MOLECULAR GENETICS OF PLASMID-DETERMINED BETA-LACTAMASE

质粒测定的 β-内酰胺酶的分子遗传学

基本信息

批准号：
3130090
负责人：
GEORGE A JACOBY
金额：
$ 23.04万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-09-30 至 1991-08-31
项目状态：
已结题

项目摘要

Infections caused by gram-negative bacteria are an increasing health problem, particularly in hospitalized patients. Plasmids in these organisms determine a variety of beta-lactamases, enzymes that are responsible for resistance to penicillin, cephalosporin, and related beta-lactam antibiotics. More than 30 plasmid-mediated beta-lactamases have been distinguished, and in some cases new enzymes with expanded specificity seem to be evolving from established types. As the major biochemical mechanism for resistance to beta-lactam antibiotics it is important to be able to follow the distribution of these enzymes in gram-negative pathogens and to understand how these enzymes are related genetically, what structural features are responsible for their ability to hydrolyze particular beta-lactams, and what is their further potential for evolution in response to the changing selection pressure imposed by the introduction of new beta-lactam drugs. The aims of this proposal are: 1. To establish the nucleotide sequence for PSE-l and OXA-7 beta- lactamases, two enzymes with distinctive properties whose structural basis will be clarified by comparison with the sequence of FSE-2 which we have determined and with OXA enzymes sequenced by others. 2. To construct enzyme hybrids between PSE-2 and OXA-5 or OXA-2 such that properties of the chimeric beta-lactamases, will aid in localization of particular enzyme functions. 3. To establish the structural basis for extended spectrum beta- lactamase activity in mutants and in a plasmid-mediated enzyme isolated at our hospital that confers resistance to aztreonam and ceftazidime. 4. To perfect conditions for selecting beta-lactamase mutants resistant to inhibition by clavulanic acid, sulbactam, or YTR 830 and to characterize those we have already obtained for the particular amino acid changes involved in diminished inhibitor affinity. 5. To analyze the contribution of amino acids making up the walls of the enzyme active site to substrate specificity by systematic site-directed mutagenesis.

由革兰氏阴性菌引起的感染日益增多健康问题，特别是住院患者。质粒在这些生物体中决定多种β-内酰胺酶、酶负责对青霉素、头孢菌素、和相关的β-内酰胺抗生素。超过30种质粒介导的 β-内酰胺酶已被区分，并且在某些情况下是新的具有扩展特异性的酶似乎是从既定类型。作为主要的生化机制对 β-内酰胺抗生素的耐药性重要的是能够跟踪这些酶在革兰氏阴性菌中的分布病原体并了解这些酶之间的关系从遗传上讲，哪些结构特征决定了它们的水解特定 β-内酰胺的能力，以及它们是什么响应变化的进一步进化潜力新β-内酰胺的引入带来的选择压力药物。该提案的目的是： 1. 建立 PSE-1 和 OXA-7 beta- 的核苷酸序列内酰胺酶，两种具有独特性质的酶，其通过与序列的比较，可以明确结构基础我们已经测定并用 OXA 酶测序的 FSE-2 由其他人。 2. 构建PSE-2和OXA-5或OXA-2之间的酶杂交体这样嵌合β-内酰胺酶的特性将有助于特定酶功能的定位。 3. 建立扩展频谱β的结构基础突变体和质粒介导的酶中的内酰胺酶活性在我们医院被隔离，对氨曲南产生耐药性头孢他啶。 4. 完善β-内酰胺酶突变体的筛选条件对克拉维酸、舒巴坦或 YTR 830 的抑制具有抗性并描述我们已经获得的那些与抑制剂减少有关的特定氨基酸变化亲和力。 5. 分析构成细胞壁的氨基酸的贡献通过系统分析酶活性位点对底物的特异性定点诱变。