Radical SAM-dependent methylation in antibiotic resistance

项目总结 越来越多的抗生素耐药感染是对人类健康的主要威胁，需要 了解产生抵抗力的机制，并制定对抗它们的策略。 与细菌核糖体的肽基转移酶中心(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突变来修饰PTC区域。 核糖体RNA(RRNA)的转录修饰，导致核糖体不再与抗生素结合。 多药耐药酶CFR是自由基SAM酶家族的成员，催化23S的甲基化 PTC区域的rRNA。这种酶对许多抗生素产生抗药性，如苯尼考， 林可酰胺类、恶唑烷酮类、胸膜多菌素类和链霉菌素A。CFR的抗药性 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位置甲基化腺苷A2503。有趣的是，A2503也是甲基化的 在R1mN的C2位，一个高度保守的自由基SAM酶是原核生物。C2 A2503甲基化 与核糖体翻译准确性的调节有关。生理性RlmN甲基化缺失， 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A2503甲基化异常--既有生理性甲基化缺失所致的失活 RlmN和获得CFR引起的高甲基化-深刻影响细菌的敏感性 从核糖体到抗生素。 在这个应用中，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。使用定向进化和抗生素选择，我们已经进化出 防止A2503甲基化并对田氨蛋白产生抵抗力的RlmN。我们将确定分子基础 RlmN变异体的显性负效应。此外，我们将研究C2甲基化缺失是如何 核糖体中的A2503的突变可产生抗药性。通过实验室进化或分离获得的CFR变异体 来自临床的抗生素耐药菌株，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的敏感性。我们会 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 影响翻译的规范。我们的工作将定义PTC的自由基SAM依赖的甲基化如何 调节核糖体的功能并调节其对抗生素的敏感性。

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.