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。我们现在正处于一个关键时刻，大多数有用的抗生素都已知，有时还存在广泛的耐药性，但几乎没有新的替代品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，这些药物对关键的人类病原体具有保留的活性，但耐药性问题继续增加。特别令人关切的是，在过去十年中，越来越多的病原菌含有异常广谱和高水平的氨基糖苷类耐药性决定因素。这些16S核糖体RNA(RRNA)甲基转移酶化学修饰细菌核糖体小亚基(30S)16S rRNA上的氨基糖苷结合部位。已鉴定出两个不同的氨基糖苷抗性甲基转移酶家族，它们分别修饰核苷鸟苷1405或腺苷1408上的16S rRNA，从而分别产生m7G1405和m1A1408修饰。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。我们实验室和其他实验室最近的研究揭示了每个酶的分子结构，既有药物产生又有致病来源，并提供了与其底物结合的m1A1408甲基转移酶的第一个视图。然而，更多的证据表明，该酶家族可能使用不同的机制来完成底物的专一性和修饰。此外，关于m7G1405如何实现同等的分子功能还知之甚少。这项提案描述了两个相互关联的目标的实验，这些实验将直接解决这些不足。在目标1中，我们将使用生化、分子生物学和结构生物学相结合的方法来确定S-腺苷-L-蛋氨酸共底物和30S亚单位底物控制m1A1408甲基转移酶活性的分子基础。AIM 2将首次对m7G1405甲基转移酶的30S识别和修饰提供详细的分子见解，使用创新的、跨学科的方法组合，包括合成化学来生产新的SAM类似物，这将首次使这些临床重要耐药酶的基本生化和结构研究成为可能。总之，这些研究将为氨基糖苷类耐药甲基转移酶的结构、活性和作用机制提供新的和根本上重要的见解，并将直接为未来制定新的策略来对抗它们带来的耐药性奠定可靠的基础。

 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 juncture where the majority of useful antibiotics 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 retained activity against key human pathogens but where the problem of resistance continues to increase. Of particular concern is the increasing identification over the last decade of pathogenic bacteria harboring exceptionally broad- spectrum and high-level aminoglycoside resistance determinants. These 16S ribosomal RNA (rRNA) methyltransferases chemically modify the aminoglycoside binding site on the 16S rRNA of the small (30S) bacterial ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S rRNA on nucleoside guanosine 1405 or adenosine 1408, to create m7G1405 and m1A1408 modifications, respectively. 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. Recent studies from our lab and others revealed the molecular structures of each enzyme, of both drug producer and pathogenic origin, and provided a first view of an m1A1408 methyltransferase bound to its substrate. However, additional evidence suggests that this enzyme family may employ diverse mechanisms to accomplish substrate specificity and modification. Further, comparatively little is known about how m7G1405 achieve the equivalent molecular functions. This proposal describes experiments in two connected aims that will directly address these deficiencies. In Aim 1 we will define the molecular mechanisms employed by the m1A1408 methyltransferases using a combination of biochemical, molecular biological and structural biology approaches to determine the molecular bases for control of m1A1408 methyltransferase activity by the S-adenosyl-L-methionine cosubstrate, and by the 30S subunit substrate. Aim 2 will provide the first detailed molecular insights into 30S recognition and modification by the m7G1405 methyltransferases using an innovative, interdisciplinary combination of approaches including synthetic chemistry to produce novel SAM analogs that will enable for the first time essential biochemical and structural studies of these clinical important resistance enzymes. Together these studies will provide novel and fundamentally important insights into the structures, activities and mechanisms of action of the aminoglycoside-resistance methyltransferases and will directly lay a secure foundation for future development of new strategies to counter the resistance they confer.