Atomic-level probing of the peptidoglycan biosynthetic machinery in bacterial cell wall biogenesis

即使对最强大的抗生素也有抗药性的微生物的出现，对 全球公共卫生。肽聚糖(PG)合成的细菌机制的协调作用 对细菌生存至关重要的过程，代表了新抗生素开发的明显目标。 在这个拟议的项目中，我们的目标是定义PG降解组分的分子相互作用 由参与糖链水解或修饰的酶组成的装置。我们之前在 脑膜炎奈瑟菌显示，以单个裂解转糖基酶(LgtA)上的热点为靶点也会禁用 PG修饰酶APE1的功能导致PG组装中断，并导致 异常的肽多聚糖组成，使这种病原体无法在宿主中存活。我们的研究将 在分子细节上揭示这些肽聚糖降解酶是如何协同工作来组装 细菌细胞壁。具体地说，我们将以全面方式定义一个裂解网络是如何 转糖基酶(Ltd A、Ltd D、Ltd E)和它们的蛋白结合伙伴促进肽聚糖 细胞器的降解和插入细菌的细胞膜。在这个项目中，我们将利用 生物化学和生物学方法探索蛋白质-蛋白质和酶-底物相互作用 各种裂解的转糖基酶，结合活性的分子基础的测定 PG代谢中的多酶复合体。PG生物合成中各组分的遗传修饰 纳米机器将被用来检验我们从结构研究中观察到的结果。我们的方法，利用高 分辨率x射线结晶学工具和冷冻-EM单粒子分析将使可视化 酶在PG组装和降解中的作用，并应提供对其机制的见解 在细胞壁组装和细菌细胞分裂期间，在插入新PG的过程中精心安排的活动。我们的 研究将引领针对多种肽聚糖代谢的新疗法的开发 酵素。

The emergence of microbes resistant to even the most powerful antibiotics represents a serious threat to global public health. The coordinated action of the bacterial machinery of peptidoglycan (PG) synthesis, a process essential for bacterial viability, represents an obvious target for the development of new antibiotics. In this proposed project, we aim to define the molecular interactions of the components of the PG degradation apparatus, consisting of enzymes involved in glycan chain hydrolysis or modification. Our previous studies in Neisseria meningitidis showed that targeting a hot spot on a single lytic transglycosylase (LgtA) also disables the function of the PG-modifying enzyme, Ape1, leading to a disruption of PG assembly, and results in an aberrant peptidoglycan composition, making this pathogen unable to survive in the host. Our studies will reveal, in molecular detail, how these peptidoglycan degrading enzymes work in concert to assemble the bacterial cell wall. Specifically, we will define, in a comprehensive way, how a network of lytic transglycosylases (LtgA, LtgD, LtgE) and their protein binding partners work to facilitate peptidoglycan degradation and the insertion of organelles into the bacterial cell envelope. In this project, we will utilize biochemical and biological approaches to probe protein-protein and enzyme-substate interactions of the various lytic transglycosylases, combined with determination of the molecular basis of activity of the multienzyme complexes in PG metabolism. Genetic modifications of the components of the PG biosynthetic nanomachine will be used to test the observations from our structural studies. Our approach, utilizing high resolution x-ray crystallographic tools along with cryo-EM single particle analysis, will allow visualization of the action of enzymes in PG assembly and degradation and should provide mechanistic insights into their orchestrated activity during the insertion of new PG during cell wall assembly and bacterial cell division. Our studies will lead the way towards the development of new therapies targeting multiple peptidoglycan metabolic enzymes.