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，控制不当的使用和细菌种群对这些药物产生耐药性的显著能力相结合，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时刻，大多数这些药物已经知道，有时广泛的耐药，很少有新的替代品或策略来对抗耐药问题。氨基糖苷类抗生素是在临床实践中广泛应用但耐药性问题持续增加的一大类药物的一个重要例子。在过去的几年里，人们非常关注的是鉴定出具有广谱高水平耐药性的致病菌，这种耐药性是由修饰小核糖体亚基RNA上氨基糖苷结合位点的酶赋予的。已经鉴定出两个不同的氨基糖苷抗性甲基转移酶家族，它们修饰核苷酸G1405或A1408上的16S核糖体RNA。早前在氨基糖苷产生细菌中发现了功能类似的两种类型的自我保护抗性酶，并且认为这些基因已横向转移到致病菌中。然而，在这两种情况下，抗性酶的结构和生物化学特征都很差。本提案描述了分为两个相关目标的实验，这将直接解决这一缺陷。在Aim 1中，我们将从氨基糖苷产生者和致病菌中确定氨基糖苷耐药甲基转移酶（即靶向G1405和A1408的甲基转移酶）两个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耐药甲基转移酶家族的潜在成员，这可能为这些酶在致病细菌群体中的起源提供更详细的见解。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）来表征和比较必需甲基供体分子s -腺苷- l-蛋氨酸（SAM）的结合。在Aim 2中，我们将全面剖析甲基转移酶-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点。我们将利用位点定向诱变结合结合和功能分析来确定每种酶中用于靶特异性的关键氨基酸，利用结构探测和交联实验绘制小核糖体亚基上的对接位点，并利用x射线晶体学提供与小核糖体亚基结合的抗生素耐药性甲基转移酶的第一个高分辨率视图。总之，这些研究将为氨基糖苷耐药甲基转移酶的结构和功能提供新颖而重要的见解，并将为未来开发新的策略来对抗它们所赋予的耐药性奠定坚实的基础。

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.