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：
10736491
负责人：
Danica Galonic Fujimori
金额：
$ 52.68万
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 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10736491
关键词：
Adenosine Amino Acyl Transfer RNA Antibiotic Resistance Antibiotics Antimicrobial Resistance Bacteria Bacterial Infections Binding Binding Sites Biological Process Chloramphenicol Clinical Complex Consensus Cryoelectron Microscopy DNA Sequence Alteration Directed Molecular Evolution Elements Enzymes Escherichia coli Family Family member Funding Future Gene Expression Genes Geographic Distribution Gram-Negative Bacteria In Vitro Investigation Linezolid Macrolides Messenger RNA Methods Methylation Mobile Genetic Elements Modification Mutation Nature Nucleotides Open Reading Frames Orphan Oxazolidinones Peptides Peptidyltransferase Positioning Attribute Property Protein Family Protein Synthesis Inhibition Proteins Public Health RNA RNA methylation RNA, Ribosomal, 23S Regulation Resistance Resolution Ribosomal Proteins Ribosomal RNA Ribosomes Sequence Analysis Site Streptogramins Testing Translational Repression Validation Variant Work antimicrobial drug cost crosslink enzyme reconstitution enzyme substrate fitness florfenicol hygromycin A improved in vivo innovation lincosamide member methicillin resistant Staphylococcus aureus mutant next generation sequencing nucleoside analog pathogenic Escherichia coli reconstitution resistance gene resistance mechanism

项目摘要

PROJECT SUMMARY More than 40% of clinically used antibiotics act by binding to the ribosome and inhibiting protein synthesis. One of the major mechanisms of resistance to these antibiotics results from the modification of the ribosome catalyzed by Cfr, an enzyme encoded by chloramphenicol-florfenicol resistance gene. By methylating the C8 position of a conserved adenosine nucleotide in the peptidyl transferase center of the bacterial ribosome, this enzyme confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutillins, streptogramin A, hygromycin A, nucleoside analog A201A and 16-member macrolides. This broad cross-resistance is unique to Cfr, and represents a major clinical challenge, one that is further exacerbated by the presence of cfr on mobile genetic elements, low fitness cost of its acquisition and its broad geographic distribution, as well as the ability to cause resistance in both gram-positive (eg, methicillin-resistant S. aureus) and gram-negative bacteria (eg, pathogenic E. coli). Cfr family is represented by over 600 unique sequences, with some member of the family sharing only ~50% sequence identity with the commonly investigated Cfr(A) enzyme from a clinical MRSA isolate. To date, only a handful of Cfr enzymes have been functionally characterized. Recent work on the structural basis of inhibition of translation by chloramphenicol and linezolid, an oxazolidinone antibiotic, shows that both antibiotics inhibit protein synthesis by binding to the ribosome-nascent peptide complexes containing specific nascent peptide residues. Sequence-specific stalling mechanisms have been exploited in nature to regulate inducibility of antibiotic resistance genes. Since cfr is often accompanied by upstream elements that may regulate its expression, we will investigate if antibiotic-induced ribosome stalling mechanisms may be involved in regulation of the expression of cfr resistance genes. Using directed evolution under antibiotic selection, we have generated variants of Cfr with improved antibiotic resistance properties. By improving enzyme expression and stability, these enzyme variants increase ribosomal RNA methylation, leading to an increase in the proportion of the ribosomes that carry the protective modification. Improved methylation of the ribosome has enabled structural determination of the Cfr-modified ribosome, which we achieved using cryo-electron microscopy. The directed evolution mutants also provide a roadmap for our future efforts to functionally annotate additional putative members of the vast and sequence-diverse Cfr enzyme family. This will be achieved through in vitro reconstitution and in vivo validation of methylation of the conserved adenosine nucleotide. Additionally, we will deploy an innovative strategy that relies on mechanism-based crosslinking of Cfr with its substrates and next-generation sequencing of crosslinked RNAs to identify, with nucleotide resolution, the sites of RNA methylation. Together, these studies have a potential to de-orphan additional Cfr enzymes and lead to identification of new substrates and biological functions of this protein family.

项目概要超过40%的临床使用抗生素通过与核糖体结合并抑制蛋白质合成而发挥作用。一对这些抗生素产生耐药性的主要机制是由核糖体催化的修饰引起的 Cfr，一种由氯霉素-氟苯尼考抗性基因编码的酶。通过甲基化a的C8位置细菌核糖体肽基转移酶中心的保守腺苷核苷酸，该酶赋予对苯酚类、林可酰胺类、恶唑烷酮类、截短侧耳素、链霉素A、潮霉素A、核苷类似物 A201A 和 16 元大环内酯。这种广泛的交叉耐药性是 Cfr 所独有的，并且代表了一项重大的临床挑战，而移动遗传上 CFR 的存在进一步加剧了这一挑战要素，其收购适应成本低，地理分布广泛，以及引起革兰氏阳性菌（例如耐甲氧西林金黄色葡萄球菌）和革兰氏阴性菌（例如致病性金黄色葡萄球菌）均具有耐药性大肠杆菌）。 Cfr 家族由 600 多个独特序列代表，其中一些家族成员仅共享与临床 MRSA 分离株中常见研究的 Cfr(A) 酶具有约 50% 的序列同一性。迄今为止，只有少数 Cfr 酶的功能得到了表征。氯霉素和利奈唑胺（一种恶唑烷酮）抑制翻译的结构基础的最新研究抗生素，表明两种抗生素都通过与核糖体新生肽结合来抑制蛋白质合成含有特定新生肽残基的复合物。序列特定的停顿机制已被在自然界中被用来调节抗生素抗性基因的诱导能力。由于cfr经常伴随着可能调节其表达的上游元件，我们将研究抗生素是否诱导核糖体停滞机制可能涉及 CFR 抗性基因表达的调节。利用抗生素选择下的定向进化，我们生成了具有改进抗生素的 Cfr 变体电阻特性。通过改善酶的表达和稳定性，这些酶变体增加了核糖体 RNA 甲基化，导致携带保护性修饰的核糖体比例增加。核糖体甲基化的改善使得能够确定 Cfr 修饰核糖体的结构，这我们使用冷冻电子显微镜实现了这一点。定向进化突变体也为我们提供了路线图未来努力对庞大且序列多样化的 Cfr 酶的其他假定成员进行功能注释家庭。这将通过体外重构和体内保守甲基化验证来实现腺苷核苷酸。此外，我们将部署基于机制的创新战略。 Cfr 与其底物的交联以及交联 RNA 的新一代测序以鉴定核苷酸分辨率，RNA甲基化位点。总之，这些研究有可能消除孤儿现象额外的 Cfr 酶，并导致该蛋白质家族的新底物和生物功能的鉴定。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

miCLIP-MaPseq Identifies Substrates of Radical SAM RNA-Methylating Enzyme Using Mechanistic Cross-Linking and Mismatch Profiling.

DOI：
10.1007/978-1-0716-1374-0_7
发表时间：
2021
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Stojković V;Weinberg DE;Fujimori DG
通讯作者：
Fujimori DG

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics.

DOI：
10.1038/s41594-022-00723-9
发表时间：
2022-03
期刊：
Nature structural & molecular biology
影响因子：
16.8
作者：
Tsai K;Stojković V;Lee DJ;Young ID;Szal T;Klepacki D;Vázquez-Laslop N;Mankin AS;Fraser JS;Fujimori DG
通讯作者：
Fujimori DG

miCLIP-MaPseq, a Substrate Identification Approach for Radical SAM RNA Methylating Enzymes.