Mechanisms Of Mycolic Acid Generation In Mycobacteria

结核病(TB)是一种影响人和牛的细菌性传染病。人类形式的结核病是由结核分枝杆菌引起的，由于全球高死亡率和难治性菌株的增加和传播，结核病一直是全球关注的健康问题，这种病原体对大多数用于治疗结核病的药物具有耐药性。由牛分枝杆菌引起的牛型结核病也对当地农业产生了重大影响，每年花费英国纳税人超过1亿英镑。结核杆菌的细胞膜是一层独特的“蜡状”层，由独特的脂类(脂肪)组成。一类这样的脂类，称为霉菌酸，是细胞被膜的关键成分。真菌酸在病原体致病能力中发挥着重要作用，但它们也是细菌的致命弱点。分枝杆菌没有分枝杆菌酸就不能生存，因此，参与制造分枝杆菌酸的基因对维持细菌的生存至关重要。这使我们有机会针对霉菌酸生物合成基因进行未来的药物开发。在这项研究中，我们的目标是了解真菌酸是如何产生的最后的完成阶段，重点是在细胞外运输和沉积之前的两个完成步骤中涉及的两种酶。第一种酶，聚酮合成酶13(Pks 13)将两个大单位结合在一起，并将它们浓缩成最终产品的前体。在这里，我们的初步工作提出了Pks 13的新的、以前未知的部分，它们可能形成一个新的结构域，驱动Pks 13的两个分子形成二聚体，从而对这种酶的工作方式提出了新的机械见解。我们的主要方法包括纯化蛋白质和单个结构域，并使用一种名为冷冻电子显微镜的技术，通过验证我们预测的Pks 13结构域在3D中的外观模型来证明这些结构的存在。我们还将探索Pks 13与我们在本提案中旨在研究的另一种酶的潜在相互作用：MMRA的基因已被证明是完成霉酚酸生物合成所必需的。我们将使用纯化的蛋白质以及能够测试蛋白质-蛋白质相互作用的遗传系统来探索Pks 13与MMRA的潜在相互作用。MMRA如何发挥作用的机制也不清楚，在这项研究中，我们的目标是通过确定MMRA的底物，通过测试一种有助于我们测量其活性的纯化酶的测定来阐明这一过程。总体而言，我们的研究将概述霉酚酸生产的最后阶段，并强调这些步骤作为未来药物开发的独特目标的潜力。

Tuberculosis (TB) is a bacterial infectious disease affecting humans and cattle. The human form of tuberculosis is caused by Mycobacterium tuberculosis, and has been a global health concerns due to the high global mortality rates and the rise and spread of difficult-to-treat, strains of the causative agent that are resistant to most drugs used for treating TB. The bovine version of TB, caused by Mycobacterium bovis, also has a significant impact on local agriculture costing the UK taxpayer over £100 million each year. The cell envelope of the TB bacillus is a unique and 'waxy' layer made up of unique lipids (fats). One class of such lipids, termed mycolic acids, are a critical component of the cell envelope. Mycolic acids play an important role in the ability of the pathogen to cause disease, however they are also an Achilles heel of the bacterium. Mycobacteria cannot survive without mycolic acids and thus the genes involved in making mycolic acids are essential to keep the bacteria alive. This gives us an opportunity to target mycolic acid biosynthesis genes for future drug development. In this study, we aim to understand the final, finishing stages of how mycolic acids are made, with a focus of two enzymes involved in the finishing steps, prior to transport and deposition on the outside of the cell. The first enzyme, polyketide synthase 13 (Pks13) brings together two large units and condenses them into a precursor for the finished product. Here, our preliminary work suggest new, previously unidentified sections of Pks13 that potentially form a new domain that drives two molecules of Pks13 to form a dimer, proposing new mechanistic insights into how this enzyme works. Our main approach involved purifying the protein and individual domains and use a technique called cryo electron microscopy to demonstrate the existence of these structures by validating our predicted models for how these domains of Pks13 look like in 3D. We will also probe the potential interaction of Pks13, with the another enzyme we aim to study in this proposal: the gene for MmrA has been shown to be essential for the 'completion' of mycolic acid biosynthesis. We will probe the potential interaction of Pks13 with MmrA, using purified proteins as well as genetic systems that enable the testing of protein-protein interactions. The mechanisms of how MmrA functions are also not clear, and in this study we aim to shed light on this process by determining the substrate for MmrA, by testing an assay for the purified enzyme that will help us measure its activity. Overall, our studies will outline the final stages of mycolic acid production, and highlight the potential of these steps to be targeted as unique targets for drug development in the future.