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 抗生素耐药性感染的日益增加是对人类健康的主要威胁， 了解产生耐药性的机制，并制定消除耐药性的战略。 抗生素与细菌核糖体的肽基转移酶中心（PTC）结合， 在细菌中合成。然而，一些细菌菌株可以通过突变和后修饰来修饰PTC区域。 核糖体RNA（rRNA）的转录修饰，导致核糖体不再能结合抗生素。 多药耐药酶Cfr是自由基SAM酶家族成员，催化23 S甲基化 PTC区域的rRNA。这种酶赋予对许多抗生素的抗性，如酚类， 林可酰胺、恶唑烷酮、截短侧耳素和链阳性菌素A。Cfr赋予抗 利奈唑胺是一种恶唑烷酮抗生素，特别令人担忧，因为这种抗生素用于治疗药物- 包括耐甲氧西林S.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位置甲基化腺苷A2503。有趣的是，A2503也被甲基化， 在其C2位置被RlmN取代，是原核生物中高度保守的自由基SAM酶。C2 A2503甲基化 与核糖体翻译准确性的调节有关。生理RlmN甲基化的丧失， 无论是在实验室选择实验中还是在临床环境中，都会导致抗生素耐药性。这些发现 提示异常A2503甲基化--既由于失活引起的生理甲基化的缺失 RlmN和Cfr获得引起的超甲基化-深刻影响细菌的易感性， 核糖体到抗生素。 在本申请中，我们将研究23 S rRNA中A2503的异常甲基化如何影响抗生素 抗性和细菌适应性。利用定向进化和抗生素选择，我们已经进化出了 RlmN，其防止A2503甲基化并赋予对泰妙菌素的抗性。我们将确定分子基础 RlmN变体的显性负面影响。此外，我们还将研究C2甲基化的缺乏如何影响 核糖体中A2503的表达赋予抗生素抗性。Cfr变体，通过实验室进化获得或分离 从临床抗生素耐药菌株，将被用来确定如何改变这种酶的序列， 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， 影响翻译规范。我们的工作将确定PTC的SAM依赖性甲基化 调节核糖体的功能并调节其抗生素敏感性。