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

结核病是一种由结核分枝杆菌引起的破坏性疾病。它是全球十大死因之一(世界卫生组织；2016年)。目前的治疗方法需要在较长时间内使用抗生素的鸡尾酒，但会产生令人不快的副作用；抗药性可能会使这些治疗方法失效。最近开发的一种史无前例的杀死结核病的策略是瞄准生物体的生物能量学，它为细菌提供感染细胞所需的能量。这个项目将专注于BD氧化酶，这是一种关键的生物能量酶，允许细菌在低氧环境中清除氧气，并可能分解宿主作为免疫反应产生的有害过氧化氢。目前正在积极开发化合物，作为候选药物来抑制BD氧化酶。除了结核病，BD氧化酶在各种细菌在低氧或应激条件下的生存中也起着关键作用。我们不会过度表达BD氧化酶复合体，而是通过在特定的培养条件下生长分枝杆菌来诱导它。原生表达策略意味着，在纯化之前，酶将在辅因子和结合脂类方面具有真实的组成。然后，我们将用温和的洗涤剂和无洗涤剂策略，以一种活性的形式提纯它，概括它的生理活性。分离的酶将进行低温EM结构研究，并将使用预稳态和稳态动力学来阐明其与过氧化氢和氧气的反应。重要的是，它与过氧化氢的反应可能是在病理生理情况下保护细胞的一种机制。本项目将使用尖端生物物理方法来了解这种酶是如何在结构和功能水平上发挥作用的。该项目将使用细菌学、膜蛋白生物化学、低温电子显微镜(数据收集和图像处理)以及利用NumPy/SciPy图书馆的Python语言编程语言进行动力学建模/信号处理的技术。

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.