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

屎肠球菌和粪肠球菌中β-内酰胺抗性酶的机制和活性

• 批准号: 10391315
• 负责人: Wolfgang Peti
• 金额: $ 71.92万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-09 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391315
• 关键词: Address; Affinity; Amino Acid Substitution; Antibiotic Resistance; Antibiotics; Arizona; Binding; Biochemistry; Biological Assay; Biomolecular Nuclear Magnetic Resonance; Cellular biology; Characteristics; Chemicals; Chemistry; Clinical; Communicable Diseases; Community-Acquired Infections; Complex; Coupled; Dangerousness; Data; Development; Doctor of Philosophy; Enterococcus; Enterococcus faecalis; Enterococcus faecium; Enzymes; Essential Amino Acids; Family; Future; Goals; Hospitals; In Vitro; Infection; Inferior; Kinetics; Knowledge; Label; Lipids; Mediating; Microbiology; Molecular; Monobactams; Mutation; N-terminal; NMR Spectroscopy; Nosocomial Infections; Penicillin Resistance; Penicillin-Binding Proteins; Peptidoglycan; Peptidyltransferase; Predisposition; Process; Protein Dynamics; Proteins; Publishing; Reagent; Regulation; Research Personnel; Resistance; Rhode Island; Rice; Role; Scheme; Staphylococcus aureus; Structure; Testing; Therapeutic; Time; Toxic effect; Translating; Universities; Variant; analog; antimicrobial; base; beta-Lactam Resistance; beta-Lactams; chemical synthesis; crosslink; design; experimental study; in vitro activity; in vivo; insight; interdisciplinary approach; member; methicillin resistant Staphylococcus aureus; mimetics; multidisciplinary; novel; peptide chemical synthesis; structural biology; suicide substrates; synergism; transpeptidation

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.

肠球菌（例如粪肠球菌和屎肠球菌）可引起严重且常常致命的医院和社区获得性感染 感染。肠球菌感染的治疗常常因其敏感性降低而受到影响 （增加耐药性）多种抗生素，包括 β-内酰胺类。这种反抗是压倒性的 归因于低亲和力青霉素结合蛋白 PBP4（粪肠球菌）和 PBP5（粪肠球菌）的表达， 两者都是低亲和力 PBP 家族的成员，该家族还包括来自耐甲氧西林葡萄球菌的 PBP2a。 金黄色葡萄球菌。在临床环境中，屎肠球菌菌株由于氨基而表现出广泛的高水平青霉素耐药性 酸替代，而类似的高耐药性粪肠球菌菌株很少见。建立在我们广泛的结构基础上 和功能性初步数据，我们将利用研究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 PBP 如何结合并催化转肽作用， 这些 PBP 中的序列变化如何进一步降低其对 β-内酰胺抗生素的亲和力，同时保留其作用 合成肽聚糖的能力，以及除低亲和力 PBP 替代之外的哪些细胞因素会增强 临床菌株表达的耐药水平？为了回答这些问题，我们将追求四个具体目标 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 生物学，特别是生物分子核磁共振波谱，以确定为什么 PBP5 是 β-内酰胺的次等靶标 抗生素。我们广泛的初步数据表明，这一杰作的努力（约 75 kDa，PBP5 是第二大 使用核磁共振波谱研究单链蛋白质）不仅是可行的，而且结合我们广泛的研究 晶体学数据将揭示为什么 β-内酰胺对 PBP5 的抑制作用很弱，并且推而广之，整个家族的低 亲和性 PBP。目标 2 和 3 将使用新开发的化学合成方案以及结构 和动力学（核磁共振波谱）以确定这些 PBP 在分子水平上如何催化 转肽作用。我们已经实现了 PBP5 特异性五肽前体的高产率合成 脂质 II 的变体，使我们能够使用核磁共振波谱和转肽酶测定来确定底物 结合并最终通过 PBP5 交联。 PBP5 中引起耐药性的突变的影响 转肽酶活性也将被测定。目标 4 将确定有助于 粪肠球菌的耐药性。我们的初步数据表明粪肠球菌 PBP2 可能有助于 β-内酰胺 高耐药性 LS4828 粪肠球菌菌株中的耐药性。我们将量化PBP2对LS4828的贡献 β-内酰胺耐药。同时，我们将使用 BioID（邻近标记）来识别 PBP4 和 PBP2 相互作用 蛋白质（我们最近发表的晶体学数据表明 PBP4 N 末端结构域是动态的 并且可能参与蛋白质相互作用）。这些研究将共同​​揭示结构和功能 肠球菌低亲和力 PBP 功能的详细信息，提供关键数据作为未来策略的基础 抑制这些重要的酶。