Radical SAM-dependent methylation in antibiotic resistance

抗生素耐药性中自由基 SAM 依赖性甲基化

• 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
• 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

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.

项目摘要 抗生素耐药感染的发生越来越多是对人类健康的主要威胁，必要 了解会议抵抗和制定战略来抵制它们的机制。 结合细菌核糖体干扰蛋白质的肽基转移酶中心（PTC）的抗生素 细菌中的合成。但是，某些细菌菌株可以通过突变和 - 核糖体RNA（RRNA）的转录修饰，导致核糖体无法再结合抗生素。 多药抗性酶CFR是自由基SAM酶家族的成员，催化23S的甲基化 PTC区域中的rRNA。这种酶赋予了对许多抗生素的抗药性，例如苯酚， Lincosamides，黄唑啉酮，胸膜素和链霉素A. CFR对会议抗性的能力 linezolide是一种抗生素，尤其令人担忧，因为该抗生素用于治疗药物 包括耐甲氧西林的金黄色葡萄球菌（MRSA）和万古霉素抗肠球菌的抗性病原体 （vre）。在病原体中，CFR甲基化腺苷A2503处于C8位置。有趣的是，A2503也是甲基化的 在RLMN的C2位置，高度保守的自由基SAM酶是原核生物。 C2 A2503甲基化 在调节核糖体的翻译准确性中暗示。物理RLMN甲基化的损失， 在实验室选择实验和临床环境中，都会引起抗生素耐药性。这些发现 表明异常A2503甲基化 - 既没有由失活引起的物理甲基化 RLMN和由CFR获得引起的高甲基化 - 深刻影响细菌的敏感性 核糖体至抗生素。 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 耐药性和细菌适应性。使用定向进化和抗生素选择，我们已经进化了变体 防止A2503甲基化和对tiamulin的会议抗性的RLMN。我们将确定分子基础 RLMN变体的主要负面影响。此外，我们将研究缺乏C2甲基化 核糖体中的A2503承认抗生素耐药性。 CFR变体，通过实验室进化或孤立获得 从临床抗生素抗性菌株中，将用于确定该酶序列的变化如何变化 调节A2503的甲基化以及甲基化的这些变化如何改变抗生素易感性。我们将 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 影响翻译的调节。我们的工作将定义PTC的根治性SAM依赖性甲基化 调节核糖体的功能，并调节其抗生素易感性。