The making of a Superbug. Understanding the mechanisms of high level antibiotic resistance in Methicillin Resistant Staphylococcus aureus (MRSA)

超级细菌的形成。

• 批准号: 2289038
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2289038
• 关键词: making; Superbug.; Understanding; mechanisms; level

Background Information:Antibiotic resistance is a global challenge to modern human healthcare. Methicillin Resistant Staphylococcus aureus (MRSA) is endemic in many countries with huge associated human and financial costs. If we are to effectively develop new control regimes these must be based on knowledge of the pathogen and how it interacts with the host. The molecular basis of MRSA is via the acquisition of a novel Penicillin Binding Protein (MecA) that shows a low affinity for the range of beta-lactam antibiotics. MecA can substitute for the native PBPs and allow bacterial cell wall synthesis in the presence of drug. Extraordinarily we understand very little as to how MecA fulfills its function and can act with the rest of the cell wall synthesis machinery. The supervisory team have discovered that the presence of MecA in S. aureus does not itself lead to high level AMR but stresses the cells. This paradox that is resolved by the bacteria acquiring mutations in the genes encoding RNA polymerase with a concomitant massive increase in AMR. RNA polymerase has been isolated from the parental and high level AMR strains. This has revealed alterations to overall RNA polymerase properties. This background data has established a firm framework for the proposed project bringing together expertise in molecular microbiology and host:pathogen interaction with protein biochemistry and structural biology to address a problem of great societal importance.Experimental Approach:1. How does MecA work?MecA itself leads to metabolic stress and only gives high level resistance in rpo backgrounds. Molecular interactions of MecA with other proteins will be determined by bacterial 2 hybrid analysis, fluorescence microscopy and protein analysis. A combination of biochemical (HPLC) and biophysical (AFM force curves) will elucidate the effect of AMR on cell wall properties.2. What is the role of RNA polymerase in high level AMR?We have shown, using RNA-seq that the rpo mutations lead to changes in expression of a set of genes involved in metabolism. Our hypothesis is that AMR is controlled by a sensing mechanism. We will purify RNA polymerase and determine the effect of the mutations on signal transduction, using a combination of protein chemistry and transcriptional assays.3. What are the fitness costs and the role of the host environment?For the first time, we have a defined set of strains only carrying the mecA and rpo mutations and thus can be directly compared in models of disease with and without antibiotic intervention to determine the cost of AMR in vivo. Importantly we have defined that high-level AMR is linked to the ability to grow anaerobically. However, the effect of antibiotics has never been routinely tested under this in vivo relevant condition. The role of anaerobiosis drug efficacy will be tested.Training:The project will provide an exciting and interdisciplinary training spanning from molecular genetics, protein chemistry, advanced microscopy approaches, biophysics through to in vivo analysis. The studentship will be set within a wider initiative in Sheffield (http://www.floreyinstitute.com) that brings together researchers from the basic science to clinical application to address the challenge of AMR.

背景信息：抗生素耐药性是现代人类医疗保健面临的全球性挑战。耐甲氧西林金黄色葡萄球菌（MRSA）在许多国家流行，相关的人力和财力成本巨大。如果我们要有效地开发新的控制机制，这些机制必须基于对病原体及其如何与宿主相互作用的了解。MRSA的分子基础是通过获得一种新的青霉素结合蛋白（MecA），该蛋白对β-内酰胺类抗生素的亲和力较低。MecA可以替代天然的PBPs，并允许细菌细胞壁在药物存在下合成。特别是，我们对MecA如何实现其功能以及如何与其他细胞壁合成机制一起发挥作用知之甚少。监督小组发现，在S。金黄色葡萄球菌本身不会导致高水平的AMR，但会对细胞产生应激。这种矛盾通过细菌获得编码RNA聚合酶的基因突变以及伴随的AMR的大量增加来解决。已从亲本和高水平AMR菌株中分离出RNA聚合酶。这揭示了总体RNA聚合酶性质的改变。这些背景数据为拟议的项目建立了一个坚实的框架，将分子微生物学和宿主：病原体相互作用的专业知识与蛋白质生物化学和结构生物学结合起来，以解决具有重大社会意义的问题。MecA如何工作？MecA本身导致代谢应激，并且仅在rpo背景下产生高水平抗性。MecA与其他蛋白质的分子相互作用将通过细菌双杂交分析、荧光显微镜和蛋白质分析来确定。生物化学（HPLC）和生物物理（AFM力曲线）的结合将阐明AMR对细胞壁性质的影响. RNA聚合酶在高水平AMR中的作用是什么？我们已经使用RNA-seq证明，rpo突变导致参与代谢的一组基因表达的变化。我们的假设是，AMR是由传感机制控制。我们将纯化RNA聚合酶，并使用蛋白质化学和转录分析的组合来确定突变对信号转导的影响。什么是健身成本和东道国环境的作用？这是我们第一次有一组只携带mecA和rpo突变的菌株，因此可以在有和没有抗生素干预的疾病模型中直接进行比较，以确定体内AMR的成本。重要的是，我们已经确定高水平AMR与厌氧生长的能力有关。然而，抗生素的作用从未在这种体内相关条件下进行过常规测试。培训：该项目将提供令人兴奋的跨学科培训，涵盖分子遗传学，蛋白质化学，先进的显微镜方法，生物物理学以及体内分析。该奖学金将在谢菲尔德（http：//www.example.com）的一个更广泛的倡议中设置，该倡议将基础科学和临床应用的研究人员聚集在一起，以应对AMR的挑战。www.floreyinstitute.com