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（如粪肠球菌和粪肠球菌）引起严重的，往往是致命的医院感染和社区感染

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

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

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