MOLECULAR MECHANISMS OF AMINOGLYCOSIDE RESISTANCE

描述：自从引入抗生素以来，细菌一直 对所有这些病毒产生抵抗力。制药公司紧随其后 开发新的抗菌剂，以便当病原体对 其中一个是有效的，另一个是有效的。不过，最近， 出现了许多对其具有多重耐药性的病原菌 大多数抗生素都是无效的。因此，两国之间的竞争 病原体的耐药性和制药业的发展 变得更糟。随着细菌耐药性问题的增加， 治疗感染性疾病的有效抗菌剂的可用性 削减了成本，成本也变高了。一种处理毒品的方法 阻力问题就是研究不同方面的机理。 细菌对抗生素的耐药性和设计合理的药物策略 在所学知识的基础上克服问题。 氨基糖苷类药物在严重革兰氏阴性菌治疗中的重要作用 感染。细菌抗药性的一种非常常见的机制 氨基糖苷类化合物是通过酶作用消除抗菌活性的。 通过修饰酶的作用使其失活。一个临床上很重要的 N-乙酰转移酶是AAc(6‘)-Ib，它对几种 氨基糖苷类药物，包括半合成的阿米卡星，是最难治疗的 使酶失活。这项调查的长期目标是 在分子水平上理解不同的事件导致 这种酶介导的细菌对氨基糖苷类药物的耐药性。这个 本项目的具体目标是：1)确定亚细胞 AAc(6‘)-Ib的定位氨基糖苷类修饰药物的定位 酶在确定细菌的抗药性水平方面起着重要作用。 细菌细胞对某种氨基糖苷类药物的抑制作用。细胞的亚细胞位置 AAc(6‘)-Ib酶的测定方法是：a)用 蛋白水解酶，b)免疫电子显微镜，c)基因融合。这些 这些方法将补充那些已经使用物理方法完成的分析 释放周质蛋白的方法。2)氨基酸的鉴定 和对Aac(6‘)-Ib功能至关重要的区域。不是所有的氨基酸 蛋白质的残基和区域对功能和 酶的结构。因此，这一特定目标的目标是确定 蛋白质Aac(6‘)-Ib的关键区域和氨基酸残基 因为它的结构和酶功能。这将使用随机 替代诱变，这是一种允许[一个]评估 小部分蛋白质的重要性。3)晶化 Aac(6‘)-Ib.生物学特性的重要一步 过程是生化和遗传数据与 所涉及的分子的详细结构。因此，一个长期的 该项目的目标是确定三维结构 Aac(6‘)-Ib.这项提议的目标是产生适合于 高分辨率X射线衍射分析。这将通过以下方式实现：a) 纯化足量的AAc(6‘)-Ib和b)建立 产生晶体的条件。

DESCRIPTION: Since the introduction of antibiotics, bacteria kept developing resistance to all of them. Drug companies kept pace by developing new antimicrobial agents so when a pathogen became resistant to one of them there was another that would be effective. However, recently many multiple-drug resistance pathogenic bacteria have emerged towards which most antibiotics are ineffective. As a consequence, the race between development of resistance by pathogens and the pharmaceutical industry is becoming worse. As the problem of bacterial resistance increases, the availability of effective antimicrobial agents to treat infections gets curtailed and the costs grow higher. An approach to deal with the drug resistance problem is to study different aspects of the mechanisms of bacterial resistance to antibiotics and design rational drug strategies to overcome the problem based on the knowledge gained. Aminoglycosides are important for the treatment of serious Gram-negative infections. A very common mechanism of bacterial resistance to aminoglycosides is to eliminate the antibacterial activity through enzymatic inactivation by the action of modifying enzymes. A clinically important N-acetyltransferase enzyme is AAC(6')-Ib which confers resistance to several aminoglycosides including the semisynthetic amikacin, the most refractory to inactivating enzymes. The long term goal of this investigation is to understand at the molecular level the different events that result in bacterial resistance to aminoglycosides mediated by this enzyme. The specific aims for this project are: 1) Determination of the subcellular localization of AAC(6')-Ib. The location of an aminoglycoside-modifying enzyme is instrumental in the determination of the level of resistance of a bacterial cell to a certain aminoglycoside. The subcellular location of the enzyme AAC(6')-Ib will be determined by: a) treatment of spheroplasts with proteases, b) imunoelectron microscopy, and c) gene fusions. These approaches will complement those analysis already done using physical methods that release periplasmic proteins. 2) Identification of amino acids and regions critical for the function of AAC(6')-Ib. Not all amino acid residues and regions of a protein contribute equally to the function and structure of an enzyme. Thus, the goal of this specific aim is to identify regions and amino acids residues of the protein AAC(6')-Ib that are critical for its structure and enzymatic function. This will be done using random replacement mutagenesis, a technique that allows [one] to assess the importance of small portions of the protein. 3) Crystallization of AAC(6')-Ib. An important step in the characterization of biological processes is the correlation of biochemical and genetic data with the detailed structure of the molecules involved. Therefore, a long-term objective of this project is to determine the three dimensional structure of AAC(6')-Ib. The goal for this proposal is to generate crystals suitable for high-resolution X-ray diffraction analysis. This will be achieved by: a) purification of sufficient amounts of AAC(6')-Ib and b) establishment of the condition to produce the crystals.