Construction Of Two-Component Signal Transduction System KdpD/E Gene Deletion Mutants And Its Preliminary Functional Study In Mycobacterium Tuberculosis

Mycobacterium tuberculosis is the causative agent of human tuberculosis(TB) that currently infects approximately one-third of the global population. Previous studies have demonstrated that the two-component signal transduction system Kdp D/E plays an important role in the process of infection to macrophages, and is also involved in the regulation to virulence of M. tuberculosis, but the mechanism remains unknown. In this study, we constructed the KdpD/E gene deletion mutants in M. tuberculosis and focused on the effect of KdpD/E on M. tuberculosis invasion into macrophages, intracellular survival and virulence regulation. 1. Construction and identification of KdpD/E gene deletion mutantsThe allelic exchange substrates(AES) were constructed and cloned into shuttle vector phAE159 which contains temperature sensitive mycobacteriophage element. The AES were efficiently delivered to wild type strain by recombinant mycobacteriophage. The positive clones grown on hygromycin resistant solid medium were further identified by PCR. The results showed that the KdpD/E gene deletion mutants ΔkdpD, ΔkdpE and ΔkdpDE in M. tuberculosis were successfully constructed. 2. Comparison of biological characteristics between wild type and mutantsThe growth curve of wild type and mutants have been determined by measurement OD600 and CFU, the results showed that KdpD/E-deletion has no significant effection on the growth of strains in vitro. Moreover, the colony morphology of wild type and mutants grown on solid medium were compared and found that ΔkdpE and ΔkdpDE formed more cording, indicating the hypervirulent of mutants. 3. Comparison on the ability of invasion into macrophages and intracellular survival between wild type and mutantsThe intracellular CFU of wild type and mutants in the process of infection to human monocyte macrophages(THP-1) were compared. The results showed that there was no significant differential between the wild type and mutants in invasion into macrophages, but the ΔkdpDE showed stronger ability in intracellular survival at different time post infection(24h, 48 h, 72h). 4. Expression of KdpE protein and preparation of polyclonal antibodyThe response regulator protein KdpE was successfully expressed by pET-28 a vector, and the rabbit anti-KdpE protein polyclonal antibody were produced. The ELISA results showed that the titre of antibody reached 1:64,000. This provided the foundation for further study on the function of KdpD/E in M. tuberculosis. 5. Identification of differentially expressed genes between wild type and mutantsThe results of quantification real-time PCR showed that the kdpA, kdpF were up-regulated in the Kdp D/E gene deletion mutants, which participates in the formation of Kdp-ATPase potassium ion pump. In addition, it was found that the lprF, lprJ, lat, ponA2, espA were up-regulated, and the furA, glyA2, Rv3161 c were down-regulated in the mutants at the same time, the differential expression of these genes may be an important cause of stronger intracellular survival ability and hypervirulent of mutants.