis.Part two.Proteomic analysis of the exosomes derived from MTB and NTM-infected macrophageObjective To study the protein constitutions of the exosomes secreted by macrophages infected with standard strains of Mycobacterium tuberculosis or the aforementioned five most isolated pathogenic Non-tuberculous Mycobacteria(NTM)species,so as to screen candidate markers for the diagnosis of tuberculosis and non-tuberculous mycobacterial diseases.Methods THP-l macrophages were infected with standard strains of MTB and five most isolated pathogenic NTM(M.kansasii,M.avium,M.intracellulare,M fortuitum and M.abscessus).Exosomes in supernatant of macrophage culture were collected and enriched by ultracentrifugation,then the protein constitutions of the exosomes were analyzed by Label-free mass spectrometry.The proteins originated from the macrophage were screened for further analysis.Subcellular localization of exosome proteins specifically expressed in each infection group according to the UniProt database was analyzed to screen the membrane proteins with cell adhesion functions.At the same time,bioinformatics assay was conducted to elucidate the functions of the exosome proteins that differentially expressed among different groups,and its involvement in different KEGG pathways were also analyzed.Results 1.Comprehensive comparison of the exosome proteins among all the mycobacterial infection groups and the blank group indicated that the number of mutual proteins of each group accounted for 51.14%(1771/3461)of the total number of proteins identified among all groups,which was a high proportion.2.In the exocytosis of the MTB and the three SGM strains infected groups,1-3 host proteins related with cell adhesion were isolated,but none of such protein was identified among the two RGM infection groups.3.Exosomal proteins Icd2,pepA,DnaK,lprA,GlcB and KatG from bacterial sources have been reported in similar studies.4.In the enrichment of the KEGG pathway,29 important proteins involved in receptor signaling of some important pathways were obtained,including JAK-STAT signaling pathway,Toll-NF?B/MAPK pathway,and signal transduction for inhibition of host cell apoptosis and phagocytic lysosome maturation.Myd88 expression was significantly elevated in MTB and M.kansasii infection groups,and the expression level was significantly higher in group(P<0.01),whereas Myd88 expression was not detected in other 4 NTM infection groups.Conclusion 1.When infected with different mycobacteria species,the constitution pattern of macrophage-derived exosomal proteins were different.The anchoring function of exosomal membrane proteins plays an important role in cell signaling and may be related to extrapulmonary dissemination of mycobacteria.2.The proteins screened in macrophage in vitro models are infection-related indicators with clear biological significance,which can be further screened and validated in the diagnosis.3.Mycobacterial infection of macrophages hinders the binding of phagosomes and lysosomes in all groups,and is a common mechanism for intracellular survival of mycobacteria.Compared with the NTM infection group,Mycobacterium tuberculosis infection is more dependent on Myd88 signal transduction.Part 3.Cytokine identification post Macrophage infection by Mycobacterium tuberculosis or non-tuber culo us mycobacteriaObjective To identify the different expression levels of cytokines derived from mycrophage infected with standard strains of Mycobacterium tuberculosis and 5 most isolated pathogenic NTM species,so as to identify the possible marker for MTB and NTM species identification.The mechanism of pathogenesis of Mycobacterium tuberculosis and non-tuberculous mycobacteria was explored by analyzing the cytokine network.Methods Eleven cytokines including IFN-?,IL-2,IL-4,IL-6,IL-10,IL-12/23 p40,IL-27,TNF-?,IP-10 and MCP-1 were selected in this study.Macrophages were infected with standard strains of MTB and 5 NTM species(M.kansasii,M.avium,M.intracellulare,M.fortuitum and M.abscessus).Cell culture supernatants were collected at 4-hour,8-hour,24-hour,and 48-hour post infection,and cytokine concentrations were measured by the Luminex liquid phase chip method.At the same time,the survival rate of macrophages was determined by LDH assay and the growth of mycobacteria in macrophages was determined by CFU numeration.The results were validated with plasma collected from patients and healthy volunteers.Comprehensive analysis and screening of cytokine combinations that may be used in diagnosis and differential diagnosis of tuberculosis and NTM disease were performedResults 1.The expression pattern of each cytokine in different infection groups was very different.The expression levels of IFN-? and IP-10 in all the mycobacterial infection group were higher than those of the uninfected group,and the IP-10 elevated levels were different for different infection groups.The expression level of TNF-a was decreased in the infected groups,especially in.intracellular group.The expression of IL-10 and IL-12/23p40 released by M tb infection was significantly higher than the five NTM groups.The expression levels of IL-6 and IL-27 released by MTB,M.Kansasii and M.intracellulare infection groups were increased compared with the control group,while.avium and two RGM groups did not increase evidently.The expression of IL-2 and IL-2-Ra were undetectable,while IL-4 and MCP-1 were not significantly different between the infected groups and uninfected group.2.TNF-?,MCP-1,IL-2R-a and IL-6 levels were significantly higher in patients of TB or NTM diseases than in healthy volunteers,and TNF-levels were also significantly different among the 3 groups.IL-2,IL-4,IL12/23p40 and IFN-y were significantly higher in the TB group than in the healthy volunteers.Conclusion 1.The expression of IP-10,IL-10,IL-6,IL-12/23p40 and TNF-a can distinguish between mycobacterial infection and uninfected groups.IP-10 may be used to distinguish between RGM and SGM.IL-6 and IL-12/23p40 increased after tuberculosis infection,and there was a significant difference between the NTM infection groups.2.The cytokine response patterns of macrophages against different species of mycobacterial infections are different,which may related to the virulence and pathogenicity of the bacteria.