Harnessing tuberculosis toxins to manipulate bacterial growth

Background. Mycobacterium tuberculosis is the biggest infectious disease killer worldwide, with 1.6 million deaths per annum and increasing rates of antimicrobial-resistant infections. Strategies to manage tuberculosis require fundamental research to drive innovation in therapeutics, but the World Health Organisation has highlighted the scarcity of new approaches. An opportunity exists to address these points by harnessing "toxin-antitoxin" (TA) systems in bacteria that target essential steps such as translation, DNA replication and cell wall synthesis. The abundance of TA systems throughout bacteria, and M. tuberculosis in particular, suggests these systems represent a rich source of new biochemistry and antibacterial targets.

TA systems encode two components, a toxic protein that targets an essential cellular process, and an antagonistic antitoxin, which blocks toxin activity when cells are growing under favourable conditions. Although the processes that lead to toxin activation remain under debate, it has been proposed that under certain stress conditions, increased toxin transcription and synthesis may lead to activation. This in turn reduces growth rate, which can provide a means to survive with minimal metabolic burden until favourable conditions return. TA systems are remarkably abundant in M. tuberculosis, which encodes more than 80 putative systems that are thought to contribute to the success of M. tuberculosis as a human pathogen. Many of the putative M. tuberculosis toxins tested thus far were shown to inhibit bacterial growth, and the highly toxic nature of some toxins suggests that their antibacterial mechanisms could be developed into antimicrobials.

Research Plan. In our recent work, we established and characterised the MenAT TA family of nucleotidyltransferases, which were proven to be active in killing tuberculosis. Toxin MenT3 targets and modifies all four mycobacterial serine tRNAs. The MenAT family also represents a new class of type VII TA systems, regulated through phosphorylation by the cognate MenA antitoxin.

These discoveries prompt two new routes for controlling the growth of M. tuberculosis; (i) modulating toxin activity and (ii) inhibiting amino-acyl charging of serine tRNAs. This project aims to explore these opportunities through a combination of biochemistry, structural biology and in silico methods encompassing protein dynamics modelling, docking studies and structure-based drug design.

Objective 1 - Toxin inhibition. Use our high resolution structures of toxin MenT3 and MenT4 with VirtualFlow, to screen and identify potential small molecule inhibitors. Hits will be screened for toxin inhibition through established biochemical assays, microbiology and X-ray crystallographic studies.

Objective 2 - Dysregulation of toxin-antitoxin interactions. Structural and biophysical studies will be performed to build models for the control of MenT toxins through phosphorylation by cognate MenA antitoxins.

Objective 3 - Target toxin targets. MenT3 modifies serine tRNAs, indicating the seryl-tRNA synthetase as a potential antibiotic target. Structural studies of the seryl-tRNA synthetase from M. tuberculosis will be combined with in silico screening methods to identify and characterise potential inhibitors.

Strategic vision. Studying the recently identified MenAT toxin-antitoxin family is timely and addresses an unmet research need, by characterising new potential antibiotic targets in a relevant human pathogen through a combination of biochemical and computational methodologies.

The CDT has five primary beneficiaries:
The CDT cohort
Our students will receive an innovative training experience making them highly employable and equipping them with the necessary knowledge and skillset in science and enterprise to become future innovators and leaders. The potential for careers in the field is substantial and students graduating from the CDT will be sought after by employers. The Life Sciences Industrial strategy states that nearly half of businesses cite a shortage of graduates as an issue in their ability to recruit talent. Collectively, the industrial partners directly involved in the co-creation of the proposal have identified recruitment needs over the next decade that already significantly exceed the output of the CDT cohort.
Life science industries
The cohort will make a vital contribution to the UK life sciences industry, filling the skills gap in this vital part of the economy and providing a talented workforce, able to instantly focus on industry relevant challenges. Through co-creation, industrial partners have shaped the training of future employees. Additional experience in management and entrepreneurship, as well as peer-to-peer activities and the beginning of a professional network provided by the cohort programme will enable graduates to become future leaders. Through direct involvement in the CDT and an ongoing programme of dissemination, stakeholders will benefit from the research and continue to contribute to its evolution. Instrument manufacturers will gain new applications for their technologies, pharmaceutical and biotech companies will gain new opportunities for drug discovery projects through new insight into disease and new methods and techniques.
Health and Society
Research outputs will ultimately benefit healthcare providers and patients in relevant areas, such as cancer, ageing and infection. Pathways to such impact are provided by involvement of industrial partners specialising in translational research and enabling networks such as the Northern Health Science Alliance, the First for Pharma group and the NHS, who will all be partners. Moreover, graduates of the CDT will provide future healthcare solutions throughout their careers in pharmaceuticals, biotechnology, contract research industries and academia.
UK economy
The cohort will contribute to growth in the life sciences industry, providing innovations that will be the vehicle for economic growth. Nationally, the Life Sciences Industrial Strategy Health Advanced Research Programme seeks to create two entirely new industries in the field over the next ten years. Regionally, medicines research is a central tenet of the Northern Powerhouse Strategy. The CDT will create new opportunities for the local life sciences sector, Inspiration for these new industries will come from researchers with an insight into both molecular and life sciences as evidenced by notable successes in the recent past. For example, the advent of Antibody Drug Conjugates and Proteolysis Targeting Chimeras arose from interdisciplinary research in this area, predominantly in the USA and have led to significant wealth and job creation. Providing a cohort of insightful, innovative and entrepreneurial scientists will help to ensure the UK remains at the forefront of future developments, in line with the aim of the Industrial Strategy of building a country confident, outward looking and fit for the future.
Institutions
Both host institutions will benefit hugely from hosting the CDT. The enhancement to the research culture provided by the presence of a diverse and international cohort of talented students will be beneficial to all researchers allied to the theme areas of the programme, who will also benefit from attending many of the scientific and networking events. The programme will further strengthen the existing scientific and cultural links between Newcastle and Durham and will provide a vehicle for new collaborative research.