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

即使是对最强大的抗生素也有抗药性的微生物的出现，对人类的健康构成了严重威胁。 全球公共卫生。肽聚糖（PG）合成的细菌机制的协调作用， 细菌生存力所必需的过程，代表了新抗生素开发的明显目标。 在这个项目中，我们的目标是确定PG降解组分的分子相互作用 由参与聚糖链水解或修饰的酶组成的装置。我们以前的研究 脑膜炎奈瑟氏菌表明，靶向单一裂解性转糖基酶（LgtA）上的热点也使 PG修饰酶Ape 1的功能导致PG组装的破坏，并导致 异常的肽聚糖组成，使这种病原体无法在宿主中存活。我们的研究将 揭示，在分子细节，这些肽聚糖降解酶如何协同工作，以组装 细菌细胞壁具体地说，我们将全面地定义溶解性蛋白质网络是如何 糖基转移酶（LtgA、LtgD、LtgE）和它们的蛋白质结合配偶体起作用以促进肽聚糖 降解和细胞器插入细菌细胞包膜。在这个项目中，我们将利用 生物化学和生物学方法来探测蛋白质-蛋白质和酶-底物的相互作用， 各种溶解性转糖基酶，结合确定活性的分子基础， 多酶复合体参与PG代谢。PG生物合成酶组分的遗传修饰 纳米机器将用于测试我们结构研究的观察结果。我们的方法，利用高 分辨率X射线晶体学工具沿着冷冻EM单颗粒分析，将允许可视化 酶在PG组装和降解中的作用，并应提供对其 在细胞壁组装和细菌细胞分裂期间插入新PG期间的协调活动。我们 这些研究将为开发靶向多种肽聚糖代谢的新疗法开辟道路。 内切酶

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.