Cell Wall Formation in Rod Shaped Bacteria

研究目的细菌被网状细胞壁包围，由一种称为肽聚糖的聚合物制成，这赋予了它们的形状，并保护它们不会因内部高膨压而破裂。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物质来生长。这主要是由一个多蛋白质复合物，称为延长体，它通过精确插入新的细胞壁材料来保持细胞的杆状。长体的失活导致杆状的损失和严重受损的细胞适应性。抗生素耐药性现在是现代医学的一个主要威胁，促使人们迫切寻找从根本上攻击细菌细胞壁形成的新方法。延长体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，并且一起，延长体和分裂体蛋白代表细菌细胞壁的主要建筑师。因此，对延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，而且还可能确定新的路线，以下一代抗生素为靶点，针对延长体和分裂体细胞壁合成蛋白，并对广泛的细菌有效。在这个项目中，我们将确定从原子到大分子尺度的延长体功能和调节的基础。这将通过先进的分子成像技术、先进的生物化学和结构生物学分析、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来实现，这是一个从根本上综合的团队科学研究计划。coli RodA-PBP 2核心的延伸体，为理解延伸体功能的分子机制提供了基础性的基础-我们已经开发了一系列新的方法来实现这个项目，包括基于延伸体底物模拟物的新的化学生物学工具，多蛋白质复合物的创新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，使我们的理解的延伸体和细菌细胞壁合成的分子基础的重大进展。物有所值高度集成的团队科学工作包在这里提出将使细菌细胞壁生物学的重大进展，根本不可能在一个普通的响应模型的建议。直接附加值是由持续的战略投资提供在沃里克建立一个卓越的细菌细胞包膜生物学的国家中心，包括：-招聘Stansfeld（2019年）和霍尔顿（2022年）- £ 5400万投资在新的实验室综合体所占用的所有沃里克申请人，-一个£ 170万的慈善捐款为霍华德道尔顿研究中心提供了基础支持细胞壁研究和资金£ 120 K smFRET仪器在此大量使用本提案。团队科学极大地促进了沃里克申请人在同一新建筑的位置，并建立了所有沃里克和贝尔法斯特申请人之间的合作。结果-基本知识的获得：该提案方法将揭示对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建筑师。- 在生物医学和生物技术中的潜在应用：这将为未来通过SEDS蛋白质靶向细胞壁合成的抗菌疗法奠定基础。- 工作人员培训：我们将培养一批跨学科的博士前和博士后科学家，他们专注于微生物学领域，迫切需要新的人才，并使未来几代人能够在学术界和工业界进行抗菌研究。

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.