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:
    10391315
  • 负责人:
  • 金额:
    $ 71.92万
  • 依托单位:
  • 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 2024-04-30
  • 项目状态:
    已结题

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(如粪肠球菌和粪肠球菌)可引起严重的、往往是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易感性降低而受到影响。 (耐药性增加)对许多类别的抗生素,包括β-内酰胺类。这种抵抗是压倒性的 由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, 这两个基因都是一个低亲和力多溴联苯多酚家族的成员,该家族还包括来自耐甲氧西林葡萄球菌的多溴联苯并2 a。 金星。在临床环境中,由于氨基对青霉素的高度耐药,粪肠球菌菌株表现出广泛的高水平耐药性。 酸替代,而类似的高度耐药粪肠球菌株是罕见的。建立在我们广泛的结构基础上 和功能初步数据,我们将利用调查人员的科学专业知识的独特协同作用来 回答以下关键的基本问题:低亲和力多酚是如何结合和催化转肽的, 这些PBP的序列变化如何进一步降低它们对β-内酰胺类抗生素的亲和力,同时保留它们的 合成肽多糖的能力,以及除了低亲和力的PBP替代外,哪些细胞因子能增强 临床菌株表现出的耐药性水平?为了回答这些问题,我们将追求四个具体目标 它融合了结构生物学、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光谱学,以确定为什么PBP5是β-内酰胺的劣质靶标 抗生素。我们广泛的初步数据表明,在~75 kDa时,PBP5是第二大 用核磁共振光谱学研究单链蛋白质)不仅是可行的,而且结合我们广泛的 结晶学数据将揭示为什么β-内酰胺类药物对Pbp5的抑制效果很差,进而对整个Low家族的抑制作用也很差。 亲和力PBPS。AIMS 2和AIMS 3将使用新开发的与结构相结合的化学合成方案 和动力学(核磁共振波谱),以在分子水平上确定这些多溴联苯如何催化 转氨酶。我们已经实现了PBP5特异性五肽前体的高产率合成和 脂质II的变种,使我们能够使用核磁共振波谱和转肽酶分析来确定底物如何 结合并最终通过PBP5交联化。PBP5耐药突变对人类免疫功能的影响 转肽酶活性也将被测定。目标4将确定促成以下因素的正交因素 粪肠球菌的耐药性。我们的初步数据表明粪肠球菌PbP2可能与β-内酰胺类有关 高抗性粪肠球菌LS4828株的抗药性。我们将量化PBP2对LS4828的贡献 β-内酰胺类耐药。同时,我们将使用BioID(邻近标记)来鉴定PBP4和PBP2的相互作用 蛋白质(我们最近发表的结晶学数据表明,PBP4的N-末端结构域是动态的 并可能参与蛋白质的相互作用)。总而言之,这些研究将揭示结构和功能 肠球菌低亲和力PBP功能的细节,提供关键数据,作为未来战略的基础 抑制这些重要的酶。

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

Wolfgang Peti其他文献

Wolfgang Peti的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ truncateString('Wolfgang Peti', 18)}}的其他基金

Serine/Threonine Phosphatases in Neurological Diseases
神经系统疾病中的丝氨酸/苏氨酸磷酸酶
  • 批准号:
    10583671
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
Protein Phosphatase 1 Holoenzyme Formation
蛋白磷酸酶 1 全酶形成
  • 批准号:
    10441693
  • 财政年份:
    2022
  • 资助金额:
    $ 71.92万
  • 项目类别:
Protein Phosphatase 1 Holoenzyme Formation
蛋白磷酸酶 1 全酶形成
  • 批准号:
    10671729
  • 财政年份:
    2022
  • 资助金额:
    $ 71.92万
  • 项目类别:
Protein Phosphatase 1 Holoenzyme Formation
蛋白磷酸酶 1 全酶形成
  • 批准号:
    10793305
  • 财政年份:
    2022
  • 资助金额:
    $ 71.92万
  • 项目类别:
Shared Tundra screening cryo-EM for New England
新英格兰共享 Tundra 冷冻电镜筛查
  • 批准号:
    10413473
  • 财政年份:
    2022
  • 资助金额:
    $ 71.92万
  • 项目类别:
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
  • 财政年份:
    2019
  • 资助金额:
    $ 71.92万
  • 项目类别:
Protein Phosphatase 1 Holoenzyme Formation and Subunit Exchange
蛋白磷酸酶 1 全酶形成和亚基交换
  • 批准号:
    9985412
  • 财政年份:
    2019
  • 资助金额:
    $ 71.92万
  • 项目类别:
Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis
屎肠球菌和粪肠球菌β-内酰胺抗性酶的机制和活性
  • 批准号:
    9927573
  • 财政年份:
    2019
  • 资助金额:
    $ 71.92万
  • 项目类别:
Dynamics & energetics of p38a kinase regulation by ligands
动力学
  • 批准号:
    8608555
  • 财政年份:
    2013
  • 资助金额:
    $ 71.92万
  • 项目类别:
Dynamics & energetics of p38a kinase regulation by ligands
动力学
  • 批准号:
    8436569
  • 财政年份:
    2013
  • 资助金额:
    $ 71.92万
  • 项目类别:

相似海外基金

Construction of affinity sensors using high-speed oscillation of nanomaterials
利用纳米材料高速振荡构建亲和传感器
  • 批准号:
    23H01982
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Affinity evaluation for development of polymer nanocomposites with high thermal conductivity and interfacial molecular design
高导热率聚合物纳米复合材料开发和界面分子设计的亲和力评估
  • 批准号:
    23KJ0116
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Grant-in-Aid for JSPS Fellows
Platform for the High Throughput Generation and Validation of Affinity Reagents
用于高通量生成和亲和试剂验证的平台
  • 批准号:
    10598276
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
Development of High-Affinity and Selective Ligands as a Pharmacological Tool for the Dopamine D4 Receptor (D4R) Subtype Variants
开发高亲和力和选择性配体作为多巴胺 D4 受体 (D4R) 亚型变体的药理学工具
  • 批准号:
    10682794
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
  • 批准号:
    2233343
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Standard Grant
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
  • 批准号:
    2233342
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Standard Grant
Molecular mechanisms underlying high-affinity and isotype switched antibody responses
高亲和力和同种型转换抗体反应的分子机制
  • 批准号:
    479363
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Operating Grants
Deconstructed T cell antigen recognition: Separation of affinity from bond lifetime
解构 T 细胞抗原识别:亲和力与键寿命的分离
  • 批准号:
    10681989
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
CAREER: Engineered Affinity-Based Biomaterials for Harnessing the Stem Cell Secretome
职业:基于亲和力的工程生物材料用于利用干细胞分泌组
  • 批准号:
    2237240
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Continuing Grant
ADVANCE Partnership: Leveraging Intersectionality and Engineering Affinity groups in Industrial Engineering and Operations Research (LINEAGE)
ADVANCE 合作伙伴关系:利用工业工程和运筹学 (LINEAGE) 领域的交叉性和工程亲和力团体
  • 批准号:
    2305592
  • 财政年份:
    2023
  • 资助金额:
    $ 71.92万
  • 项目类别:
    Continuing Grant
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了