Investigation On The Polymorphism Of TA System And Its Relative Pathogenicity Of Mycobacterium Tuberculosis

Tuberculosis (TB) is a chronic infectious disease which seriously endangers human health, which is caused by Mycobacterium tuberculosis (M. tuberculosis). When it infects its host, the bacterium lives freely inside and outside of the cell, and may encounter a variety of microbicidal environment, such as lack of nutrition and essential elements, hypoxia, low pH, antimicrobial proteins and so on. To escape those, M. tuberculosis is persisting in non proliferation state without causing disease, this characteristic is called the persistence. It is estimated that one-third of the world population are infected with MTB. However, most of new cases are caused by the actived latent M. tuberculosis. Althought the chemotherapy regimens does great work in treating active tuberculosis, it does little in treating the latent, which is the main cause of its long treatment period and difficult to cure radical.In recent years, concerning the study of the mechanisms of M. tuberculosis persistence, the toxin-antitoxin (TA) system are believed to be involved in the bacterial persistence. TA systems are widespread in prokaryotic genomes and consist of a stable toxin protein and an unstable antitoxin protein. In normal growth conditions, the toxin protein and antitoxin protein co-express to maintain the stable growth of the bacteria. In response to stress, the antitoxin proteins are specifically degraded by some proteases, and toxin proteins are released to engender the effects of inhibiting the bacterial growth. Five TA families have been identified in M. tuberculosis H37Rv including VapBC, MazEF, RelBE, HigBA and ParDE.To investigate the single nucleotide polymorphism (SNP) in the TA system of M. tuberculosis among different genotypes, we selected 183 clinical M. tuberculosis isolates and performed spoligotyping of them. Drug susceptibility testing was performed with the Lowenstein-Jensen proportion method. The sequences of TAS genes were obtained by PCR and DNA sequencing method. We compared the sequences to look for mutations. Functional consequences of nonsynonymous SNPs were predicted by I-Mutant 2.0 servers. Several TAS genes with bigger DDG value were amplified and cloned into a shuttle vector pMV261. The recombinant vector was transformed into Mycobacterium smegmatis (M smegmatis) by electroporation method. To further explore the role of this system in the pathogenicity of M. tuberculosis, we compared the M. smegmatis which contained recombinant vector in growth curve, heat pressure, nitrate pressure and acid pressure.As the results showed, among the 183 M. tuberculosis isolates,138 (75.41%) belonged to Beijing family, while 45 (24.59%) belonged to non-Beijing family. Analysis of the polymorphisms of 63 TA system genes, we discovered 127 nonsynonymous SNPs,4 nucleotide deletion and 44 synonymous mutations and 100 of the SNPs were restricted to Beijing family strains.32 IGR SNPs were founded, including base substitutions and deletions, the majority of mutations occurred in Beijing family. In the exploration of the relationship between TA system and drug resistance, there were 6 SNPs might be associated with rifampicin resistance,4 SNPs might be related to streptomycin resistance,1 SNP might be associated with ethambutol resistance, but the relevant mutation related to isoniazid phenotype resistance was found. The recombinant expression plasmids pMV261 which respectively contained the gene vapC2, vapBC2, vapC38, vapBC38, vapBC18 and vapBC47, were successfully constructed and transformed into M. smegmatis. The growth curve of recombinant strains showed that there was no significant difference among the recombinant bacteria. This indicated that M. smegmatis was less affected by exogenous gene under normal growth conditions. Under heat, acid and nitrate stress, the toxin protein vapC2 and vapC38 inhibited the growth of bacteria and antitoxin protein could alleviate the inhibitory effects of toxin protein. Missense mutations caused amino acid changes, which finally had effects on the function of the protein.In summary, the SNPs in the coding sequences of TA system in clinical isolates can be relatively high and most of the mutations occurred in Beijing family strains, it might be better for M. tuberculosis Beijing family strains to adapt to different host environment. The result suggests a possible role for TA system in the resistance outer pressure for bacterial growth.