Understanding the Evolutionary Origins and Molecular Mechanisms of Antimicrobial Peptide Resistance

全球抗生素耐药性的增加使治疗细菌感染变得困难，感染性疾病的死亡率正在以惊人的速度上升。每年有70万人死于耐甲氧西林金黄色葡萄球菌等耐药细菌，一些细菌现在对所有可用的药物都有抗药性。2014年，世界卫生组织发布了第一份关于抗菌素耐药性的全球报告，敦促世界各国政府联合起来应对这一卫生紧急情况。一个目标是确定新的抗菌剂，但30年来没有开发出新的主要类别的抗生素。目前的研究正在探索抗菌肽(AMPs)的临床应用。AMPS针对的是一种基本的细菌结构(细胞壁)，这种结构不容易通过突变而改变，因此被认为是关于耐药性的“安全”。20世纪50年代末S引入万古霉素时也提出了同样的假设，但环境细菌耐药基因的转移导致了目前的一种“超级细菌”--万古霉素耐药肠球菌。在环境细菌中已经存在对AMPs的耐药性，许多人类病原体，如葡萄球菌，都含有相关基因。因此，为了防止类似万古霉素的发展，我们必须了解这些AMP耐药系统，并利用这些发现设计出抵消它们的策略。一种创新的方法是阻断细菌检测抗生素并激活其耐药性的途径。干扰这一过程的药物将恢复抗生素的效力，提供长期解决方案。类似的治疗方法已经用于癌症治疗，但尚未用于治疗抗生素耐药性。近年来，在许多革兰氏阳性细菌中发现了一种新型的AMP耐药性，包括。像金黄色葡萄球菌这样的人类病原体。这些所谓的BCE类系统包括一个可能将抗生素从细胞中移除的转运体，以及一个控制转运体生产的调控系统。它们的关键特征是，转运蛋白充当AMP传感器，并控制监管系统，从而间接控制自身。有两个方面使这些系统与详细的探索高度相关：(I)它们与几乎在所有细菌中发现的其他耐药转运蛋白共享一个保守的结构域；(Ii)它们独特的调控途径是阻断耐药性的主要药物靶点。由于病原菌很难处理，我们将使用与枯草芽孢杆菌紧密相关的杆菌抗菌素BCE系统作为我们的实验模型，我们的首要目标是确定这些系统是如何进化的，以便了解它们与其他耐药系统的关系。BCE类转运蛋白包含一个名为FtsX的结构域，我们预计它对耐药性很重要，在许多致病细菌中都可以找到。我们将使用计算和实验方法来确定这个结构域的功能。这将提供有关BCE类系统以及拥有FtsX域的其他运输商的信息。第二个目标是解决运输商如何控制监管系统的问题。我们将使用分子生物学技术来找出蛋白质在哪里相互作用，以及信息是如何从转运体传递到调控系统的。阻断这一途径将阻止耐药的激活，我们将提供所需的信息来探索它作为新的药物靶点。耐药途径的第一步是由细胞检测AMP，但尚不清楚BCE样转运蛋白是如何实现这一点的。在我们的第三个目标中，我们将使用蛋白质生化方法来研究AMP结合。了解药物是如何结合的，将允许设计防止检测到从而产生耐药性的修饰。我们的项目将通过类似BCE的系统提供对AMP耐药性的详细了解，并确定作为抗药性靶点探索的重要过程。

The global increase in antibiotic resistance has made it difficult to treat bacterial infections, and mortality from infectious disease is rising at an alarming rate. Each year 700,000 people die from resistant bacteria such as MRSA, and some bacteria are now resistant to all available drugs. In 2014, the World Health Organisation issued its first Global Report on Antimicrobial Resistance, urging governments world-wide to join forces in tackling this health emergency. One goal is to identify new antimicrobials, but no new major class of antibiotics has been developed in 30 years. Current studies are exploring antimicrobial peptides (AMPs) for clinical use. AMPs target an essential bacterial structure (the cell wall), which cannot be easily changed by mutation, and are thus considered as "safe" regarding resistance. The same assumption was made with the introduction of vancomycin in the late 1950's, but transfer of resistance genes from environmental bacteria has resulted in one of the current "superbugs", Vancomycin Resistant Entercocci (VRE). Resistance against AMPs in environmental bacteria already exists, and many human pathogens, e.g. Staphylococci, contain related genes. To prevent a similar development as was seen for vancomycin, it is therefore imperative that we understand these AMP resistance systems and use the findings to devise strategies to counteract them. One innovative approach is to block the pathway by which bacteria detect the antibiotic and activate their resistance. A drug that interferes with this process would restore the efficacy of the antibiotic, providing a long-term solution. Similar treatments are already used in cancer therapy, but not yet to tackle antibiotic resistance.In recent years, a new type of AMP resistance has been identified in many Gram-positive bacteria, incl. human pathogens like S. aureus. These so-called Bce-like systems consist of a transporter that presumably removes the antibiotic from the cell, and a regulatory system that controls production of the transporter. Their key feature is that the transporter acts as an AMP sensor and controls the regulatory system and thus indirectly itself. Two aspects make these systems highly relevant for detailed exploration: (i) they share a conserved domain with other resistance transporters found in nearly all bacteria; (ii) their unique regulatory pathway presents a prime drug target for blocking resistance. Because pathogenic bacteria are difficult to handle, we will use the closely related bacitracin resistance Bce system of Bacillus subtilis as our experimental model.Our first aim is to determine how these systems evolved, in order to understand their relationship to other resistance systems. Bce-like transporters contain a domain, called FtsX, we expect to be important for resistance and which can be found in many disease-causing bacteria. We will use computational and experimental methods to determine the function of this domain. This will provide information on Bce-like systems as well as on the other transporters possessing an FtsX domain.The second aim addresses the question how the transporter controls the regulatory system. We will use molecular biology techniques to find out where the proteins interact, and how information is passed from the transporter to the regulatory system. Blocking this pathway will prevent activation of resistance, and we will provide the information needed to explore it as a novel drug target.The first step of the resistance pathway is detection of AMPs by the cell, yet it is unknown how Bce-like transporters accomplish this. In our third aim, we will use protein biochemistry methods to study AMP binding. Knowledge of how a drug is bound will allow the design of modifications that prevent detection and thus resistance.Our project will provide detailed understanding of AMP resistance by Bce-like systems and identify important processes to explore as drug targets in combatting resistance.