REGULATION OF AMPICILLIN RESISTANCE IN E FACEIUM

描述(逐字摘自申请者摘要)： 多重耐药肠球菌在美国医院的流行情况 过去十年的治疗选择有限，影响了发病率和死亡率 增加了重病住院患者的护理成本。这个 对万古霉素耐药的表达在 这一次。然而，同样有问题的是，几乎所有 万古霉素耐药肠球菌(VRE)是一种表达 对高浓度氨苄西林的抗药性。虽然很明显氨苯西林 粪肠球菌的耐药性需要表达低亲和力的青霉素结合 蛋白5(PBP5)，可检测到的PBP5与 氨苄西林的耐药性水平并不准确。中的几个点突变 在表达高水平氨苄青霉素的菌株中发现了pbp5。 抗性，但这些突变对水平的具体贡献 抵抗力从未被评估过。我们已经确定了第一个可转让的 东北地区一株VRE粪肠球菌对氨苄西林耐药性的研究 俄亥俄州。该菌株的抗病基因pbp5有几个 与氨苄西林高水平耐药有关的突变 其他粪便埃希菌分离株。奇怪的是，氨苯西林的抗药性水平 通过转接，粪肠球菌菌株不等同于由 捐赠者，尽管有文件表明生产了等量的PBP5。 在过去的两年里，我们获得了证据表明氨苄西林的水平 表达的抗性与转录相关(但不一定 翻译)指定为ftsWEf的上游开放阅读框。具体的 本方案的目的是：1)对E. 确定粪便杆菌的功能(MIC，亲和力)和结构 特定突变的重要性。与法国和法国的合作 瑞士，我们现在拥有创造突变体和 分析它们的功能影响并确定其晶体结构；2) 研究推测的上游抑制因子PSR在调节中的作用 氨苄西林耐药基因在粪肠球菌中的表达 FtsWEf转录影响氨苄西林水平的机制 表达的抗性；4)评估上游开放读数 名为Nane-EF和ywrF-EF的框架会影响氨苄西林耐药水平 表达和5)以确定肽聚糖前体是否在 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具抗药性的医院病原体 为PBP5、相关性提供重要的结构-功能相关性 这可能对新的和更好的抑制性的发展很重要 化合物。它们还将提供有关细胞机制的重要新信息。 粪肠球菌和其他革兰氏阳性细菌的壁面合成以及 粪肠球菌对氨苄西林耐药性的调控机制。

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.