Cell Wall Formation in Rod Shaped Bacteria

研究目的细菌被网状细胞壁包围，这种细胞壁由一种名为肽聚糖的聚合物制成，这种聚合物赋予细菌形状，防止它们因内部膨胀压力过高而破裂。在许多杆状细菌中，包括大肠杆菌、沙门氏菌和铜绿假单胞菌等病原体，细胞通过在细胞侧壁周围添加新材料来生长。这主要是由一种名为细长体的多蛋白质复合体完成的，该复合体通过精确插入新的细胞壁材料来保持细胞的杆状。伸长体失活会导致杆状结构的丧失，严重损害细胞的适合性。抗菌素耐药性现在是现代医学的主要威胁，促使人们迫切寻求从根本上新的方法来攻击细菌细胞壁的形成。Elongasome蛋白在结构上与分裂细胞的分裂体细胞壁合成蛋白非常相似，细长体和分裂体蛋白一起代表细菌细胞壁的主要构建者。因此，对伸长体功能的分子基础的研究不仅将促进我们对细菌细胞壁是如何构建的理解，而且还可能发现针对伸长体和分裂体细胞壁合成蛋白的下一代抗生素的新途径，并且对广泛的细菌有效。在本项目中，我们将从原子到大分子的尺度确定伸长体的功能和调节的基础。这将利用先进的分子成像技术、先进的生化和结构生物学分析、尖端的计算分析和模拟方法以及定制的化学探针之间的协同作用，在一个根本上一体化的团队-科学研究计划中实现。TIMELINESS-我们最近解决了细长体的E.ColiRodA-PBP2核心的结构，为理解细长体功能的分子机制提供了基础-我们开发了一系列新的实现该项目的方法，包括基于细长体底物模拟的新化学生物工具，创新的多蛋白质复合体的分子模拟和细胞壁合成蛋白质的单分子生物物理学的新方法-这使我们处于独特的地位，可以在我们对细长体和细菌细胞壁合成的分子基础的理解方面取得重大进展。MONEY的价值这里提出的高度集成的团队科学工作包将使细菌细胞壁生物学的重大进展成为可能，这在普通的响应模型建议中是不可能的。直接附加值是由华威持续的战略投资提供的，以建设一个国家细菌细胞被膜生物学的卓越中心，包括：-招募Stansfeld(2019年)和Holden(2022年)-在所有Warwick申请者占用的新实验室综合体上投资5400万GB，-为Howard Dalton研究中心提供170万GB的慈善捐赠，该中心为细胞壁研究提供支持，并为本提案中大量使用的GB 120K smFRET仪器提供资金。团队科学大大促进了沃里克申请者在同一座新建筑中的位置，以及所有沃里克和贝尔法斯特申请者之间建立的合作。结果-基础知识的获得：这一提议的方法将揭示对SEDS-PBP蛋白的分子功能和调控的广泛见解，SEDS-PBP蛋白是细菌细胞壁的主要构造者。-在生物医学和生物技术方面的潜在应用：这将为未来针对SEDS蛋白质合成细胞壁的抗菌疗法奠定基础。-工作人员培训：我们将培训一批跨学科的博士后和博士后科学家，专注于迫切需要新人才的微生物学领域，并使学术界和工业界的未来几代抗菌研究成为可能。

PURPOSE OF RESEARCHBacteria are surrounded by a mesh-like cell wall, made out of a polymer called peptidoglycan, which gives them their shape and protects them from bursting due to high internal turgor pressure. In many rod-shaped bacteria, including pathogens such as Escherichia coli, Salmonella species and Pseudomonas aeruginosa, cells grow by adding new material around the cell sidewall. This is principally performed by a multi-protein complex called the elongasome which maintains the cell's rod shape by precise insertion of new cell wall material. Inactivation of the elongasome leads to loss of rod-shape and severely impaired cell fitness. Antimicrobial resistance now represents a major threat to modern medicine, motivating the urgent search for fundamentally new ways to attack the formation of the bacterial cell wall. Elongasome proteins are structurally very similar to the divisome cell wall synthesis proteins that divide the cell, and together, elongasome and divisome proteins represent the primary architects of the bacterial cell wall. Therefore, the research into the molecular basis of elongasome function proposed will not only advance our understanding of how the bacterial cell wall is built but may also identify new routes to next generation antibiotics that target elongasome and divisome cell wall synthesis proteins and are effective against a wide range of bacteria.In this project we will determine the basis of elongasome function and regulation from an atomic to macromolecular scale. This will be achieved using synergy between advanced molecular imaging techniques, advanced biochemical and structural biology analysis, cutting-edge computational analysis and simulation methods and bespoke chemical probes, in a fundamentally integrative team-science research programme.TIMELINESS- We recently solved the structure of the E. coli RodA-PBP2 core of the elongasome, providing a foundational basis for understanding the molecular mechanism of elongasome function- We have developed a repertoire of new approaches to realise this project including new chemical biology tools based on elongasome substrate mimetics, innovative molecular simulations of multi-protein complexes and new methods for single molecule biophysics of cell wall synthesis proteins- This makes us uniquely placed to make major advances in our understanding of the elongasome and the molecular basis of bacterial cell wall synthesis.VALUE FOR MONEYThe highly integrated team science work packages proposed here will enable major advances in bacterial cell wall biology that are simply not possible in an ordinary responsive model proposal. Direct added value is provided by sustained strategic investment at Warwick to build a national centre of excellence in bacterial cell envelope biology, including:- Recruitment of Stansfeld (2019) and Holden (2022) - £54M investment in the new laboratory complex occupied by all Warwick applicants, - A £1.7M charitable donation for a Howard Dalton research centre which provides underpinning support for cell wall research and funds a £120K smFRET instrument heavily used in this in this proposal. Team science is greatly facilitated by location of Warwick applicants in the same new building and established collaborations between all Warwick and Belfast applicants. OUTCOMES-Fundamental knowledge gain: This proposal approach will reveal broad insights into the molecular function and regulation of the SEDS-PBP proteins that are the major architects of the bacterial cell wall. -Potential applications in biomedicine and biotechnology: This will lay the foundations for future antimicrobial therapies targeting cell wall synthesis by SEDS proteins. -Staff training: We will train a cohort of interdisciplinary pre and postdoctoral scientists orientated in an area of microbiology that desperately needs new talent and enable future generations of antimicrobial research in academia and industry.