Modulation of antibiotic resistance and protein synthesis by disrupting Elongation Factor G dynamics

抗生素耐药性是一个严重的公共卫生问题;英格兰首席医疗官警告说，它构成了与气候变化或恐怖主义同等的“灾难性威胁”，预计到2050年，与抗生素耐药性有关的死亡人数将超过癌症。如果没有协调一致的努力，我们将面临一个未来，即常规可治疗的感染可能由于缺乏有效的抗生素治疗而变得致命。由于新抗生素的可用性很少，因此克服对现有抗生素的耐药性以扩大这些重要治疗方法的有效性非常重要。要做到这一点，我们必须了解所涉及的蛋白质是如何介导抗性的。这项工作旨在了解对一种重要的临床抗生素夫西地酸（FA）的耐药性机制，并确定控制其作用方式的相关蛋白质的关键区域。这可以为开发克服这种耐药性的药物提供资源，并恢复这种重要抗生素的有效性。FA用于治疗金黄色葡萄球菌感染，是少数几种对医院“超级细菌”MRSA有效的口服抗生素之一。FA与一种名为EF-G的蛋白质（制造蛋白质的重要组成部分）相互作用，并阻止其工作，这意味着细菌不能制造蛋白质，因此不能生长。近年来，通过另一种蛋白质FusB与EF-G的相互作用将其从FA的影响中拯救出来，或者EF-G本身的突变，对FA的抗性急剧增加。最近研究了FusB和EF-G之间的相互作用，表明FusB引起EF-G的长程变化，产生EF-G中的运动，这对引起FA阻力很重要。我有新的数据表明，我可以通过改变EF-G来破坏这些运动，当我这样做时，蛋白质对FA的抵抗力就会降低。我的目标是确定EF-G的哪些部分在控制这些运动中很重要，试图找到EF-G的关键区域，这些区域可以作为开发药物的目标，这些药物可以阻止FA耐药性，从而延长这种抗生素的有效性。我将对EF-G进行一系列改变，并通过一种称为核磁共振（NMR）的技术对EF-G中的运动进行结构研究，以监测这些改变的影响。然后，我将研究这些变化对FusB引起FA抗性的能力有什么影响，确定控制其对FusB反应的EF-G的重要区域。我将利用这些重要领域的知识，看看在已经对FA具有抗性而不需要FusB的EF-G中改变它们是否也可以防止它产生FA抗性，从而提出可能控制两种类型抗性的任何领域。为了补充这一点，我将研究EF-G中的类似运动是否对EF-G在细菌内制造蛋白质的功能很重要，以更好地了解这种蛋白质是如何工作的。这可以为药物发现研究提供进一步的信息，试图阻止这种必需蛋白质的工作，为抗生素开发研究提供潜在的靶点。这些研究将使我们了解细菌如何对这种重要的抗生素产生耐药性，提供可用于设计新的抗生素治疗方法以绕过这种耐药性的信息。随着抗生素耐药性的增加和新抗生素的发现越来越少，利用现有的耐药机制来开发绕过耐药性的药物或设计可添加到抗生素中以克服耐药性的药物是非常重要的。了解抗生素耐药性的结构基础可以导致开发这样的药物，这些药物可以与抗生素一起使用，以克服现有的耐药性，使我们能够继续使用抗生素治疗感染。

Antibiotic resistance is a serious public health concern; the Chief Medical Officer for England warned it poses a 'catastrophic threat' on a par with climate change or terrorism and deaths related to antibiotic resistance are predicted to outnumber those from cancer by 2050. Without concerted efforts, we face a future in which routinely treatable infections may become fatal due to a lack of effective antibiotic treatments. As few new antibiotics are becoming available it is important to overcome resistance to existing antibiotics to extend the usefulness of these important treatments. To do this we must understand how the proteins involved mediate resistance. This work aims to understand the mechanism of resistance to an important clinical antibiotic, fusidic acid (FA), and identify key regions of the proteins involved that control how they act. This could provide resources for the development of drugs to overcome this resistance and rejuvenate the usefulness of this important antibiotic.FA is used against infections by the bacteria Staphylococcus aureus and is one of few remaining oral antibiotics active against the hospital 'superbug' MRSA. FA interacts with a protein called EF-G (an important part of the machinery for making proteins) and prevents it from working, meaning bacteria cannot make proteins and so cannot grow. Resistance to FA has increased dramatically in recent years either by the interaction of another protein, FusB, with EF-G that rescues it from the effects of FA, or mutations in EF-G itself. The interaction between FusB and EF-G has recently been studied, showing FusB causes long-range changes in EF-G that produce motions in EF-G important for causing FA resistance. I have new data showing that I can disrupt these motions by altering EF-G and that when I do, the proteins become less resistant to FA. I aim to determine which parts of EF-G are important in controlling these motions to try to find key areas of EF-G that could act as targets for the development of drugs that can stop FA resistance and so extend the usefulness of this antibiotic. I will make a series of changes to EF-G and monitor the effects of those changes using structural investigations of motions in EF-G by a technique called nuclear magnetic resonance (NMR). I will then study what effects these changes have on the ability of FusB to cause FA resistance, identifying important areas of EF-G that control its response to FusB. I will use knowledge of these important areas to see if changing them in EF-G that is already resistant to FA without needing FusB can prevent it from producing FA resistance too, suggesting any areas that might control both types of resistance. To complement this, I will study whether similar motions in EF-G are important in the function of EF-G in making proteins within bacteria to try to get a better understanding of how this protein works. This could provide further information for drug discovery studies to try to stop this essential protein from working, providing a potential target for antibiotic development studies. These studies will allow us to understand how bacteria become resistant to this important antibiotic, providing information that can be used to design new antibiotic treatments to bypass this resistance. They also help us to understand the role of EF-G in the essential process of making proteins, which may show more potential drug targets for development of new antibiotics.With the increase in antibiotic resistance and few new antibiotics being discovered, it is important to use knowledge of current resistance mechanisms to develop drugs that bypass resistance or to design drugs that can be added to antibiotics to overcome the resistance. Understanding the structural basis of antibiotic resistance can lead to the development of such drugs that can be administered with the antibiotic to overcome the existing resistance, allowing us to continue to use the antibiotic to treat infections.