Energy metabolism and antibiotic action in the tuberculosis pathogen

Tuberculosis is one of the top 10 causes of death worldwide. In 2017, 10 million people developed the disease; it caused 1.6 million deaths (World Health Organisation). The disease is caused by the infectious mycobacterium Mycobacterium tuberculosis and current treatment strategies rely on taking a combination of drugs over an extended period with unpleasant and damaging side-effects; in addition, growing antibiotic-resistance threatens to compromise even these treatments.

A new strategy uses drugs which target energy metabolism, an approach which has not previously been used to target bacteria. However, the new clinical candidates were discovered without an understanding of the target or mechanism of action, as the details of mycobacterial energy metabolism remain to be elucidated. We need this fundamental knowledge to design more effective antibacterial drugs, and understand the synergy arising from using antibacterials together.

Energy metabolism comprises the reactions that generate ATP, the energy currency of the cell. Most of this energy comes from passing electrons from food molecules, such as sugars, to oxygen. As the electrons are passed down an electron transport chain, the released energy is used to synthesise ATP. The large enzyme complexes that catalyse these reactions in mycobacteria are little understood, as is their organisation within the cell.

This work will combine cell-level biophysical techniques on living bacteria with molecular-level structural and functional investigations of individual components to answer critical questions on how mycobacterial energy metabolism works and how it can be exploited. Our overarching questions are:

-How do some of the unusual enzymes found in mycobacteria convert energy and allow the organism to respond to lethal stresses? -How are energy-converting systems organised in mycobacteria? -What are the mechanisms that allow mycobacteria to survive in different oxygen concentrations in the body? -What happens when these essential processes are attacked by antibiotics?

A number of non-invasive biophysical techniques will be brought together in a single device to create a 'bioenergetic chamber'; these techniques have not been previously used together. This device will allow us to measure key cellular molecules without needing to break open or disrupt the bacteria, providing a unique window into the workings of the cell.

It is important to measure these parameters noninvasively as even very mild perturbations to the cell, with an antibiotic for example, lead to rapid changes so that 'quench-and-measure' techniques are often compromised. These noninvasive measurements will be complemented by studies on the in vivo organisation of the electron transport chain. Once we have an understanding of how the systems work in untreated bacteria, we will add clinical antibiotics, such as bedaquiline and isoniazid, to observe how these drugs work to disrupt the cell.

By clarifying their mechanism of action, we should be able to offer insights into how to make more effective antibiotics.

These measurements made on the cellular level will be expanded with molecular-level biophysical investigations of how key enzymes function using electron cryomicroscopy (cryoEM) and specialised functional assays. CryoEM has recently undergone a revolution and now allows us to image enzymes and computationally reconstruct their structure at near atomic resolution.

We will focus on the 'bd oxidase', which has an important role in allowing tuberculosis to survive in the low oxygen conditions found deep inside the tubercles that grow in patients' lungs; the enzyme also breaks down hydrogen peroxide, a molecule often made by the body to kill invading bacteria. We will complement these studies by examining the three related enzymes at the 'succinate:quinone' junction, which are also critical in allowing tuberculosis to adapt to different oxygen conditions.