Structural and functional study of the Mycobacterium RbpA protein: a master regulator of gene expression implicated in drug resistance – MycoMaster
The RbpA protein from M. tuberculosis, a master regulator of gene expression, implicated in drug resistance
RbpA is a transcriptional factor essential for M. tuberculosis growth and implicated in tolerance to the first-line anti-tuberculosis drug, rifampicin. Our project aims to understand the RbpA-controlled mechanisms of gene regulation which allow M. tuberculosis to survive antibiotic treatment and to cause recurrent infection.
Molecular mechanism of the RbpA action and its role in gene regulation
Mycobacterium tuberculosis is a pathogen killing ~1.5 million people yearly. Development of a new therapeutic strategies and identification of the new drug targets necessitates understanding of the molecular mechanisms employed by bacteria to invade host and to survive antibiotic treatment. Virulence of M. tuberculosis, its ability to acquire the antibiotic-resistant, persistent state, as well as its tolerance to drugs are controlled at the level of gene transcription. Bacterial multi-subunit RNA polymerase (RNAP) is the central enzyme of transcription. RNAP is composed of the catalytic core and dissociable subunit, sigma, which is required for promoter recognition and initiation of RNA synthesis. In M. tuberculosis, the housekeeping sigma-A controls expression of ~70% of genes during active growth while sigma-B controls genes in the stationary phase, persistent state and during stress response. RbpA is an essential transcriptional regulator which stimulates transcription by RNAPs harboring either the sigma-A or the sigma-B subunits. Our findings indicate that activation of the RbpA-controlled genes may play a pivotal role in tuberculosis pathogenesis and virulence. Molecular mechanism through which RbpA controls transcription is poorly understood. In this context, our project aims to decipher molecular mechanism of action of RbpA from M. tuberculosis and to elucidate it's role in gene regulation. The objectives of our project are to map genes controlled by RbpA genome-wide; to define promoter DNA motifs that modulate RbpA activity; to characterize structure of the M. tuberculosis RNAP holoenzyme and its complex with RbpA and to determine residues of RNAP essential for RbpA function.
Cartography of the RbpA-dependent genes is performed by using in vitro Run-Off-assay on total M. tuberculosis genomic DNA followed by microarray and RNA-seq analysis of RNA transcripts (ROR-seq). The RbpA targets defined by the in vitro approach will be validated by ChIP-seq analysis using non-pathogenic Mycobacterium strains. Thus, majority of the promoter regions bound by the activator in a live cell could be retrieved. In parallel, the synthetic promoter DNA fragments containing different combinations of the promoter elements and their mutant derivatives are used in biochemical assays to assess the effect of RbpA on RNAP activity. Structures of RNAP harboring sigma-B, its complex with RbpA and DNA are explored by using high resolution Cryo-EM analysis. As a complementary approach we use the micro-electron diffraction (micro-ED) which is a good alternative to X-ray diffraction. To determine specific residues of the RNAP subunits essential for RbpA function, the RbpA binding sites are explored by alanine-scanning mutagenesis followed by the analysis of the mutants in biochemical assays.
We found that RNAP harboring sigma-B subunit adopts unusual inactive conformation which is converted to the active conformation upon RbpA binding. Our data provide structural basis for the stress-response gene regulation in M. tuberculosis.
The control and prevention of tuberculosis was defined by European Center for Disease Prevention and Control (ECDC) as one of the priority work areas. Our study will build structural model of the M. tuberculosis RNAP in complex with unusual transcriptional activator and will help to characterize specific features of the mycobacterial transcriptional machinery which determine its high tolerance to the drugs. The outcomes of our project will contribute to understanding of the mechanisms of pathogenesis and persistence in M.tuberculosis. In particular, we expect to get insight on the role of RbpA in regulation of the genes implicated in resistance to clinical drugs like rifampicin, isoniazid and aminoglycosides.
1. Vishwakarma,R.K., Cao,A.M., Morichaud,Z., Perumal,A.S., Margeat,E. and Brodolin,K. (2018) Single-molecule analysis reveals the mechanism of transcription activation in M. tuberculosis. Sci Adv. 4(5):eaao5498. doi: 10.1126/sciadv.aao5498.
2. Perumal, A.S., Vishwakarma, R.K., Hu, Y., Morichaud Z. Brodolin, K. (2018) RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase. Nucl Acids Res. doi: 10.1093/nar/gky714
Mycobacterium tuberculosis, the human pathogen that cause tuberculosis, is responsible for ~1,5 million of deaths every year. Spreading of the muti-drug-resistant forms of tuberculosis becomes a threat for public health worldwide. Development of new therapeutic strategies requires deep understanding of the molecular mechanisms conferring resistance. Our project aims to understand specific mechanisms of gene regulation that allow M. tuberculosis to survive antibiotic treatment and to cause recurrent infection. We employ a multidisciplinary approach combining genomics, biochemical and biophysical methods to explore function of the novel transcriptional regulator, RbpA, essential for replication of M. tuberculosis and increasing its tolerance to antibiotics. The outcomes of our project will help to understand mechanisms of pathogenesis and will provide novel concepts and targets in drug discovery.
Project coordination
Konstantin BRODOLIN (CPBS FRE3689 - CNRS Université de Montpellier)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
CBS Centre de Biochimie Structurale de Montpellier
CPBS FRE 3689 CPBS FRE3689 - CNRS Université de Montpellier
Help of the ANR 429,783 euros
Beginning and duration of the scientific project:
September 2016
- 48 Months
