Structural and mechanistic insights into antibiotic resistance in Staphylococcus aureus

背景：抗生素耐药性（AR）破坏了有效的抗菌化疗，并对全球公共卫生构成重大威胁。对这一问题的全面回应包括对AR介导机制的详细了解。该项目旨在描述在“超级细菌”金黄色葡萄球菌中发现的几种抗生素抗性蛋白的结构和分子机制；抗性系统目前还没有得到很好的理解。首先，我们将重点关注能够介导氨基糖苷类或莫匹罗星耐药的非特征决定因素。目的：借助结构表征，阐明葡萄球菌耐药蛋白介导对作用于细菌翻译机制的临床重要抗生素耐药的分子机制。新颖性：尽管它们对抗生素的临床使用有不利影响，但目前尚不存在本文将要研究的耐药蛋白的结构或机制细节。这些信息对于制定克服或规避这些抵抗机制的战略至关重要。及时性：抗生素耐药性是目前全球关注的一个主要问题，被世界卫生组织认为是对人类健康的最大威胁之一，也是包括BBSRC在内的研究委员会优先资助的一个突出问题。结构方面的研究将采用低温电子显微镜，因此将利用国家的最先进的设施最近建立在利兹。实验方法：对于这两个最初的目标，我们已经可以获得纯化的可溶性蛋白质。对于两者中较大的，晶体学已经被证明是不成功的，因此我们将使用低温电镜进行结构表征。对于较小的蛋白质，我们在初步研究中已经能够生成需要优化的晶体-因此，在这种情况下，最初的努力将集中在获得x射线结构上。在这些研究中获得的结构见解将为下游诱变和生化实验提供信息，以询问功能。

Background: Antibiotic resistance (AR) undermines effective antibacterial chemotherapy and constitutes a major threat to global public health. A comprehensive response to this problem includes gaining a detailed understanding of the mechanisms by which AR is mediated. This project seeks to characterize the structural and molecular mechanism of several antibiotic resistance proteins found in the 'superbug', Staphylococcus aureus; resistance systems that are not currently well understood. In the first instance, we will focus on uncharacterized determinants capable of mediating resistance the aminoglycosides or mupirocin. Objectives: To elucidate, with the aid of structural characterization, the molecular mechanism by which staphylococcal resistance proteins mediate resistance to clinically-important antibiotics acting on the bacterial translation machinery.Novelty: Despite their adverse impact on the clinical use of antibiotics, no structural or mechanistic detail currently exists for the resistance proteins that will be studied here. Such information will be crucial to inform the development of strategies to overcome or circumvent these resistance mechanisms.Timeliness: Antibiotic resistance is a major global concern at present, considered by the WHO to be one of the greatest threats to human health, and features prominently in the funding priorities of the research councils, including the BBSRC. The structural aspects of the study will employ cryo-EM, and will therefore make use of the state-of-the art facilities recently established at Leeds.Experimental approach: For both initial targets of interest we already have access to purified, soluble protein. For the larger of the two, crystallography has already proven unsuccessful, and we will therefore use cryo-EM for structural characterization. For the smaller protein, we have in preliminary studies been able to generate crystals that require optimization - hence, the initial effort in this case will focus on obtaining an X-ray structure. The structural insights gained in these studies will inform downstream mutagenesis and biochemical experiments to interrogate function.