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

细菌细胞壁生物发生中肽聚糖生物合成机制的原子水平探测

• 批准号: 10685947
• 负责人: Allison H Williams
• 金额: $ 41.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685947
• 关键词: Acetylesterase; Affinity Chromatography; Antibiotics; Bacteria; Bacterial Physiology; Binding; Binding Proteins; Biochemical; Biochemical Reaction; Biogenesis; Biological; Biological Assay; Biophysics; Cell Wall; Cell division; Cells; Communication; Complement; Complex; Coupled; Cryoelectron Microscopy; Data; Development; Drug resistance; Enzymes; Future; Genetic; Goals; Gram-Negative Bacteria; Hot Spot; Hydrolysis; Infection; Intracellular Transport; Lytic; Maps; Mass Spectrum Analysis; Membrane; Metabolic; Metabolism; Microbe; Modification; Molecular; Molecular Conformation; Multienzyme Complexes; Nature; Neisseria; Neisseria gonorrhoeae; Neisseria meningitidis; Organelles; Penicillin-Binding Proteins; Peptidoglycan; Peptidoglycan glycosyltransferase; Pilum; Polymerase; Polysaccharides; Positioning Attribute; Process; Productivity; Protein-Protein Interaction Map; Proteins; Public Health; Resistance; Resolution; Role; Site; Structure; Testing; Time; Validation; Visualization; Work; X-Ray Crystallography; analog; antimicrobial; biochemical tools; cell envelope; cell motility; drug development; fighting; human pathogen; insight; macromolecule; member; nanobodies; nanomachine; new therapeutic target; next generation; particle; pathogen; pathogenic bacteria; protein complex; structural biology; tool

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.

即使对最强大的抗生素也产生抗药性的微生物的出现对人类构成了严重威胁。 全球公共卫生。肽聚糖（PG）合成细菌机制的协调作用， 对于细菌活力至关重要的过程，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 降解成分的分子相互作用 装置，由参与聚糖链水解或修饰的酶组成。我们之前的研究 脑膜炎奈瑟菌表明，针对单个裂解性转糖基酶 (LgtA) 的热点也会使 PG 修饰酶 Ape1 的功能会破坏 PG 组装，并导致 异常的肽聚糖组成，使得这种病原体无法在宿主体内生存。我们的研究将 以分子细节揭示这些肽聚糖降解酶如何协同工作以组装 细菌细胞壁。具体来说，我们将以全面的方式定义裂解网络如何 转糖基酶（LtgA、LtgD、LtgE）及其蛋白质结合伴侣有助于促进肽聚糖的形成 降解以及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-底物相互作用 各种裂解性转糖基酶，并结合其活性的分子基础的测定 PG代谢中的多酶复合物。 PG生物合成成分的基因修饰 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 射线晶体学工具与冷冻电镜单粒子分析一起，将允许可视化 酶在 PG 组装和降解中的作用，并应提供对其的机制见解 在细胞壁组装和细菌细胞分裂期间插入新 PG 期间精心策划的活动。我们的 研究将引领针对多种肽聚糖代谢的新疗法的开发 酶。