RNA modification and antibiotic resistance

描述(申请人提供)：应用抗生素治疗细菌感染使现代医疗实践发生了革命性的变化。在此后的几十年里，不适当的使用控制和细菌群体对这些药物产生抗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。我们现在正处于这样一个关键时刻，这些药物中的大多数都已知，有时还存在广泛的耐药性，几乎没有新的替代药物或战略来应对耐药性问题。氨基糖苷类抗生素是一大类药物中的一个重要例子，在临床实践中得到广泛应用，但耐药问题仍在不断增加。在过去的几年里，人们非常关注的是对具有广谱高水平耐药性的病原菌的鉴定，这些细菌是由修饰核糖体小亚基RNA上的氨基糖苷结合部位的酶所产生的。已鉴定出两个不同的氨基糖苷类耐药甲基转移酶家族，它们修饰核苷酸G1405或A1408上的16S核糖体RNA。在氨基糖苷类细菌中发现了两种功能相似的自我保护耐药酶，并认为这些基因已横向转移到病原菌。然而，在这两种情况下，涉及的抗性酶在结构和生化特征上都很差。这项提案描述了将直接解决这一缺陷的两个相互关联的目标的实验。在目标1中，我们将确定两个氨基糖苷耐药甲基转移酶家族成员(即针对G1405和A1408的成员)的高分辨X射线晶体结构。我们还将表征新发现的这个耐药甲基转移酶家族的潜在成员，这可能会为这些酶在病原细菌种群中的起源提供进一步的详细见解。最后，对于每个酶家族，我们将使用诱变和恒温滴定量热法(ITC)来表征和比较必需的甲基供体分子S-腺苷-L-蛋氨酸(SAM)的结合。在目标2中，我们将全面剖析甲基转移酶-核糖体小亚基识别的机制，这是未来特异性酶抑制剂的最佳靶点。我们将使用结合和功能分析相结合的定点突变来确定每个酶中的关键氨基酸，使用结构探测和交联实验绘制小核糖体亚单位上的对接位置，并使用X射线结晶学提供与小核糖体亚单位结合的抗生素抗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类耐药甲基转移酶的结构和功能提供新的和根本上重要的见解，并将为未来任何新策略的开发奠定可靠的基础，以对抗它们带来的耐药性。

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.