Developing a better understanding of expression and function of PBP5-R of Enterococcus faecium for future development of therapeutic modalities for VRE infections

摘要 抗生素耐药性现在得到了世界各国领导人的认可，包括美国国会（以及至少一个最近 作为对人类最严重的公共卫生威胁之一。我们的提案集中在肠球菌 屎肠球菌（Efm），一种极耐药的医院相关（HA）革兰氏阳性球菌，目前占 40%的院内肠球菌感染，其中一些是医学上无法治疗的。>80%的临床Efm分离株 万古霉素耐药（VRE），几乎所有这些都是氨苄青霉素耐药（Amp-R）， 选择;对较新药物（利奈唑胺、达托霉素）的耐药性已有充分记录，并有越来越多的报告。 我们现在知道，大多数临床Efm分离株属于一个独特的Efm进化枝（HA进化枝A1）， 与社区相关（CA）分支B（主要由人类祖先组成）分离多年。一 医学上非常重要的表型差异是CA人类进化枝（进化枝B）容易被CA抑制， (MICs 0.125-2 mg/L），而HA分离株为Amp-R（MIC通常>64 mg/L）。Efm的PBP 5是一种低亲和力青霉素- 结合蛋白（PBP），其是BXR所必需的;它有两种主要形式，称为PBP 5-S（CA进化枝B， Amp-S菌株）和PBP 5-R（HA进化枝A1，Amp-R菌株）; PBP 5-S/R似乎是在 进化枝A2（动物进化枝）与进化枝A1密切相关。pbp 5-R和pbp 5-S等位基因相差约5%，并且存在一个 20-21个氨基酸（aa）的共有序列（一些PBP 5-R具有额外的aa），其可以区分-S和-R蛋白; 使用多拷贝质粒，最接近预测活性位点的4个氨基酸的工程改变显著降低了 相应的重组PBP 5对[14 C]青霉素的亲和力和增加的MIC。 天然染色体pBP 5位点中天然存在的等位基因或突变基因尚未报道。 PBPs是众所周知的抗生素靶点，通常适用于结构研究。我们的长期 总体目标是使用PBP 5结构：功能关系来开发针对Efm（包括VRE）的治疗剂。 在这里，我们建议为未来的药物设计，将目标Amp-R EFM提供背景。1）。我们将使用X- X射线晶体学来确定各种PBP 5-S、PBP 5-R和中间体PBP 5-R的高分辨率结构。 影响功能的S/R蛋白，如β-内酰胺MIC（在将pbp 5等位基因克隆到一种蛋白的天然pbp 5位点后， recPBP 5 HA Efm），以及由等位基因编码的recPBP 5蛋白对β-内酰胺的亲和力。比较高- PBP 5形式的分离结构加上与β-内酰胺+/-肽聚糖组分的共结晶，功能性 检测和特异性定点pbp 5突变，应该为理解特异性pbp 5基因的作用提供基础。 在自然界中观察到的PBP 5形式对β-内酰胺的亲和力的变化，并提供了如何抑制这种PBP的见解。 功能未来项目（例如，R33）然后将使用这些结构来合理地设计或提高 PBP 5形式的抑制剂，赋予Amp-R以开发有效的抗Efm疗法。2）。我们将测试我们的假设 进化枝B Efm的Psr，而不是在进化枝A1中发现的截短的Psr，导致PBP 5表达的抑制， 即使在pbp 5-R存在下也能阻止Amp-R，未来可能的目标是设计Psr模拟物。

ABSTRACT Antimicrobial resistance is now recognized by world leaders, including the US Congress (and at least one recent President) as one of the most serious public health threats to mankind. Our proposal focuses on Enterococcus faecium (Efm), an extremely resistant, hospital-associated (HA) Gram-positive coccus that now accounts for almost 40% of nosocomial enterococcal infections, some of which are medically untreatable. >80% of clinical Efm isolates are vancomycin resistant (VRE) and almost all of these are ampicillin resistant (Amp-R), the traditional drugs of choice; resistance to newer agents (linezolid, daptomycin) is well documented and increasingly reported. We now know that most clinical Efm isolates belong to a distinct Efm clade (the HA clade A1) that is thousands of years separated from the community-associated (CA) clade B (comprised mostly of human commensals). A medically very important phenotypic difference is that CA human commensals (clade B) are readily inhibited by Amp (MICs 0.125-2 mg/L), while HA isolates are Amp-R (MICs often >64 mg/L). PBP5 of Efm is a low-affinity penicillin- binding protein (PBP) that is essential for Amp R; it comes in two main forms referred to as PBP5-S (CA clade B, Amp-S strains) and PBP5-R (HA clade A1, Amp-R strains); PBP5-S/R appears to be a transitional form found in clade A2 (animal clade) that is closely related to clade A1. pbp5-R and pbp5-S alleles differ by ~ 5% and there is a consensus of 20-21 amino acids (aa) (some PBP5-Rs have an extra aa) that can distinguish -S from -R proteins; using a multi-copy plasmid, engineered changes of 4 aa, most near the predicted active site, significantly lowered affinity of the corresponding recombinant PBP5 for [14C]penicillin and increased Amp MICs. However, studies of naturally occurring alleles or of mutated genes in the native chromosomal pbp5 site have not been reported. PBPs are well known targets for antibiotics and are generally amenable to structural studies. Our long-term overarching goal is to use PBP5 structure:function relationships to develop therapeutics against Efm, including VRE. Here, we propose to provide the background for future drug design that would target Amp-R Efm. 1). We will use X- ray crystallography to determine the high-resolution structures of various PBP5-S, PBP5-R and intermediate PBP5- S/R proteins that impact function, such as β-lactam MICs (after cloning pbp5 alleles into the native pbp5 site of a ∆pbp5 HA Efm), and affinity of recPBP5 proteins encoded by the alleles for β-lactams. A comparison of high- resolution structures of PBP5 forms plus co-crystallization with β-lactams +/- peptidoglycan components, functional assays and specific site-directed pbp5 mutations, should provide the foundation for understanding the role of specific aa changes seen in nature on affinity of PBP5 forms for β-lactams and provide insights into how to inhibit this PBP's function. Future projects (e.g., an R33) would then use these structures to rationally design or improve the efficacy of inhibitors of PBP5 forms that confer Amp-R to develop an effective anti-Efm therapy. 2). We will test our hypothesis that Psr of clade B Efm, but not the truncated Psr found in clade A1, results in repression of PBP5 expression, preventing Amp-R even in the presence of pbp5-R, with a possible future goal of designing Psr mimickers.