Radical SAM-dependent methylation in antibiotic resistance

抗生素耐药性中自由基 SAM 依赖性甲基化

基本信息

批准号：
10228618
负责人：
Danica Galonic Fujimori
金额：
$ 44.06万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-14 至 2023-05-21
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10228618
关键词：
Address Adenosine Affect Antibiotic Resistance Antibiotic susceptibility Antibiotics Antimicrobial Resistance Bacteria Bacterial Antibiotic Resistance Bacterial Infections Binding Centers for Disease Control and Prevention (U.S.)Cessation of life Characteristics Clinic Clinical Collaborations Defect Development Directed Molecular Evolution Dominant-Negative Mutation Enzymes Evolution Family Genetic Genetic Transcription Health Human Hypermethylation Impairment Infection Infection prevention Institutes Knowledge Laboratories Methylation Microbe Modification Molecular Multi-Drug Resistance Mutation Nucleotides Oxazolidinones Peptidyltransferase Phenotype Physiological Positioning Attribute Predisposition Prokaryotic Cells Protein Biosynthesis RNA, Ribosomal, 23S Regulation Resistance Resistance development Ribosomal RNA Ribosomes Role Site Streptogramins Testing Translation Process Translational Regulation Translations Treatment Failure Vancomycin resistant enterococcus Variant Work antibiotic resistant infections bacterial fitness diagnostic platform drug resistant pathogen experimental study fitness improved lincosamide member methicillin resistant Staphylococcus aureus pathogen pathogenic bacteria pathogenic microbe pleuromutilin prevent public health relevance resistance mechanism resistant strain

项目摘要

PROJECT SUMMARY The increasing occurrence of antibiotic resistant infections is a major threat to human health, necessitating understanding of mechanisms that confer resistance and development of strategies to counteract them. Antibiotics that bind to the peptidyltransferase center (PTC) of the bacterial ribosome interfere with protein synthesis in bacteria. However, some bacterial strains can modify the PTC region through mutations and post- transcriptional modifications of ribosomal RNA (rRNA), resulting in a ribosome that can no longer bind antibiotics. The multi-drug resistance enzyme Cfr, a member of radical SAM enzyme family, catalyzes methylation of 23S rRNA in the PTC region. This enzyme confers resistance to a number of antibiotics, such as phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A. The ability of Cfr to confer resistance to linezolide, an oxazolidinone antibiotic, is particularly worrisome as this antibiotic is used for the treatment of drug- resistant pathogens including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE). In pathogens, Cfr methylates adenosine A2503 at the C8 position. Interestingly, A2503 is also methylated at its C2 position by RlmN, a radical SAM enzyme that is highly conserved is prokaryotes. C2 A2503 methylation is implicated in the regulation of translational accuracy of the ribosome. A loss of physiological RlmN methylation, both in laboratory selection experiments and in clinical settings, causes antibiotic resistance. These findings suggest that aberrant A2503 methylation – both the absence of physiological methylation caused by inactivation of RlmN and the hypermethylation caused by acquisition of Cfr – profoundly impacts susceptibility of the bacterial ribosome to antibiotics. In this application, we will investigate how aberrant methylation of A2503 in 23S rRNA impacts antibiotic resistance and bacterial fitness. Using directed evolution and antibiotic selection, we have evolved variants of RlmN that prevent A2503 methylation and confer resistance to tiamulin. We will determine the molecular basis of the dominant negative effect of RlmN variants. Furthermore, we will investigate how the lack of C2 methylation of A2503 in ribosomes confers antibiotic resistance. Cfr variants, obtained by laboratory evolution or isolated from clinical antibiotic resistant strains, will be used to determine how changes in the sequence of this enzyme modulate methylation of A2503 and how these changes in methylation alter antibiotic susceptibility. We will further assess the impact of aberrant methylation on bacterial fitness and evaluate how changes in methylation influence the regulation of translation. Our work will define how radical SAM-dependent methylation of the PTC regulates the function of the ribosome and modulates its antibiotic susceptibility.

项目概要抗生素耐药性感染的日益增多是对人类健康的主要威胁，因此需要了解产生阻力的机制并制定应对策略。与细菌核糖体肽基转移酶中心 (PTC) 结合的抗生素会干扰蛋白质然而，一些细菌菌株可以通过突变和后处理来改变 PTC 区域。核糖体 RNA (rRNA) 的转录修饰，导致核糖体不再结合抗生素。多重耐药酶 Cfr 是自由基 SAM 酶家族的成员，催化 23S 甲基化 PTC 区域的 rRNA 赋予酶对多种抗生素（如苯酚）的抗性。林可酰胺类、恶唑烷酮类、截短侧耳素和链霉素 A。 Cfr 赋予耐药性的能力利奈唑胺是一种恶唑烷酮类抗生素，尤其令人担忧，因为这种抗生素用于治疗药物- 耐药病原体，包括耐甲氧西林金黄色葡萄球菌 (MRSA) 和耐万古霉素肠球菌 (VRE) 在病原体中，Cfr 在 C8 位点使腺苷 A2503 甲基化。在其 C2 位置被 RlmN（一种高度保守的自由基 SAM 酶）C2 A2503 甲基化。与核糖体翻译准确性的调节有关。生理性 RlmN 甲基化的丧失，在实验室选择实验和临床环境中，这些发现都会导致抗生素耐药性。表明异常的 A2503 甲基化 – 失活引起的生理甲基化缺失 RlmN 和获得 Cfr 引起的高甲基化——深刻影响细菌的敏感性核糖体到抗生素。在此应用中，我们将研究 23S rRNA 中 A2503 的异常甲基化如何影响抗生素利用定向进化和抗生素选择，我们进化出了不同的变体。 RlmN 阻止 A2503 甲基化并赋予泰妙菌素抗性，我们将确定其分子基础。此外，我们将研究 C2 甲基化的缺乏是如何产生的。核糖体中的 A2503 赋予抗生素抗性，通过实验室进化获得或分离。来自临床抗生素耐药菌株，将用于确定这种酶的序列如何变化调节 A2503 的甲基化以及这些甲基化的变化如何改变抗生素敏感性。进一步评估异常甲基化对细菌适应性的影响，并评估甲基化的变化如何我们的工作将定义 PTC 的 SAM 依赖性甲基化的彻底性。调节核糖体的功能并调节其抗生素敏感性。