Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages.

Mycobacterium tuberculosis (Mtb) survives inside the macrophages by modulating the host immune responses in its favor. The 6-kDa early secretory antigenic target (ESAT-6; esxA) of Mtb is known as a potent virulence and T-cell antigenic determinant. At least 23 such ESAT-6 family proteins are encoded in the genome of Mtb; however, the function of many of them is still unknown. We herein report that ectopic expression of Mtb Rv2346c (esxO), a member of ESAT-6 family proteins, in non-pathogenic Mycobacterium smegmatis strain (MsmRv2346c) aids host cell invasion and intracellular bacillary persistence. Further mechanistic studies revealed that MsmRv2346c infection abated macrophage immunity by inducing host cell death and genomic instability as evident from the appearance of several DNA damage markers. We further report that the induction of genomic instability in infected cells was due to increase in the hosts oxidative stress responses. MsmRv2346c infection was also found to induce autophagy and modulate the immune function of macrophages. In contrast, blockade of Rv2346c induced oxidative stress by treatment with ROS inhibitor N-acetyl-L-cysteine prevented the host cell death, autophagy induction and genomic instability in infected macrophages. Conversely, MtbΔRv2346c mutant did not show any difference in intracellular survival and oxidative stress responses. We envision that Mtb ESAT-6 family protein Rv2346c dampens antibacterial effector functions namely by inducing oxidative stress mediated genomic instability in infected macrophages, while loss of Rv2346c gene function may be compensated by other redundant ESAT-6 family proteins. Thus EsxO plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Study on the construction and virulence observation of Rv2346 c gene knockout strains of Mycobacterium tuberculosis mediated by bacteriophage / 中华传染病杂志

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 .Themice infected with Rv2346c KO strain had reduced mortality (53％ vs 20％ ,χ2 =6 .1112 ,P<0 .05) ,lung tissue inflammation (1040 ± 89 vs 1960 ± 56 ,t=7 .1016 ,P<0 .05) and CFU count in vitro (15 .0 ± 0 .8 vs 90 .0 ± 1 .5 ,t=23 .0361 , 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 .