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

结核病是由结核分枝杆菌引起的一种毁灭性疾病。它是全球十大死亡原因之一（世界卫生组织; 2016）。目前的治疗方法需要长时间服用抗生素鸡尾酒，副作用令人不快;耐药性甚至有可能使这些治疗无效。最近开发的一种前所未有的杀死结核病的策略是针对生物体的生物能量学，它提供了细菌感染细胞所需的能量。这个项目将集中在bd氧化酶上，这是一种重要的生物能酶，可以使细菌在低氧环境中吸收氧气，并可能分解宿主产生的有害的过氧化氢作为免疫反应。化合物目前正在积极开发，以抑制作为候选药物的bd氧化酶。除了结核病，bd氧化酶在低氧或压力条件下对多种细菌的生存也有重要作用，我们将通过在特定的培养条件下培养分枝杆菌来诱导它，而不是过度表达bd氧化酶复合物。天然表达策略意味着酶在纯化之前将具有辅因子和结合脂质方面的真实组成。然后，我们将以活性形式纯化它，使用温和的洗涤剂和无洗涤剂策略来重现其生理活性。将对分离的酶进行CryoEM进行结构研究，并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病理生理情况下细胞保护的机制。该项目将使用尖端的生物物理方法来了解这种酶在结构和功能水平上的工作原理。该项目将使用细菌学，膜蛋白生物化学，cryoEM（数据收集和图像处理）以及动力学建模/信号处理技术，使用Python编程语言和NumPy/SciPy库。

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.