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.

Our work aims to answer fundamental questions on the energy metabolism of tuberculosis, which are relevant to both novel and established antibiotics that either directly affect these processes (for example bedaquiline) or are affected by them (isoniazid). Tuberculosis treatment relies on taking a combination of drugs over an extended period, often with damaging side effects. Resistance to these drugs is growing. The challenge, therefore, is to develop drugs which remain effective and have fewer side effects. By understanding the interactions between tuberculosis biology and antibiotics we aim to aid those developing new compounds to ultimately help those suffering with this terrible disease.

We will develop a device, the bioenergetic chamber, which will measure the energetic status of bacterial cells without breaking them open or disrupting them. Once we have built and validated the system, we will use it to understand how antibiotics such as bedaquiline work to kill tuberculosis, allowing us to design new drugs in the future. Building partnerships with workers on the front-line of tuberculosis drug development will be an important route to ensuring our work has real world impact. These include those in the charitable sector, the pharmaceutical industry, and publicly funded academic efforts. The key to these partnerships will be offering a unique tool which measures parameters that cannot be directly measured in any other way, enabling us to clarify the effects of developmental compounds and drugs on the bacterial cells and allowing further development in possibly unexpected directions. The TB Alliance, a charity that has the largest pipeline of preclinical compounds against tuberculosis and that we have already engaged with, is often willing to make their developmental compounds available to researchers who apply for them, and this is one route we will take. Other charities and pharmaceutical labs have similar policies.

Beyond supporting work to develop drugs that directly target tuberculosis, our work will help us understand how tuberculosis infects cells in the body by surviving the reactive hydrogen peroxide generated as a defence mechanism by the host. This is a growing area of research that may in the future lead to novel vaccines or antibiotics. Once infection has taken hold within the granulomas (tubercles) in the lungs, the interior has a low oxygen concentration. Tuberculosis uses several of the enzymes we are working on in this project to survive and enter a dormant persister state where they are difficult to kill. Later, these cells can reactivate and cause further infection. By understanding how these processes we may be able to develop drugs that target these cells directly and shorten treatment.

Electron cryo-microscopy (cryoEM) is a fast-growing technique for resolving the structures of biological molecules using an electron microscope. It is excellent for imaging membrane proteins, which are very hard to resolve in any other way. Membrane proteins are important biologically and the target for about half of all drugs. UK pharmaceutical companies such as AstraZeneca, GSK, Astex, and Heptares are investing heavily in cryoEM, but there is a severe lack of trained scientists to support the new microscopes. This project will use cryoEM to resolve membrane protein structures, providing the postdoctoral associate working on this project with extensive experience in the skills required to contribute to this workforce.

To assist other groups working in drug discovery and development we will upload our full cryoEM datasets to publicly accessible databases so they can reprocess them with their own targets in mind. The datasets resulting from the bioenergetic chamber will also be released. We will also make use of preprint servers, so our paper drafts are publicly accessible immediately once they are written.