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 在细菌中合成。然而，一些细菌菌株可以通过突变和后处理来修改 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。这种酶赋予对多种抗生素的抗性，例如苯酚、 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 位点将腺苷 A2503 甲基化。有趣的是，A2503 也被甲基化 在其 C2 位置被 RlmN 取代，RlmN 是原核生物中高度保守的自由基 SAM 酶。 C2 A2503 甲基化 涉及核糖体翻译准确性的调节。生理学 RlmN 甲基化的丧失， 在实验室选择实验和临床环境中，都会导致抗生素耐药性。这些发现 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 抵抗力和细菌适应性。利用定向进化和抗生素选择，我们进化出了以下变体 RlmN 可防止 A2503 甲基化并赋予泰妙菌素抗性。我们将确定分子基础 RlmN 变体的显性负面影响。此外，我们将研究 C2 甲基化的缺乏如何 核糖体中 A2503 的存在会赋予抗生素耐药性。 Cfr 变异体，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。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如何 影响翻译的规范。我们的工作将定义 PTC 的 SAM 依赖性甲基化是如何激进的 调节核糖体的功能并调节其抗生素敏感性。