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

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，伴随着巨大的人力和经济成本。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知识。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，在药物存在的情况下允许细菌细胞壁的合成。特别的是，我们对MecA如何发挥其功能以及如何与细胞壁合成机器的其余部分一起发挥作用知之甚少。监督小组发现，金黄色葡萄球菌中MecA的存在本身并不会导致高水平的AMR，但会对细胞造成压力。这一悖论是通过细菌获得RNA聚合酶编码基因的突变并伴随着AMR的大量增加来解决的。从亲本株和高水平的AMR菌株中分离到RNA聚合酶。这揭示了RNA聚合酶的整体性质发生了变化。这些背景数据为拟议的项目建立了坚实的框架，将分子微生物学和宿主：病原体与蛋白质生物化学和结构生物学的相互作用结合起来，以解决一个具有重大社会意义的问题。实验方法：1.MecA是如何工作的？MecA本身会导致代谢压力，并且只在rpo背景下产生高水平的抗性。MecA与其他蛋白质的分子相互作用将通过细菌2杂交分析、荧光显微镜和蛋白质分析来确定。生化(高效液相色谱)和生物物理(AFM力曲线)相结合将阐明AMR对细胞壁特性的影响。RNA聚合酶在高水平的AMR中的作用是什么？我们已经使用RNA-SEQ表明，rpo突变导致一系列参与新陈代谢的基因表达的变化。我们的假设是AMR是由一种感知机制控制的。我们将纯化RNA聚合酶，并结合蛋白质化学和转录分析来确定突变对信号转导的影响。宿主环境的适应成本和作用是什么？我们第一次有了一组明确的只携带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进行直接比较，以确定体内AMR的成本。重要的是，我们已经定义了高水平的AMR与厌氧生长的能力有关。然而，在这种与体内相关的条件下，抗生素的效果从未被常规测试过。培训：该项目将提供从分子遗传学、蛋白质化学、高级显微镜方法、生物物理学到活体分析的令人兴奋的跨学科培训。该奖学金将在谢菲尔德(http://www.floreyinstitute.com))的一个更广泛的倡议范围内设立，该倡议将从基础科学到临床应用的研究人员聚集在一起，以应对AMR的挑战。

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.