Abstract
Mycobacterium tuberculosis has the ability to survive inside macrophages under acid-nitrosative stress. M. tuberculosis Rv1685c and its ortholog in M. smegmatis, MSMEG_3765, are induced on exposure to acid-nitrosative stress. Both genes are annotated as TetR transcriptional regulators, a family of proteins that regulate a wide range of cellular activities, including multidrug resistance, carbon catabolism and virulence. Here, we demonstrate that MSMEG_3765 is co-transcribed with the upstream genes MSMEG_3762 and MSMEG_3763, encoding efflux pump components. RTq-PCR and GFP-reporter assays showed that the MSMEG_3762/63/65 gene cluster, and the orthologous region in M. tuberculosis (Rv1687c/86c/85c), was up-regulated in a MSMEG_3765 null mutant, suggesting that MSMEG_3765 acts as a repressor, typical of this family of regulators. We further defined the MSMEG_3765 regulon using genome-wide transcriptional profiling and used reporter assays to confirm that the MSMEG_3762/63/65 promoter was induced under acid-nitrosative stress. A putative 36 bp regulatory motif was identified upstream of the gene clusters in both M. smegmatis and M. tuberculosis and purified recombinant MSMEG_3765 protein was found to bind to DNA fragments containing this motif from both M. smegmatis and M. tuberculosis upstream regulatory regions. These results suggest that the TetR repressor MSMEG_3765/Rv1685c controls expression of an efflux pump with an, as yet, undefined role in the mycobacterial response to acid-nitrosative stress.
Highlights
Tuberculosis (TB) is still endemic in many low and middle-income countries and the high incidence of Mycobacterium tuberculosis multi-drug resistant strains continues to plague the control of TB worldwide (Ayabina et al, 2016; Malone and Gordon, 2016; Dheda et al, 2017)
We characterize the TetR regulator MSMEG_3765 (Rv1685c) using a combination of mutagenesis, local and global gene expression analyses, and DNA binding studies to show that it regulates the MSMEG_3762/63/65 operon encoding an efflux pump. This system is conserved in M. tuberculosis and our experiments suggest that this TetR regulator plays a novel role in the mycobacterial response to the intracellular environment
MSMEG_3765 and the Surrounding Region Is Conserved in M. tuberculosis and Other Pathogenic Mycobacteria
Summary
Tuberculosis (TB) is still endemic in many low and middle-income countries and the high incidence of Mycobacterium tuberculosis multi-drug resistant strains continues to plague the control of TB worldwide (Ayabina et al, 2016; Malone and Gordon, 2016; Dheda et al, 2017). Bacilli are able to grow in intra- and extra-cellular environments in spite of the presence of anti-microbial effectors such as reactive oxygen and nitric oxide intermediates, acidification or metal accumulation (Butler et al, 2010; Awuh and Flo, 2017). This is due to the low permeability of the mycobacterial cell envelope for many toxic molecules, to the ability of mycobacteria to detoxify reactive oxygen and reactive nitrogen molecules, and to the maintenance of a neutral intra-bacterial pH within acidic environments. Model systems, including the fast-growing non-pathogenic M. smegmatis, have been widely used to highlight relevant aspects of M. tuberculosis physiology, exemplified by the modulation of gene expression by the DosR and Lsr regulators involved in dormancy and adaptation to hypoxic stress (Colangeli et al, 2009; Trauner et al, 2012; Agrawal et al, 2015)
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