Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis

肠球菌（如E. faecalis和E.粪菌）引起严重的和通常致命的医院和社区获得性 感染.肠球菌感染的治疗经常受到其敏感性降低的影响 （增加的耐药性）对许多种类的抗生素，包括β-内酰胺。这种阻力是压倒性的 可归因于低亲和力青霉素结合蛋白PBP 4（E. faecalis）和PBP 5（E.屎）， 它们都是低亲和性PBPs家族的成员，该家族还包括来自耐甲氧西林链球菌的PBP 2a。 金黄色。在临床上，E.屎肠菌菌株显示出广泛的高水平的青霉素耐药性， 酸取代，而类似的高抗E.粪菌属菌株很少见。基于我们广泛的结构 和功能性初步数据，我们将利用调查人员科学专业知识的独特协同作用， 回答以下关键的基本问题：低亲和力PBPs如何结合和催化转肽作用， 这些PBPs中的序列变化如何进一步降低它们对β-内酰胺抗生素的亲和力， 合成肽聚糖的能力，以及除了低亲和力PBP取代之外的细胞因子增加了什么 临床菌株的耐药水平？为了回答这些问题，我们将追求四个具体目标 它整合了结构生物学、化学合成、生物化学和微生物学。目标1将使用结构性 生物学，特别是生物分子NMR光谱学，以确定为什么PBP 5是β-内酰胺的次要靶标 抗生素我们广泛的初步数据表明，这种巡回演出的努力（在~75 kDa，PBP 5是第二大 单链蛋白质研究使用核磁共振光谱）不仅是可行的，但结合我们广泛的 晶体学数据，将揭示为什么β-内酰胺只能很差地抑制PBP 5，并通过扩展， 亲和PBPs。目标2和3将使用新开发的化学合成方案， 和动力学（核磁共振光谱），以确定如何，在分子水平上，这些PBPs催化 转肽作用我们已经实现了PBP 5特异性五肽前体的高产率合成， 脂质II的变体，使我们能够使用NMR光谱和转肽酶测定，以确定如何底物 结合并最终被PBP 5交联。PBP 5中引起耐药的突变对 还将测定转肽酶活性。目标4将确定正交因素，有助于 E.粪便我们的初步数据表明，E.粪杆菌PBP 2可能导致β-内酰胺 高抗菌株LS 4828 E.粪菌株我们将量化PBP 2对LS 4828的贡献 β-内酰胺耐药。同时，我们将使用BioID（邻近标记）来鉴定PBP 4和PBP 2相互作用， 蛋白质（我们最近发表的晶体学数据显示，PBP 4 N-末端结构域是动态的， 并且可能参与蛋白质相互作用）。总之，这些研究将揭示出 肠球菌低亲和力PBP功能的详细信息，为未来的战略提供了关键数据， 抑制这些重要的酶。

Enterococci (e.g. E. faecalis and E. faecium) cause severe and often fatal nosocomial and community-acquired infections. Therapy of enterococcal infections is frequently compromised by their decreased susceptibility (increased resistance) to many classes of antibiotics, including β-lactams. This resistance is overwhelmingly attributable to the expression of low-affinity penicillin-binding proteins PBP4 (E. faecalis) and PBP5 (E. faecium), both of which are members of a family of low-affinity PBPs that also includes PBP2a from methicillin-resistant S. aureus. In the clinical setting, E. faecium strains show widespread high-level penicillin resistance due to amino acid substitutions, while similar highly-resistant E. faecalis strains are rare. Building on our extensive structural and functional preliminary data, we will leverage the unique synergy of scientific expertise of the investigators to answer the following key fundamental questions: how do low affinity PBPs bind and catalyze transpeptidation, how do sequence changes in these PBPs further reduce their affinity for β-lactam antibiotics while retaining their ability to synthesize peptidoglycan, and what cellular factors beyond low affinity PBP substitutions augment levels of resistance expressed by clinical strains? To answer these questions, we will pursue four specific aims that integrate structural biology, chemical synthesis, biochemistry and microbiology. Aim 1 will use structural biology, especially biomolecular NMR spectroscopy, to determine why PBP5 is an inferior target of β-lactam antibiotics. Our extensive preliminary data shows that this tour-de-force effort (at ~75 kDa, PBP5 is the second largest single-chain protein studied using NMR spectroscopy) is not only feasible but, combined with our extensive crystallographic data, will reveal why β-lactams only poorly inhibit PBP5 and, by extension, the entire family of low affinity PBPs. Aims 2 and 3 will use newly developed chemical synthesis schemes coupled with structure and dynamics (NMR spectroscopy) to determine how, at a molecular level, these PBPs catalyze transpeptidation. We have achieved high-yield syntheses of PBP5-specific pentapeptide precursors and variants of lipid II, enabling us to use NMR spectroscopy and transpeptidase assays to determine how substrates bind and ultimately become cross-linked by PBP5. The impact of resistance-causing mutations in PBP5 on transpeptidase activity will also be determined. Aim 4 will identify the orthogonal factors that contribute to resistance in E. faecalis. Our preliminary data suggest that E. faecalis PBP2 likely contributes to β-lactam resistance in the highly resistant LS4828 E. faecalis strain. We will quantify the contribution of PBP2 to LS4828 β-lactam resistance. In parallel, we will use BioID (proximity labeling) to identify PBP4 and PBP2 interacting proteins (our recently published crystallographic data revealed that the PBP4 N-terminal domains are dynamic and are likely involved in protein interactions). Together, these studies will reveal the structural and functional details of enterococcal low-affinity PBP function, providing critical data upon which to base future strategies for inhibiting these important enzymes.