In-depth Characterization Of The Activity And Mechanisms Underlying The Rv0621, A Mycobacterium Tuberculosis H37Rv Gene Involved In The SDS Tolerance

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a widespread disease that remains a leading cause of mortality around the world. One-third of the global population is currently reported to harbor latent M. tuberculosis, and10%of those are predicted to be reactivated in some later stage, which pose a formidable threat to global public health. M. tuberculosis can adapt to a wide range of stresses such as nutrient limitation, hypoxia and alveolar surfactant during host infection. Alveolar surfactant is a mild detergent with antibacterial activity and could damage the fatty acid-rich cell envelope of M. tuberculosis. It has been demonstrated that sodium dodecyl sulfate (SDS) can produce an effect similar to that of alveolar surfactant. Two sigma factor genes (sigB and sigE) underlying response to SDS exposure have been identified. The sigB gene was upregulated11folds and the sigE gene was upregulated6folds after SDS treatment. In addition, sixty-two genes were induced in M. tuberculosis after SDS exposure.This study mainly focus on the cloning, expression and the functions of Rv0621on the SDS and anti-tuberculosis drugs stress response and the fatty acids alterations in Mycobacterium smegmatis mc2155. M. tuberculosis H37Rv genome was used as a template and Rv0621gene was amplified by PCR. The resulting PCR product was gel purified and ligated with digested pET32a (+) and pNIT (myc) vector. The ligated products pET32a (+) and pNIT (myc)-Rv0621were transformed into Escherichia coli strain DH5a. The correct sequence was verified by DNA sequencing. Then, the recombinant plasmids were transformed into Escherichia coli BL21and M. smegmatis mc2155separately. Identification of the recombinant strains was performed by plasmid PCR. The fusion proteins were expressed. The effect of RvO621overexpression on the host bacteria, including the growth, SDS stress tolerance and antituberculosis drug tolerance, was explored.We found that the overexpression of RvO621had no significant influence on the growth of M. smegmatis mc2155. Introduction of RvO621can increase the M.smegmatis tolerance to SDS stress and the rifampicin. To gain a mechanistic appreciation of this phenomenon, the effect of RvO621on the fatty acids compositions of M. smegmatis was assayed. A decrease in saturated fatty acids and an increase in unsaturated fatty acids were noted in recombinant M. smegmatis. Together, these results suggest that RvO621might endow recombinants stronger resistance to stress by selectively increasing the ratio of unsaturated fatty acids of cellular wall. Further studies on the mechanism of how this selectivity was accomplished are needed.