The mechanism of autolysin regulation in Vibrio cholerae

霍乱弧菌自溶素调节机制

• 批准号: 10000951
• 负责人: Tobias Doerr
• 金额: $ 31.68万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000951
• 关键词: Active Sites; Affect; Aftercare; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Autolysin; Bacteria; Biochemical; Biogenesis; Biological Assay; Biological Models; Carbapenems; Cations; Cell Death; Cell Wall; Cells; Cephalosporins; Cholera; Complex; Crystallization; Cytolysis; Data; Dependence; Development; Diarrhea; Endopeptidases; Ensure; Enzymes; Equilibrium; Exposure to; Family; Genetic; Goals; Growth; Homeostasis; Housekeeping; In Vitro; Individual; Knowledge; LytA enzyme; Lytic; Maintenance; Maps; Mediating; Metals; Molecular Conformation; Monobactams; Pathway interactions; Penicillins; Peptidoglycan; Physiological; Play; Polysaccharides; Process; Regulation; Research; Resistance development; Rod; Role; Starvation; Structure; Structure-Activity Relationship; System; Testing; Up-Regulation; Vibrio cholerae; Virulence Factors; Zinc; amidase; antimicrobial; base; beta-Lactams; cell growth; cell killing; chemical bond; design; experimental study; genome-wide; human pathogen; in vivo; insight; new therapeutic target; novel; paralogous gene; pathogen; response

Project Summary Most bacteria maintain a cell wall, an essential, mesh-like structure mainly comprising the polysaccharide peptidoglycan (PG). Some of our most powerful antibiotics, the beta lactams (penicillins, carbapenems and cephalosporins) target enzymes required for cell wall synthesis and derive their efficacy from their ability to not only inhibit cell wall biogenesis, but also to actively cause its disruption. Cell wall disruption after exposure to beta lactams is mediated by “autolysins”, a group of enzymes (amidases, lytic transglycosylases and endopeptidase) with the capacity to cut a variety of chemical bonds within the PG mesh. Under normal growth conditions, autolysins engage in important cell wall remodeling functions, such as PG mesh expansion during cell elongation; how these functions are regulated to ensure proper PG maintenance is poorly understood. We have shown that in the diarrheal pathogen Vibrio cholerae, the endopeptidases (EPs) ShyA and ShyC are required for cell elongation during normal growth (the physiological functions of another paralogue, ShyB, are unknown), but are also key factors mediating cell wall breakdown after exposure to beta lactam antibiotics. How ShyA and ShyC are regulated to ensure proper cell wall maintenance in the absence of antibiotics is unknown. Here, we propose experiments to build a thorough understanding of mechanisms of endopeptidase regulation in V. cholerae on multiple levels. Since M23 EPs are well-conserved throughout Bacteria, our experiments will likely yield insights with broad relevance to other pathogens. In Aim 1, we will dissect the functional relationship between PG synthesis and degradation processes. We will also test the hypothesis that ShyA and ShyC's direct interaction with cell wall synthesis complexes regulates their activity. In Aim 2 we will precisely map structure-function relationships in EPs and discover additional regulators of their activity. In Aim 3 we will determine the mechanism of EP regulation by metal homeostasis. Taken together, these experiments will provide us with an extensive framework of how an important human pathogen maintains the balance between cell wall synthesis and remodeling, with the goal of discovering new potential targets for antibiotics that modulate autolysin activity.

项目概要 大多数细菌都有细胞壁，这是一种重要的网状结构，主要包含多糖 肽聚糖（PG）。我们的一些最强大的抗生素，β-内酰胺类（青霉素、碳青霉烯类和 头孢菌素）靶向细胞壁合成所需的酶，并从其不合成细胞壁的能力中获得功效。 不仅抑制细胞壁的生物发生，而且还主动引起其破坏。暴露后细胞壁破裂 β-内酰胺由“自溶素”介导，自溶素是一组酶（酰胺酶、裂解性转糖基酶和 肽链内切酶）能够切割 PG 网格内的各种化学键。正常生长情况下 条件下，自溶素参与重要的细胞壁重塑功能，例如 PG 网格扩张 细胞伸长；人们对如何调节这些功能以确保适当的 PG 维护知之甚少。我们 研究表明，在腹泻病原体霍乱弧菌中，内肽酶 (EP) ShyA 和 ShyC 正常生长期间细胞伸长所需的（另一个旁系同源物 ShyB 的生理功能是 未知），但也是接触β内酰胺抗生素后介导细胞壁分解的关键因素。 如何调节 ShyA 和 ShyC 以确保在没有抗生素的情况下适当的细胞壁维持 未知。在这里，我们提出实验来全面了解肽链内切酶的机制 霍乱弧菌在多个层面上的调节。由于 M23 EP 在细菌中得到了很好的保存，我们的 实验可能会产生与其他病原体广泛相关的见解。在目标 1 中，我们将剖析 PG合成和降解过程之间的函数关系。我们还将检验以下假设： ShyA 和 ShyC 与细胞壁合成复合物的直接相互作用调节其活性。在目标 2 中，我们将 精确绘制 EP 中的结构-功能关系，并发现其活性的其他调节因子。瞄准 3我们将确定金属稳态调节EP的机制。综合起来，这些实验 将为我们提供一个关于重要的人类病原体如何维持平衡的广泛框架 细胞壁合成和重塑之间的关系，旨在发现抗生素的新潜在靶点 调节自溶素活性。