The mechanism of autolysin regulation in Vibrio cholerae

霍乱弧菌自溶素调节机制

• 批准号: 9762289
• 负责人: Tobias Doerr
• 金额: $ 33.32万
• 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/9762289
• 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; 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; retinal rods

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.

项目摘要 大多数细菌都有细胞壁，这是一种基本的网状结构，主要由多糖组成。 肽聚糖(PG)。我们一些最有效的抗生素，β-内酰胺类(青霉素类、碳青霉烯类和 头孢菌素)靶向合成细胞壁所需的酶，并通过其不 既能抑制细胞壁的生物发生，又能主动造成其破坏。暴露于空气中的细胞壁破裂 β-内酰胺类药物由“自溶素”、一组酶(酰胺酶、裂解转糖基酶和 内肽酶)，具有切断PG网状结构中各种化学键的能力。在正常生长状态下 条件下，自溶素参与重要的细胞壁重塑功能，如在 细胞伸长；如何调节这些功能以确保适当的PG维持，目前还知之甚少。我们 已经表明，在腹泻病原体霍乱弧菌中，内肽酶(EPs)ShyA和ShyC是 在正常生长过程中细胞伸长所必需的(另一个同源基因ShyB的生理功能是 未知)，但也是导致暴露于β-内酰胺类抗生素后细胞壁破裂的关键因素。 如何调控ShyA和ShyC以确保在没有抗生素的情况下适当地维持细胞壁 未知。在这里，我们建议进行实验以建立对内肽酶机制的透彻理解。 霍乱弧菌的多层次调控。由于M23 EPs在细菌中保存良好，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功能关系。我们还将检验这一假设 ShyA和ShyC与细胞壁合成复合体的直接相互作用调节它们的活性。在《目标2》中我们将 精确绘制EPs的结构-功能关系图，并发现其活动的其他调节因素。在AIM 3我们将通过金属动态平衡来确定EP的调节机制。总而言之，这些实验 将为我们提供一个广泛的框架，了解一种重要的人类病原体如何维持平衡 在细胞壁合成和重塑之间，目标是发现抗生素的新潜在靶点 调节自溶酶活性的物质。