The mechanism of autolysin regulation in Vibrio cholerae

霍乱弧菌自溶素调节机制

基本信息

批准号：
10463655
负责人：
Tobias Doerr
金额：
$ 30.48万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
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/10463655
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 experimental study genome-wide human pathogen in vivo insight new therapeutic target novel paralogous gene pathogen rational design response

项目摘要

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保持细胞壁，一个必需的网状结构主要完成多糖肽聚糖（PG）。我们一些最强大的抗生素，βlactams（青霉素，碳青霉烯和头孢菌素）靶酶是细胞壁合成所需的酶，并从其能力中得出效率仅抑制细胞壁生物发生，但也可以主动引起其破坏。暴露后的细胞壁破坏 β乳酸乳酰胺由一组酶（amidass，裂解糖基酶和内肽酶）具有切割PG网格中各种化学键的能力。在正常生长下条件，自动素从事重要的细胞壁重塑功能，例如PG网格扩展细胞伸长；如何调节这些功能以确保理解适当的PG维护。我们已经表明，在腹泻病原体弧菌霍乱中，内肽酶（EPS）SHYA和SHYC为正常生长过程中细胞伸长所必需的未知），但也是暴露于βlacTAM抗生素后介导细胞壁破裂的关键因素。如何调节Shya和Shyc以确保在没有抗生素的情况下进行适当的细胞壁维护是未知。在这里，我们提出了实验，以建立对内肽酶机制的透彻理解在多个级别上进行的霍乱弧菌调节。由于M23 EP在整个细菌中保存良好，因此实验可能会产生与其他病原体具有广泛相关性的见解。在AIM 1中，我们将剖析 PG合成与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其活性。在目标2中，我们将精确地绘制EP中的结构功能关系，并发现其活动的其他调节因子。目标 3我们将通过金属稳态来确定EP调节的机理。两者一起，这些实验将为我们提供一个广泛的框架，即重要人类病原体如何保持平衡在细胞壁合成和重塑之间，目的是发现新的抗生素潜在靶标该调节自脂蛋白活动。