SirR (Rv2788) Is A Novel Transcriptional Repressor Associated With Mycobacterium Stresses Survival And Transcriptional Regulation

Mycobacterium tuberculosis（MTB）, the etiological agent of tuberculosis, remains a leading cause of morbidity and mortality due to single bacterium infection worldwide. Despite half a century of intense chemotherapy, TB still claims the lives around 1.5 million, and there are 9.0 million new and re-emerging infections in 2014. The emergence of multidrug-resistant（MDR）, extensively drug-resistant（XDR） and even totally drug resistant（TDR） tuberculosis, HIV-coinfection, shortage of new antibiotics and vaccine, collectively contributed to the difficulty of TB treatment.TB is one of the most successful intracellular pathogen during infection,which largely rests on its remarkable ability to adapt to diverse stresses. In addition to the unique waxy and highly impermeable mycolic acid containing cell wall and efflux, exquisite transcriptional regulators of TB also play remarkable role in its success. In bacteria, metal-dependent regulatory proteins maintain metal homeostasis by controlling the expression of genes involved in metal import, storage and efflux. The diphtheria toxin repressor（Dtx R） family is one of the best characterized metalloregulatory proteins. It has representatives that sense either or both Fe2+ and Mn2+ as co-repressors to promote DNA binding. In M. tuberculosis, there are two Dtx R homologues: IdeR（iron dependent repressor） and SirR（iron repressor）. Our study was mainly focus on the role of SirR. SirR was renamed MntR, since it has been annotated as an iron-dependent repressor, functions instead as a manganese-dependent transcriptional repressor, regulating transporters that promote manganese import.To explore the role of SirR, M.smegmatis was used as the host strain for heterologous expression Rv2788. Rv2788 has no effect on the growth,but significantly change the colony morphology of Ms_Rv2788. Through simulating stresses which TB encountered during infection, we found Rv2788 can enhance the resistance of recombinants to diverse stresses,such as hydrogen peroxide, diamide exposure, surface stress SDS and acidic condition. In addition, Rv2788 improved the survival of Ms_Rv2788 when treated with multiple antibiotics including chloramphenicol, vancomycin and amikacin. GC-MS analysis shown three fatty acid C14: 1w5（t）,C14: 0, C16: 1w7（c） were up-regulated in recombinant Ms_Rv2788.To understand whether the increased drug resistance of Ms_Rv2788 is caused by decrease of cell wall permeability, we measured the accumulations of EtBr and Nile Red in bacteria by fluorescence. The results showed EtBr accumulated more slowly and lower levels in Ms_Rv2788 than Ms_Vec strains, indicating an decrease in cell wall permeability. It suggested Rv2788 may change the resisitance of mycobacteria to stresses by regulating permeability of cell wall. In our study, we found a special phenomenon that Ms_Rv2788 is hypersensitive to capreomycin. The result of RT-PCR shown Rv2788 can negatively regulate genes involved in capreomycin resistance. These data suggested that the transcriptional regulation of Rv2788 ledding to susceptibility of Ms_Rv2788 to capreomycin.To test whether Rv2788 regulate the transcription of its target genes, RT-PCR was used to monitor the transcriptional levels of their homologous genes in recombinants. Five genes covering diverse functions were downregulated in Ms_Rv2788 comparison with Ms_Vec strains, including transcription repressor WhiB1 MSMEG₁919, Glutamate-ammonia-ligase adenylyltransferase GlnE MSMEG₄293, transcriptional regulatory protein MSMEG₅074, DNA-binding protein Hup B MSMEG₂389 and oxidoreductase MSMEG₆763. Then EMSA confirmed these downregulated genes were regulated by Rv2788 directly. However, we found higher expression of lexA in recombinant Ms_Rv2788 strains.The dysregulation of the target genes of Rv2788, such as whiB1 and lexA, might underpin such phenotypes we observed.In brief, we provide here evidence that M. tuberculosis Rv2788 plays an important role in response to stresses. It’s first reported that Rv2788 may directly or indirectly involves in fatty acid metabolism and changes colony morphology and cell wall permeability, resulting in the resistance of recombinant M. smegmatis to stresses. Moreover, a role of Rv2788 in transcriptional regulation was confirmed by PT-PCR and EMSA. These results might represent ideal novel target candidate for TB treatment.