RNA modification and antibiotic resistance

描述（由申请人提供）：抗生素在细菌感染治疗中的应用彻底改变了现代医学实践。在此后的几十年里，不适当的控制使用和细菌种群对这些药物产生耐药性的显着能力的结合严重限制了许多抗生素的临床用途。我们现在正处于一个关键点，其中大多数药物都已知道，有时是广泛的耐药性，很少有新的替代品或策略来对抗耐药性问题。氨基糖苷类抗生素是在临床实践中广泛应用的一大类药物中的一个重要例子，但其中耐药性问题继续增加。非常令人关注的是，在过去几年中鉴定出具有广谱高水平耐药性的病原菌，这些耐药性是由修饰核糖体小亚基RNA上的氨基糖苷类结合位点的酶所赋予的。已经鉴定了两个不同的氨基糖苷类耐药性甲基转移酶家族，其修饰核苷酸G1405或A1408上的16 S核糖体RNA。这两种类型的功能类似的自我保护耐药酶早期在氨基糖苷类抗生素生产菌中被发现，人们认为这些基因已经横向转移到病原菌。然而，在这两种情况下，所涉及的抗性酶的结构和生物化学特征非常差。该提案描述了分为两个相关目标的实验，这些目标将直接解决这一缺陷。在目标1中，我们将确定来自氨基糖苷类生产菌和病原菌的两个氨基糖苷类耐药甲基转移酶家族（即靶向G1405和A1408的家族）成员的高分辨率X射线晶体结构。我们还将描述新发现的这种耐药性甲基转移酶家族的潜在成员，这些成员可能会提供进一步详细的了解这些酶在致病菌群中的起源。最后，对于每个酶家族，我们将使用诱变和等温滴定量热法（ITC）表征和比较必需甲基供体分子S-腺苷-L-甲硫氨酸（SAM）的结合。在目标2中，我们将充分剖析甲基转移酶-小核糖体亚基识别的机制，这代表了未来特异性酶抑制剂的最佳靶标。我们将确定关键氨基酸内的每一个酶的目标特异性结合和功能测定结合使用定点诱变，映射的对接位点上的小核糖体亚基使用结构探测和交联实验，并提供第一个高分辨率视图的抗生素抗性甲基转移酶结合到小核糖体亚基使用X射线晶体学。这些研究将为氨基糖苷类耐药甲基转移酶的结构和功能提供新的和根本性的重要见解，并将为未来开发任何新策略以对抗它们所赋予的耐药性奠定坚实的基础。 公共卫生相关性：耐药性增加是一个主要问题，可能会终止许多抗生素的临床用途，并从根本上改变我们治疗细菌感染的能力。我们的目标是确定化学改变特定RNA分子以阻止抗生素结合从而赋予耐药性的酶的分子结构和活性。这些研究对于了解细菌中抗生素耐药性的基本机制至关重要，并且有朝一日可能会支持新策略的开发，以延长当前抗生素的临床使用寿命或创造新的设计药物来对抗耐药细菌。

DESCRIPTION (provided by applicant): The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled use and the remarkable ability of bacterial populations to develop resistance to these drugs has severely restricted the clinical usefulness of many antibiotics. We are now at a critical point where the majority of these drugs have known and sometimes extensive resistance, and few novel replacements or strategies to combat the resistance problem exist. The aminoglycoside antibiotics are one important example of a large group of drugs with wide application in clinical practice but where the problem of resistance continues to increase. Of great concern is the identification over the last several years of pathogenic bacteria with broad-spectrum high-level resistance conferred by enzymes that modify the aminoglycoside binding site on the RNA of the small ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S ribosomal RNA on nucleotide G1405 or A1408. Functionally analogous self-protection resistance enzymes of both types were earlier identified in aminoglycoside-producing bacteria, and it is thought that these genes have laterally transferred to the pathogenic bacteria. However, in both cases the resistance enzymes involved are very poorly structurally and biochemically characterized. This proposal describes experiments grouped into two connected aims that will directly address this deficiency. In Aim 1, we will determine the high-resolution X-ray crystal structures of members of both families of aminoglycoside-resistance methyltransferases (i.e. those targeting G1405 and A1408) from both aminoglycoside-producers and pathogenic bacteria. We will also characterize newly identified potential members of this resistance methyltransferase family that may provide further detailed insights into the origins of these enzymes in pathogenic bacterial populations. Finally, for each enzyme family, we will characterize and compare the binding of the essential methyl group donor molecule S-adenosyl-L-methionine (SAM) using mutagenesis and isothermal titration calorimetry (ITC). In Aim 2, we will fully dissect the mechanism of methyltransferase- small ribosome subunit recognition, which represents the best target for future specific enzyme inhibitors. We will determine critical amino acids within each enzyme for target specificity using site-directed mutagenesis in combination with binding and functional assays, map the docking site on the small ribosome subunit using structure probing and cross-linking experiments, and provide the first high-resolution view of an antibiotic resistance methyltransferase enzyme bound to the small ribosome subunit using X-ray crystallography. Together these studies will provide novel and fundamentally important insights into the structure and function of aminoglycoside-resistance methyltransferases and will lay a secure foundation for any future development of new strategies to counter the resistance they confer. PUBLIC HEALTH RELEVANCE: Increasing resistance is a major problem that threatens to end the clinical usefulness of many antibiotics and fundamentally alter our ability to treat bacterial infections. Our goal is to define the molecular structures and activities of enzymes that chemically alter specific RNA molecules to prevent antibiotic binding and thereby confer resistance. Such studies are essential to understand basic mechanisms of antibiotic resistance in bacteria and may one day underpin the development of new strategies to extend the clinically useful life of current antibiotics or to create novel designer drugs to combat resistant bacteria.