| Part â… Diversity in the encoding genes of T cell epitopes in Mycobacterium tuberculosisInfection, occurrence, development and prognosis of tuberculosis (TB) are dependent on the different mechanisms of the cell immune response of the body. T lymphocytes play an important role in immunity to tuberculosis in humans. Recognition of foreign antigens by T lymphocytes depends on binding of short peptide fragments (termed epitopes), derived by proteolysis of foreign proteins, to the major histocompatibility complex (MHC) proteins on the surfaces of macrophages and dendritic cells. Studies in human pathogenic viruses, bacteria and protozoa have revealed that genes encoding antigens tend to be highly variable as a consequence of diversifying selection to evade host immunity. However, there are few studies on whether similar evolutionary mechanisms operate in TB and whether the bacteria undergo antigenic variation in response to host immune pressure. Studies on T cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) can help us further understand the mechanism of interaction between TB and host, immune response caused by TB antigen molecules and pathogenesis of TB. In addition, it benefits to improve the methods of immune diagnosis and develop new vaccines.In this study, we chose180representative strains in China for amplifying the gene sequences of480T cell epitopes in M.tuberculosis genome, comparing the differences of these epitopes in gene and amino acids levels and finding out epitopes and even proteins which probably undergo antigenic variation in response to host immune pressure. Mega5.0software was used to genotype and analyze these strains phylogeneticly. It was revealed the phylogenetic and evolutionary relationship between TB strains and human.The results showed that415epitopes were hyperconserved and65epitopes (13.54%) changes in gene level among all480epitopes. There were60epitopes changes in amino acid level, which accounted for12.5%.18proteins changed in gene sequences and6proteins (PstS1, esxL, MPT64, esxO, lppx and MT0322) had the greater changes with at least2amino acid changes. Among480epitopes in this study, the value of dN/dS is1.38. dN/dS values of12genes were above one, suggesting that these genes may undergo antigenic variation in response to host immune pressure.180strains were genotyped into9major clusters based on polymorphism in480epitopes. Some strains with certain spoligotypes like T, U, CAS, H37Rv family and BCG presented a certain degree of aggregation. However, other stains were dispersed in different clusters. Beijing family strains displayed no significant aggregation. This indicated that Beijing strains presented diversify in interaction between human T cell and pathogen. After synonymous mutations and random mutations were removed,180strains were divided into3major clusters. Genotypes based on this method reveal the diversify interaction between TB strains and human T cell. Part IIEvaluation of Four Candidate VNTR Loci for Genotyping225Chinese Clinical Mycobacterium Tuberculosis Complex StrainsThe genotyping results of Mycobacterium tuberculosis (M tuberculosis) showed that the prevalence of tuberculosis in the world mainly caused by several special M. tuberculosis genetic lineage (family) strains. Each family strains have unique molecular characteristics, regional distribution and pathogenicity. The method with variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) is one of the common methods for the genotyping M. tuberculosis, and has been widely used in epidemiological research of tuberculosis around the world. Combinations of different multiple VNTR loci can get different genotype clusters and has different discriminatory power. Currently,12-locus VNTR is the most widely used and has been integrated in TB control systems on a national scale. Later after that, the15-locus and24-locus sets have been proposed as a basis for standardized MIRU-VNTR typing of M. tuberculosis.Here, by sequencing2Chinese M. tuberculosis strains CCDC5079(CP002884) and CCDC5180(CP002885) and using published sequence data,4promising VNTR loci were identified and applied to genotype225Chinese clinical M. tuberculosis complex strains, and then the discriminatory power was evaluated.The results showed that225strains had165distinct profiles and were divided into6clusters by these4-VNTRs. The predominant cluster (cluster V) included139strains. Most of them were Beijing family (111). Hunter-Gaston Index (HGI) of BJ1, BJ2, BJ3and BJ4was0.634,0.917,0.697and0.910respectively. The HGI of combination of4loci was0.995suggested a good discriminatory power. In addition,126Beijing strains could be divided into15subculsters by these4new VNTR loci, HGI of BJ1, BJ2, BJ3and BJ4in genotyping Beijing family strains were0.447,0.878,0.315and0.850respectively. The HGI of combination of4loci in genotyping Beijing family strains was0.988.It seemed that the M. bovis strains and M. bovis-BCG family strains have the unique copy number in BJ1and BJ2of which the repeat number was1.0and5.5respectively, which may be applicable to separate M. bovis and M. bovis-BCG family strains from clinical M. tuberculosis complex strains. In addition, BJ1pattern of FJ06057presents unique high copy number8.0in225strains, which indicate a special characteristic in M. africanum strain. |
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