| Mycobacterium tuberculosis (MTB) remains one of the world’s most successful pathogens, infecting one-third of world’s population. Only in 2012, there are 8.6 million new infections and among of them,1.3 million people died. Currently, Mycobacterium bovis bacillus Calmette-Guerin (BCG) is still the only available vaccine for human. BCG has been proven efficacy against childhood TB and disseminated TB, however it is of limited efficacy against adult pulmonary tuberculosis, varying from 0 to 80%. Non-tuberculous mycobacterial infection may interfere with the efficacy of BCG; and also, during the course of passaging attenuation, some protective antigens, for example, the regions of difference (RD) were deleted. In addition, as a live attenuated vaccine BCG may cause disseminated tuberculosis for immunocompromised persons. Therefore, developing novel efficient and safety TB vaccine and exploring reasonable TB immune strategy is vital important. This requires fully understand the mechanisms of pathogenesis and immunogenicity of mycobacteria, while identification of proteins or peptides which can induce host to produce protective immune reaction is the basis for such purpose.1. Computational analysis of ESX-5 secretion system of Mycobacterium tuberculosisType VII secretion system (T7SS) is a novel bacterial secretion system recently discovered in Mycobacterium tuberculosis. T7SS is composed of a number of unique proteins, all the secreted proteins are translocated by a specific pathway, and all of them are dependent on each other during the course of secretion. The orthology search of ESX-5 components of H37Rv against 60 other mycobacterial species identified six species named after Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium rhodesiae, Mycobacterium chubuense, Mycobacterium massiliense and Mycobacterium neoaurum did not have orthologs corresponding to any of the 16 ESX-5 components. Forty-six species were observed to contain orthologs corresponding to at least seven out of the 16 ESX-5 components, among of which forty-eight species contain orthologs corresponding to at least eleven components. The region of Rv1787-Rv1792 was lack in some mycobacterial strains with ESX-5 system, which suggest that these six proteins may not be an integral or essential components of the secretion machinery. Comparison of the phylogenetic profiles of ESX-5 components indicated that Rv1786, Rv1787, Rv1788, Rv1789, Rv1791 and Rv1793 belonged to the same cluster, Rv1782, Rv1783, Rv1794, Rv1795, Rv1796, Rv1797 and Rv1798 belonged to another cluster, while Rv1785c, Rv1790, Rv1792 belonged to three individual clusters respectively. Rv1782, Rv1783, Rv1795, Rv1796 and Rv1797 were predicted to contain transmembrane helices. Rv1788, Rv 1791 and Rv1796 were predicted to contain signal peptides. Rv1782, Rv1783, Rv1785c, Rv1787, Rv1789, Rv1790, Rv1796 and Rv1798 were predicted to contain glycosylation sites. Rv1796 (MycP5) is a subtilisin-like serine protease, and Rv1795 maybe its substrate. All these data will be beneficial to understand the ESX-5 secretion working mechanism and provide a theoretical basis for the prevention, diagnosis and treatment of tuberculosis.2. T cell epitopes identification of ESX-5 secretion system associated PPE25 and PE19 proteins of Mycobacterium tuberculosisC57BL/6 mice were immunized s.c. at the base of tail with 1 X 106 CFU/mouse of Mycobacterium tuberculosis standard virulence strain H37Rv or the ESX-5 associated ppe25-pel9 region deletion strain H37Rv△ppe25-pe19. Three weeks post immunization, spleens were removed and splenocytes were generated. Splenocytes were stimulated with 10 μg/ml of individual synthetic 15-mers from PPE25 (seventy-one 15-mers, overlapping by 5 aminos) or PE19 (twenty-nine 15-mers, overlapping by 3 aminos). After 72 h of incubation, IFN-y was quantified by ELISA. As a result, PPE25:1-20, PPE25:41-65, PPE25:186-200, PPE25:201-215, PPE25:236-255, PPE25:281-295 and PPE25:321-335 were identified as the MHC-II restricted T cell epitopes of PPE25, while only PPE25:236-255, PPE25:281-295 and PPE25:321-335 were identified as ESX-5 specific epitopes. And also PE19:1-18, PE19:34-51 and PE19:82-99 were identified as the MHC-II restricted T cell epitopes of PE19, among of them only PE19:1-18 was ESX-5 specific.3. MHC-I T cell epitopes identification of PPE26 from Mycobacterium tuberculosis and CD8+T cell hybridomas generation against PPE26:44-52MHC-I restricted T cell epitopes of PE/PPE proteins located in ESX-5 region of Mycobacterium tuberculosis were predicted by the use of SYFPEITHI software on line to select 9-mers with high potential affinity. Splenocytes from H37Rv infected BALB/c mice produced IFN-γ after in vitro stimulation with these epitope peptides. CD8+T cell hybridomas specific to PPE26:44-52 were generated by the fusion of murine thymoma cell line BW5147 transfected with cd8 and the antigen specific T cells from the lymphoid organs of BCG-immunized mice by the use of PEG1500. After HAT selective medium screening,136 wells were tested and only three clones named after 1E1,4A8 and 4D8 were positive to the determined peptide.These hybridomas were able to produce IL-2 when cocultured with dendritic cells (DCs) loaded with the PPE26:44-52 peptide and were all restricted by H-2Kd, which are accordance with the prediction results. These obtained T cell hybridomas will provide biological material for quantitative determination of specific MHC-peptide complexes on the surface of antigen presenting cells.4. Prokaryotic expression of PPE25, PPE26, PPE27 and PE19 of Mycobacterium tuberculosis and their immunological characteristicsThe coding genes of ppe25, ppe26, ppe27 and pel9 were PCR amplified from Mycobacterium tuberculosis H37Rv strain genomic DNA, and then cloned into the prokaryotic expression vectors. The constructed recombinant plasmids were transformed into competent E. coli BL21 (DE3) respectively and the target proteins were expressed with IPTG induction. SDS-PAGE analysis showed that all these proteins were successfully expressed with the relative molecular mass of 40 kD,44 kD,41 kD and 15 kD respectively, and all the target proteins were expressed in the way of inclusion body. After denaturation and renaturation, fusion proteins were purified by Ni-Sepharose affinity chromatography. Western blotting assay indicated that the purified proteins could well react with anti-HIS monoclonal antibodies and proved their immunoreactivity. The fusion proteins could elicit New Zealand white rabbit to produce high titers of specific antibodies, and PPE26 was the best one. These results indicate that such PE/PPE proteins have good humoral immunogenicity. Further experiments confirmed that PPE25/27: 56-71, PPE26:370-385 and PE19:56-71 are the predominant B cell epitopes of associated proteins. Peripheral blood monouclear cells (PBMC) from TB patients produced antigen-specific IFN-y, IL-2, and TNF-a after in vitro stimulation with PPE25/PPE26/PPE27 protein mixture or PE19 protein. So these PE/PPE proteins have good cellular immunogenicity. This study will lay a foundation for exploring the potential of PPE25, PPE26, PPE27 and PE19 protein in the development of novel TB vaccines and diagnostic reagents. |
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