The mechanism of autolysin regulation in Vibrio cholerae

霍乱弧菌自溶素调节机制

• 批准号: 10238099
• 负责人: Tobias Doerr
• 金额: $ 30.52万
• 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/10238099
• 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，并从它们的能力中获得它们的功效， 不仅抑制细胞壁的生物合成，而且积极地引起其破坏。暴露于以下物质后细胞壁破裂 β-内酰胺是由“自溶素”介导的，“自溶素”是一组酶（酰胺酶、溶解性转糖基酶和 内肽酶），具有切割PG网内各种化学键的能力。在正常增长下 在某些条件下，自溶素参与重要的细胞壁重塑功能，如PG网扩张， 细胞伸长;这些功能如何调节以确保适当的PG维持知之甚少。我们 已经表明，在霍乱病原体霍乱弧菌中，内肽酶（EPs）ShyA和ShyC是 在正常生长过程中细胞伸长所需的蛋白质（另一种parasites，ShyB的生理功能， 未知），但也是暴露于β内酰胺抗生素后介导细胞壁破裂的关键因素。 ShyA和ShyC是如何调节的，以确保在没有抗生素的情况下保持适当的细胞壁， 未知在这里，我们提出了实验，以建立一个彻底的了解内肽酶的机制， 在多个水平上调节霍乱弧菌。由于M23 EP在整个细菌中非常保守，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它们的活性。在目标2中， 精确绘制EP的结构-功能关系，并发现其活性的其他调节因子。在Aim中 3.探讨金属稳态对EP的调节机制。综合来看，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， 细胞壁合成和重塑之间的联系，目的是发现抗生素的新的潜在靶点 调节自溶素活性。