Beta-Lactam Resistance Mechanisms of Staphylococcus aureus

项目摘要/摘要 金黄色葡萄球菌是一种有问题的人类致病菌，它对β-内酰胺类药物具有广泛的耐药性。 抗生素。这种耐药性是可诱导的，是由一组编码β-内酰胺类抗生素的基因产生的。 传感器/信号转导蛋白、一个基因抑制子和两个耐药决定因素(Blazβ-内酰胺酶和 一种独特的青霉素结合蛋白，命名为PBP2a)。这些过程的一个关键特征是识别 抗生素通过β-内酰胺传感器/信号转导BLAR，一种完整的膜蛋白。认识到 抗生素由BLAR的感应域通过膜释放构象变化，这 导致胞质侧的蛋白酶域被激活。这一过程最终以表达 抗生素耐药性决定因素的基因。在具体目标1中，我们描述了荧光的使用 在活的金黄色葡萄球菌中发现关闭BLAR对β-内酰胺类抗生素识别的药物的工具， 这将逆转抗性表型。提出了一个用于结构活性分析的发现漏斗 这些化合物之间的关系。在特定目标2中，我们传达了一项发现，Blaz b-内酰胺酶是 以一种依赖于脂肪和磷酸化的方式结合到细胞膜表面 举止。我们概述了一种纯化膜锚定Blaz的方法，目的是 磷酸化位点的鉴定。该蛋白质将用于动力学研究，以与非 膜锚定的Blaz，它没有被磷酸化。详细介绍了X射线结晶学的计划 结构性问题的表征。脂化和磷酸化依赖的通用性 在金黄色葡萄球菌中，我们将探索10种不同蛋白质在膜表面的锚定作用。在每一个中 在这种情况下，概述了确定磷酸化位置的计划，以阐明磷酸化和 这些蛋白质的膜隔离。这些研究将最终阐明复杂的机械结构。 金黄色葡萄球菌菌株已经进化出对β-内酰胺类抗生素的抗药性。

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.