Using cryoEM and functional biophysics to understand the mycobacterial bd oxidase, a key enzyme in tuberculosis

Tuberculosis is a devastating disease caused by Mycobacterium tuberculosis. It is one of the top 10 causes of death worldwide (World Health Organisation; 2016). Current treatments require administering a cocktail of antibiotics over an extended period with unpleasant side-effects; drug resistance threatens to render even these treatments ineffective. A recently developed and unprecedented strategy to kill tuberculosis is to target the bioenergetics of the organism, which provides the energy the bacteria needs to infect cells. This project will focus on the bd oxidase, which is a crucial bioenergetic enzyme for allowing the bacteria to scavenge oxygen in low oxygen environments and possibly to break down the harmful hydrogen peroxide generated by the host as an immune response. Compounds are currently under active development to inhibit the bd oxidase as drug candidates. Beyond tuberculosis, the bd oxidase also has a critical role in the survival of diverse bacteria in low oxygen or stressful conditions.

Rather than over-expressing the bd oxidase complex, we will induce it by growing mycobacteria in defined culture conditions. The native-expression strategy means the enzyme, before purification, will have an authentic composition in terms of cofactors and bound lipids. We will then purify it in an active form that recapitulates its physiological activity using both mild detergent and detergent-free strategies. The isolated enzyme will be subjected to CryoEM for structural studies and pre-steady-state and steady-state kinetics will be used to clarify its reactions with hydrogen peroxide and oxygen. Importantly, its reaction with hydrogen peroxide may be a mechanism for cell protection in pathophysiological situations.

This project will use cutting-edge biophysical methods to understand how this enzyme works on both structural and functional levels. The project will use techniques from bacteriology, membrane protein biochemistry, cryoEM (both data collection and image processing), and kinetic modelling/signal processing using the Python programming language with the NumPy/SciPy Libraries.