Beta-Lactam Resistance Mechanisms of Staphylococcus aureus

金黄色葡萄球菌的β-内酰胺耐药机制

• 批准号: 10586370
• 负责人: Shahriar Mobashery
• 金额: $ 66.4万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586370
• 关键词: Address; Adjuvant; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Applications Grants; Back; Bacteria; Cell membrane; Communication; Complex; Cytoplasm; Engineering; Event; Exposure to; Future; Gene Expression; Genes; Growth; Human; Integral Membrane Protein; Kinetics; Lactams; Libraries; Membrane; Membrane Proteins; Methods; Molecular Conformation; Monitor; Monobactams; Morbidity - disease rate; Natural Products; Operon; Pathway interactions; Penicillin-Binding Proteins; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphorylation Site; Process; Protease Domain; Proteins; Resistance; Side; Signal Transduction; Staphylococcus aureus; Staphylococcus aureus infection; Structure; Structure-Activity Relationship; Surface; System; Transducers; United States National Institutes of Health; Work; X-Ray Crystallography; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; design; inhibitor; mortality; novel; pathogenic bacteria; resistance mechanism; response; screening; sensor; small molecule; tool; tool development; trait

Project Summary/Abstract Staphylococcus aureus is a problematic human bacterial pathogen, which is broadly resistant to b-lactam antibiotics. This resistance is inducible and is conferred by a set of genes that encode a b-lactam antibiotic sensor/signal transducer protein, a gene repressor and two resistance determinants (the BlaZ b-lactamase and a unique penicillin-binding protein designated as PBP2a). A key feature of these processes is the recognition of the antibiotic by the b-lactam sensor/signal transduce BlaR, an integral membrane protein. Recognition of the antibiotic by the sensor domain of BlaR unleashes conformational changes through the membrane, which lead to the activation of the protease domain on the cytoplasmic side. This process culminates in expression of the genes for the antibiotic-resistance determinants. In Specific Aim 1, we describe the use of a fluorescent tool in live S. aureus for discovery of agents that shut down the BlaR recognition of the b-lactam antibiotics, which would reverse the resistance phenotype. A discovery funnel is outlined for analysis of structure-activity relationship for these compounds. In Specific Aim 2 we communicate a discovery that the BlaZ b-lactamase is incorporated to the surface of the cytoplasmic membrane in a lipidation- and phosphorylation-dependent manner. We outline a method for purification of the membrane-anchored BlaZ for the purpose of the identification of the sites of phosphorylation. The protein will be used in kinetic studies to compare to the non- membrane-anchored BlaZ, which is not phosphorylated. Plans are detailed for X-ray crystallography for characterization of the structural issues. The generality of the lipidation- and phosphorylation-dependent anchoring of proteins to the membrane surface in S. aureus will be explored for 10 distinct proteins. In each case, plans are outlined to identify the sites of phosphorylation to elucidate rules for phosphorylation and membrane sequestration of these proteins. These studies will shed definitive light on the complex machinery that S. aureus strains have evolved for resistance to b-lactam antibiotics.

项目总结/摘要 金黄色葡萄球菌（Staphylococcus aureus）是一种有问题的人类细菌病原体，其对b-内酰胺具有广泛的耐药性 抗生素这种耐药性是可诱导的，由一组编码b-内酰胺抗生素的基因赋予 传感器/信号转导蛋白，一种基因阻遏物和两种耐药决定因子（BlaZ β-内酰胺酶和 一种独特的青霉素结合蛋白，命名为PBP 2a）。这些过程的一个关键特征是认识到 抗生素的b-内酰胺传感器/信号肽BlaR，一个完整的膜蛋白。承认 通过BlaR的传感器结构域的抗生素通过膜释放构象变化， 导致细胞质侧蛋白酶结构域的活化。这一过程的高潮是表达 抗疟决定因素的基因在具体目标1中，我们描述了荧光 工具活S.金黄色葡萄球菌发现了关闭b-内酰胺抗生素的BlaR识别的试剂， 这将逆转抗性表型。一个发现漏斗概述了结构活性分析 这些化合物的关系。在具体目标2中，我们交流了一个发现，即BlaZ b-内酰胺酶是 以脂化和磷酸化依赖的方式掺入细胞质膜表面， 方式我们概述了一种用于纯化膜锚定的BlaZ的方法， 鉴定磷酸化位点。蛋白质将用于动力学研究，以与非蛋白质进行比较。 膜锚定的BlaZ，其不被磷酸化。详细介绍了X射线晶体学计划， 结构性问题的特征。脂化和磷酸化依赖性 锚定蛋白质的膜表面在S。金黄色葡萄球菌将探索10种不同的蛋白质。在每个 案例，计划概述，以确定磷酸化的网站，阐明磷酸化的规则， 这些蛋白质的膜隔离。这些研究将明确阐明复杂的机制 那辆多金黄色葡萄球菌菌株已经进化出对β-内酰胺抗生素的抗性。