Radical SAM-dependent methylation in antibiotic resistance

项目总结 超过40%的临床使用的抗生素通过与核糖体结合并抑制蛋白质合成来发挥作用。一 对这些抗生素产生耐药性的主要机制是由催化的核糖体修饰引起的 由氯霉素-氟苯尼考耐药基因编码的cfr。通过甲基化一个分子的C8位 在细菌核糖体的肽基转移酶中心保守的腺苷核苷酸，这种酶赋予 对青霉素、林可酰胺、恶唑烷酮类、截胸菌素类、链菌素A、潮霉素A、 核苷类似物A201A和16元大环内酯类化合物。这种广泛的交叉阻力是CFR独有的，而且 这是一个重大的临床挑战，移动基因上的CFR的存在进一步加剧了这一挑战 要素，其收购的低适应成本和广泛的地理分布，以及导致 革兰氏阳性菌(如耐甲氧西林金黄色葡萄球菌)和革兰氏阴性菌(如致病菌)的耐药性 E.Coli)。CFR家族由600多个独特的序列表示，该家族中的一些成员仅共享 与临床分离的耐甲氧西林金黄色葡萄球菌的CFR(A)酶有50%的同源性。到目前为止， 只有少数几种CFR酶被功能鉴定。 氯霉素和恶唑烷酮类药物抑制翻译的结构基础研究进展 抗生素，表明这两种抗生素都通过与核糖体新生多肽结合来抑制蛋白质合成。 含有特定新生多肽残基的复合体。特定于序列的拖延机制已经被 在自然界中被用来调节抗生素耐药性基因的诱导性。因为CFR通常伴随着 可能调节其表达的上游元件，我们将调查抗生素是否导致核糖体停滞 其机制可能与CFR抗性基因的表达调控有关。 使用抗生素选择下的定向进化，我们已经产生了带有改进抗生素的CFR变体 阻力特性。通过提高酶的表达和稳定性，这些酶变异体增加了核糖体 RNA甲基化，导致携带保护性修饰的核糖体比例增加。 核糖体甲基化的改进使得能够确定CFR修饰的核糖体的结构，这 我们用低温电子显微镜实现了这一点。定向进化突变体也为我们的 未来努力从功能上注释大量和序列多样化的CFR酶的其他推定成员 一家人。这将通过体外重组和体内验证保守的甲基化来实现 腺苷核苷酸。此外，我们还将部署以机制为基础的创新战略 CFR与其底物的交联化和下一代交联型RNA测序以鉴定 核苷酸解析，RNA甲基化的位点。总而言之，这些研究有可能消除孤儿 其他CFR酶，并导致鉴定新的底物和该蛋白家族的生物学功能。

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.