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: 10624757
• 负责人: Wolfgang Peti
• 金额: $ 70.12万
• 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 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10624757
• 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; Crystallography; 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; Maps; Mediating; Microbiology; Molecular; 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E.粪便和E.粪便）会导致严重且经常致命的医院和社区获得 感染。肠内感染的治疗经常因其易感性下降而损害 （增加）对包括β-内酰胺在内的许多类别的抗生素。这种抵抗是压倒性的 归因于低亲和力青霉蛋白结合蛋白PBP4（E.粪便）和PBP5（E.粪便）的表达 两者都是低亲和力PBP家族的成员，其中还包括耐甲氧西林的PBP2A。 金黄色葡萄酒。在临床环境中，粪肠菌株表现出宽度的高水平青霉素耐药性。 酸取代，虽然相似的高度抗性大肠杆菌菌株很少见。建立我们广泛的结构 和功能性初步数据，我们将利用研究人员的科学专业知识的独特协同作用 回答以下关键基本问题：低亲和力PBP如何结合和催化转肽， 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 能够合成肽聚糖的能力，以及超过低亲和力PBP替代的哪些细胞因素增加 临床菌株表达的阻力水平？要回答这些问题，我们将追求四个具体目标 AIM 1将使用结构 生物学，尤其是生物分子NMR光谱，以确定为什么PBP5是β-内酰胺的下等靶标 抗生素。我们广泛的初步数据表明，这种巡回赛的工作（在〜75 kDa，PBP5是第二大 使用NMR光谱法的单链蛋白研究不仅是可行的，而且与我们的广泛相结合 晶体学数据将揭示为什么β-内酰胺仅抑制PBP5，并且整个家庭的家庭均抑制了。 亲和力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（NMR光谱），以确定在分子水平上这些PBP催化的方式 转肽。我们已经实现了PBP5特异性五肽前体的高收益合成和 脂质II的变体，使我们能够使用NMR光谱和转肽酶测定来确定底物 绑定并最终与PBP5交联。 PBP5中引起抗性突变的影响 也将确定转肽酶活性。 AIM 4将确定有助于 大肠杆菌中的抗性。我们的初步数据表明，粪肠球菌PBP2可能有助于β-内酰胺 高度抗性的LS4828 E.粪便菌株中的抗性。我们将量化PBP2对LS4828的贡献 β-内酰胺抗性。同时，我们将使用Bioid（接近标签）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，提供了关键数据，以基于未来的策略来实现 抑制这些重要的酶。

项目成果

Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

青霉素结合蛋白和替代双 β-内酰胺组合治疗青霉素 MIC 升高的严重粪肠球菌感染。

• DOI: 10.1128/aac.00871-22
• 发表时间: 2023
• 期刊: Antimicrobial agents and chemotherapy
• 影响因子: 4.9
• 作者: Cusumano,JaclynA;Daffinee,KathrynE;Ugalde-Silva,Paul;Peti,Wolfgang;Arthur,Michel;Desbonnet,Charlene;Rice,LouisB;LaPlante,KerryL;García-Solache,Mónica
• 通讯作者: García-Solache,Mónica

Molecular basis of β-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5.

• DOI: 10.1038/s41467-023-39966-5
• 发表时间: 2023-07-17
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Hunashal, Yamanappa;Kumar, Ganesan Senthil;Choy, Meng S.;D'Andrea, Everton D.;Da Silva Santiago, Andre;Schoenle, Marta V.;Desbonnet, Charlene;Arthur, Michel;Rice, Louis B.;Page, Rebecca;Peti, Wolfgang
• 通讯作者: Peti, Wolfgang

Enterococcal Physiology and Antimicrobial Resistance: The Streetlight Just Got a Little Brighter.

• DOI: 10.1128/mbio.03511-20
• 发表时间: 2021-02-23
• 期刊: mBio
• 影响因子: 6.4
• 作者: Rice LB