Study on the construction and virulence observation of Rv2346c gene knockout strains of Mycobacterium tuberculosis mediated by bacteriophage

Objective To investigate the Rv2346c gene function through constructing Rv2346c gene knockout strains of Mycobacterium tuberculosis (M. tuberculosis) mediated by bacteriophage and observing its virulence after infecting mice lung tissue in vivo. Methods The affinal exchange sites (AES) of the target gene was built, and then integrated into the phage genomes of M. tuberculosis for harvesting the phagemids. The phagemids was imparted into Mycobacterium smegmatis to get recombinant phages with the same AES. A high titer of the recombinant phages was harvested through amplification in vitro. The M. tuberculosis was transfected and coated on solid medium with hygromycin resistance and cultured for 4 weeks at 37℃. Single clone was picked out and gene knock-out was confirmed by PCR. Then C57BL/6J mice were infected with either wild type strain (WT) or knockout strain (KO) of M. tuberculosis. Mice mortality, lung tissue inflammation and colony-forming units (CFU) counts in vitro were observed 6 to 8weeks post infection with different strains. Paired-samples t test was used for comparison between groups, chi-square test was used for comparison of rates. Results The products of PCR and inserted fragment sizes were consisted with the expectation and confirmed to be the target gene. The target fragment of Rv2346c was removed successfully and the mice were infected for 6-8 weeks. The mice infected with Rv2346c KO strain had reduced mortality (53% vs 20%, χ2=6.1112, P<0.05), lung tissue inflammation (1 040±89 vs 1 960±56, t=7.101 6, P<0.05) and CFU count in vitro (15.0±0.8 vs 90.0±1.5, t=23.036 1, P<0.05) compared with WT strain 6-8 weeks post infection. Conclusion Rv2346c gene knockout strains of M. tuberculosis mediated by bacteriophageis are successfully constructed, which establishes the foundation for the future gene function study of Rv2346c. Key words: Bacteriophages; Mycobacterium tuberculosis; Gene knockout strains; Rv2346c gene