TConditional ermination of Transcription in Mycobacterium tuberculosis

Antimicrobial resistance (AMR) is an increasing problem pertaining to all bacterial infections, including tuberculosis (TB), caused by infection with Mycobacterium tuberculosis. According to the latest WHO estimates, drug resistant TB lead to around 240 000 deaths in 2016. Unlike most other pathogens, M. tuberculosis does not rely on plasmid-borne resistance. Instead, M. tuberculosis expresses several natural drug resistance machines called efflux pumps, which excrete anti-bacterial drugs that enter the cell.
Transcription is the process of copying DNA to RNA, which is subsequently decoded to synthesise proteins. One way of controlling gene expression is by premature termination of transcription, i.e. the process of transcription is initiated but not completed, meaning there is no RNA to decode and hence no protein is expressed. However, premature termination of transcription can be regulated by physical or molecular signals, in which case it is referred to as conditional termination.
Recently, several efflux pumps were shown to be regulated by conditional termination in Bacillus subtilis, Enterococcus faecalis and Listeria monocytogenes, and similar mechanisms are likely to be widespread in other bacteria including M. tuberculosis.
The aim of this project is to apply very recently developed methods based on Next-generation sequencing to (1) define the abundance of conditional terminators in M. tuberculosis, and (2) determine to what extent natural resistance mechanisms in M. tuberculosis are controlled by such conditional terminators and (3) to what extent anti-TB drugs control overall gene expression in M. tuberculosis.
The successful completion of this project will shed light on regulatory aspects of M. tuberculosis's natural drug resistance, and further our understanding of how anti-TB drugs may contribute to and possibly enhance this drug resistance.

This project will investigate how conditional termination of transcription contributes to gene expression control in M. tuberculosis. More specifically, we will define transcriptional terminator motifs and investigate the genome-wide abundance of conditional terminators in M. tuberculosis by applying Term-seq (3' end enriched RNA-seq) to cultures of M. tuberculosis grown under standard growth conditions. We will also determine to what extent essential metabolites and anti-TB drugs regulate gene expression and in particular conditional termination in M. tuberculosis by applying Term-seq to cultures of M. tuberculosis grown in the presence of S-adenosylmethionine (SAM) and glycine (essential metabolites) or selected anti-TB drugs (Isoniazid, Ethambutol, Bedaquiline and Linezolide). The findings will be further consolidated by using traditional molecular biology methods. To validate termination and antitermination function of selected elements we will employ reporter genes fusions of wildtype and mutated elements expressed under different growth conditions. To identify genes associated with synthesis, modification or turnover, we will express these reporters and in transposon-insertion mutants. To validate predicted structures of selected elements we will perform RNA structure probing with a complement of single-stranded and double-stranded specific RNases. To probe how potential ligands modulate transcription kinetics and termination we will employ single-round in vitro transcription; to screen for putative ligands, we will modify our existing in vitro transcription assay to include the expression of a fluorophore-binding RNA aptamer (Mango), which will enable high-throughput (96-well format).

The immediate beneficiaries of this research will be scientists working on:

1. Mycobacterium tuberculosis (Mtb) basic and translational research
2. Basic transcriptional/RNA polymerase mechanisms
3. RNA processing
4. Post-transcriptional control of bacterial gene expression
5. Structure, function and evolution of regulatory RNAs (e.g. riboswitches and other cis-regulatory elements)
6. Mechanisms used by bacteria to co-ordinate different aspects of growth such as cell wall remodelling and protein synthesis (rpfB operon)
7. Mechanisms used by bacteria in response to toxic compounds/drugs
8. Developing novel anti-TB drugs.
9. Developing novel antimicrobials against other bacteria including non-tuberculous mycobacteria (NTM)
Additional beneficiaries include clinicians working with TB and pharmaceutical companies in the development of novel disease interventions. On a more long-term basis, our research will benefit TB patients, the general public and communities with a high prevalence of TB. Due to the extreme conservation of the investigated genes and elements between Mtb and Mycobacterium bovis, this research will also benefit veterinarians working with bovine TB (bTB) in the field, and this will in turn have an impact on the devastating effects that bTB has on UK farmers, wildlife and economy.
The (b)TB research community will benefit from the increased understanding on how Mtb/ Mb adapt to and survive in a hostile host environment and what role external metabolites and drugs in combination with regulatory RNA play in this adaption.
The research will provide a platform for similar studies in other mycobacteria and will benefit scientists working on emerging NTM.
The TB research community will also benefit from sharing strains, materials and models developed during the project. Generated materials will be provided to scientific community on request.

Previous efforts to characterise Mtb gene regulation have been dominated by a protein-centric view. However, we know that regulatory RNA plays an important role in other pathogens, and it is essential to include this largely uncharacterised reservoir of regulators in our efforts to understand the basic biology of Mtb.
Regulatory RNA is deeply integrated into the regulatory networks of bacterial gene expression. Novel findings from important pathogen such as Mtb will contribute to advancing our knowledge of the adaptive processes that occur upon entering the host environment and drug treatment and hence expand our understanding of fundamental regulatory mechanisms.
Scientists working on regulatory RNAs in other organisms will benefit from the added knowledge about how GC-rich regulatory RNAs regulate their cognate mRNAs. This knowledge will aid our understanding of the evolution of structure-function relationships in all types of non-coding RNAs.
The added knowledge of RNA dynamics may offer inspiration for more distantly related systems, i.e. eukaryotic miRNAs, piRNAs and siRNA.
Pharmaceutical companies will be able to tap into the enhanced knowledgebase concerning regulation of gene expression and metabolism in their efforts to develop novel drugs and vaccines. Deeper insights into RNA biology may improve methods in RNA interference/gene silencing technology, which in future will be applicable to a plethora of animal and human diseases.
The ultimate goal for research into any pathogen is eradication of the disease it causes. The long-term beneficiaries of our research are humans who will gain from improved vaccines, diagnostics and therapies. Understanding the contribution of conditional termination in persistence, reactivation, modulation of host response and transmission of Mtb will allow an assessment of the potential for targeting this aspect of Mtb biology for the development of new vaccines and therapeutics. Improved diagnostics may in turn lead to improved disease control, which will benefit global health.