| Recent years, studies on variation of human T cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) find that some of the T cell epitopes are highly variable, suggesting these variation might be involved in diversifying selection to evade host immunity.MPT64, ESAT-6 and CFP-10 are three important secretory proteins of M. tuberculosis, which are playing an important role in tuberculosis (TB) diagnosis and candidate antigen of vaccine. In our previous studies, we found that MPT64 gene are highly variable, while ESAT-6/CFP-10 genes were conservative; several studies showed that PE35 and PPE68 could effect on ESAT-6/CFP-10, and we found that the human T cell epitopes of PE35 and PPE68 genes were highly variable, which suggest that these two antigens may be involed in diversifying selection to evade host immunity. However, studies on whether the polymorphism of PE35 and PPE68 has an impact on ESAT-6/CFP-10 proteins have not been much. MPT64, which is the main component of short culture filtrate protein and can be recognised by the human Thl cell, is very helpful for TB diagnosis and new candidate antigen of vaccine. It was reported MPT64 gene existed certain variations in different countries and regions, which would have an impact on the detection of MPT64.In order to further explore the polymorphism of MPT64, PE35 and PPE68 genes and the impact on virulence of M. tuberculosis, and the mechanism of immune evasion it caused. We selected PE35, PPE68, MPT64 genes and the genes that were reported having an impact on ESX-1 system, including Rv3870, Rv3877, Rv3616c, Rv3871, Rv3615c and Rv3849 genes, in order to study the impact of these genes polymorphisms on the secretory proteins MPT64 and ESAT-6/CFP-10.A total of 161 strains were selected from 2346 MTBC strains isolated in Beijing Municipality and 12 provinces and autonomous regions, China. First, the strains were cultured, heat inactivated and extracted DNA and then used directly in polymerase chain reactions (PCRs), and comparative analysis on the sequences; second, selected the genes that showed highly variation, then compared analysis on the protein expression level between the mutation strains and the non-mutation strains or the impact of these mutations on the detection kits. Finally, analyzing the influence of these gene variations on secretory protein MPT64, PE35 and PPE68.The results showed that only Rv3871 gene had an apparent mutation among the tested six genes, while the other five genes (Rv3870, Rv3877, Rv36J6c, Rv3615c, Rv3849 and Rv3871) did not present an obvious gene mutation. For Rv3871 gene, there were six nonsynonymous mutations, six synonymous mutations and one three-base deletion. The MPT64, PE35 and PM68 present drastic changes, which would possibly induce changes in protein structure. Among 161 strains,13 present gene polymorphism of MPT64, including 8 strains harbored 63bp deletion,4 had nonsynonmous nucleotide mutation and one with a single-base insertion. For PE35,23 isolates presented polymorphism in the gene sequence of PE35, including 21 strains had an A deletion and the other two harbored two different nonsynonymous mutations. For PPE68,8 strains displayed polymorphisms, including two isolates had two different deletions and six showed five nonsynonymous mutations. After tested through detection kit, the 63bp deletion of MPT64 obtained false negative results, while single-base mutation yielded positive results, and all non-mutation strains were positive. Although PE35 and PPE68 presented gene polymorphism and T cell epitope variation, these variation had no obvious impact on the secretory proteins ESAT-6/CFP-10.In conclusion, the polymorphism of MPT64 gene have an impact on the diagnostic test kit based on mpt64 detection, as the 63-bp deletion mutation caused false negative results. Instead, the polymorphisms PE35 and PPE68 genes did not have an obvious influence on the virulence factors ESAT-6/CFP-10 of M. tuberculosis. |
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