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

基本信息

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
项目类别：
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

项目摘要

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肠球菌感染的治疗常常因其敏感性降低而受到影响。对许多类别的抗生素（包括β-内酰胺类）（耐药性增加）这种耐药性是压倒性的。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。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，而基于我们广泛的结构的类似的高耐药性粪肠球菌菌株很少见。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为了回答这些问题，我们将追求四个具体目标目标 1 将使用结构生物学、化学合成、生物化学和微生物学。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-内酰胺的次等靶标我们广泛的初步数据表明，这一伟大的努力（约 75 kDa，PBP5 是第二大的）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，并且通过扩展，整个家族的低目标 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我们已经实现了 PBP5 特异性五肽前体的高产合成。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 PBP5 结合并最终交联 PBP5 中引起耐药性的突变的影响。目标 4 将确定转肽酶活性。我们的初步数据表明，粪肠球菌 PBP2 可能有助于产生 β-内酰胺。我们将量化 PBP2 对 LS4828 的贡献。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涉及蛋白质相互作用），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、提供未来策略的关键数据抑制这些重要的酶。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

肠球菌生理学和抗菌素耐药性：路灯变得更亮了。

DOI：
发表时间：
2021-02-23
期刊：
影响因子：
6.4
作者：
Rice; Louis B
通讯作者：
Louis B

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：
发表时间：
2023-02-16
期刊：
影响因子：
4.9
作者：
Cusumano, Jaclyn A;Daffinee, Kathryn E;Ugalde;Peti, Wolfgang;Arthur, Michel;Desbonnet, Charlene;Rice, Louis B;LaPlante, Kerry L;García
通讯作者：
García

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

ESKAPE 细菌 E. faecium 青霉素结合蛋白 PBP5 β-内酰胺抗生素耐药性的分子基础。

DOI：
发表时间：
2023-07-17
期刊：
影响因子：
16.6
作者：
Hunashal, Yamanappa;Kumar, Ganesan Senthil;Choy, Meng S;D'Andréa, Éverton 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