REGULATION OF AMPICILLIN RESISTANCE IN E FACEIUM

E FACEIUM 中氨苄青霉素耐药性的调节

• 批准号: 6632105
• 负责人: Louis B. Rice
• 金额: $ 24.59万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6632105
• 关键词: Enterococcus; ampicillin; bacterial genetics; bacterial proteins; binding proteins; drug resistance; gene mutation; glucosamine; microorganism culture; open reading frames; penicillins; peptidoglycan; protein structure function; site directed mutagenesis

DESCRIPTION (Verbatim from Applicant's Abstract):The dramatic rise in prevalence of multi-resistant enterococci in United States hospitals over the past decade has limited therapeutic options, affected morbidity and mortality and increased the cost of caring for seriously ill hospitalized patients. The expression of resistance to vancomycin has received the most attention during this time. However, it is equally problematic that virtually all vancomycin-resistant enterococci (VRE) are Enterococcus faecium that express resistance to high levels of ampicillin. While it is clear that ampicillin resistance in E. faecium requires expression of low affinity penicillin-binding protein 5 (PBP5), the correlation between the amounts of detectable PBP5 and the level of ampicillin resistance is not exact. Several point mutations in pbp5 have been identified in strains expressing high-level ampicillin resistance, but the specific contributions of these mutations to the levels of resistance have never been assessed. We have identified the first transferable ampicillin resistance described from E. faecium in a VRE strain from Northeast Ohio. The pbp5 gene conferring resistance in this isolate possesses several mutations that have been associated with high-level ampicillin resistance in other E. faecium isolates. Curiously, levels of ampicillin resistance expressed by transconjugant E. faecium strains are not equivalent to those expressed by the donor, despite documentation that equivalent amounts of PBP5 are produced. In the past two years, we have acquired evidence that levels of ampicillin resistance expressed correlate with transcription (but not necessarily translation) of an upstream open reading frame designated ftsWEf. The specific aims of this proposal are to: 1) perform site directed mutagenesis of E. faecium pbp5 to determine the functional (MIC, affinity) and structural importance of specific mutations. With collaborations in France and Switzerland, we now possess the molecular expertise to create the mutants and analyze their functional impact and determine the crystal structure; 2) to investigate the role of the putative upstream repressor psr in regulating expression of ampicillin resistance in E. faecium; 3) to investigate the mechanisms by which transcription of ftsWEf impacts the levels of ampicillin resistance expressed by E. faecium; 4) to assess whether upstream open reading frames designated nanE-Ef and ywrF-Ef affect levels of ampicillin resistance expressed and 5) to determine whether the peptidoglycan precursors differ in sensitive and resistant strains. These investigations will yield new insights into what is arguably the most resistant nosocomial pathogen of our time by providing important structure-function correlations for PBP5, correlations which may be important for the development of newer and better inhibitory compounds. They will also yield important new information on mechanisms of cell wall synthesis in E. faecium and other Gram-positive bacteria as well as on the mechanisms by which ampicillin resistance in E. faecium is regulated.

描述（逐字摘自申请人摘要）： 2005年美国医院中多重耐药肠球菌的患病率 在过去的十年中，治疗选择有限，影响了发病率和死亡率 增加了重病住院患者的护理费用。的 万古霉素耐药性的表达在研究中受到了最多的关注。 这次然而，同样有问题的是， 万古霉素耐药肠球菌（VRE）是屎肠球菌， 对高浓度氨苄青霉素的抗性。很明显氨苄青霉素 E.屎肠需要表达低亲和力青霉素结合蛋白 蛋白5（PBP5），可检测的PBP5和 氨苄青霉素耐药性的水平并不准确。几个点突变， PBP5已经在表达高水平氨苄青霉素的菌株中被鉴定 耐药性，但这些突变的具体贡献， 阻力从未被评估过。我们已经确定了第一个可转移的 氨苄青霉素抗性描述自E.来自东北的VRE菌株中的屎囊 俄亥俄州。在该分离物中赋予抗性的pbp 5基因具有几个 与高水平氨苄青霉素耐药相关的突变， 其它大肠粪菌分离物。奇怪的是，氨苄青霉素耐药水平 通过接合子E.屎肠菌菌株不等同于 尽管有文件证明生产了等量的PBP5，但捐助方仍然没有提供。 在过去的两年里，我们有证据表明氨苄青霉素的含量 表达的抗性与转录相关（但不一定 翻译）指定为ftsWEf的上游开放阅读帧。具体 本发明的目的是：1）进行E. 屎菌pbp5的功能（MIC，亲和力）和结构 特定突变的重要性。与法国的合作， 瑞士，我们现在拥有创造突变体的分子技术， 分析其功能影响并确定晶体结构; 2） 研究推定的上游阻遏物psr在调节 氨苄青霉素抗性在E.（3）研究了 ftsWEf转录影响氨苄青霉素水平的机制 E. 4）评估上游是否开放阅读 命名为nanE-Ef和ywrF-Ef的框架影响氨苄青霉素抗性水平 5）确定肽聚糖前体是否与肽聚糖前体不同， 敏感和耐药菌株。这些调查将产生新的见解 可以说是我们这个时代最耐药的医院病原体， 为PBP5提供了重要的结构-功能相关性， 这可能对开发新的更好的抑制剂很重要。 化合物.它们也将产生重要的新信息的机制，细胞 壁合成E.屎肠菌和其他革兰氏阳性菌以及 氨苄青霉素耐药的机制。粪便是有规律的。