PENICILLIN INTERACTIVE PROTEINS OF STAPHYLOCOCCUS AUREUS

金黄色葡萄球菌的青霉素相互作用蛋白

• 批准号: 6349926
• 负责人: Henry F HENRY CHAMBERS
• 金额: $ 31.23万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349926
• 关键词: DNA binding protein; Staphylococcus aureus; Staphylococcus infection; bacteria infection mechanism; bacterial genetics; bacterial proteins; beta lactamase; biological signal transduction; cell cycle; enzyme activity; enzyme induction /repression; gene deletion mutation; gene expression; immunoelectron microscopy; metalloendopeptidases; methicillin; multidrug resistance; northern blottings; penicillins; protein localization; protein protein interaction; protein structure function; regulatory gene; sequence tagged sites; zymogens

Methicillin-resistant strains of Staphylococcus aureus are a major clinical problem. They are multiple drug resistant, but ineffectiveness of penicillins and beta-lactam antibiotics is the real problem, as these are drug of choice to treat staphylococcal infections. The objective of this research is to further knowledge of mechanisms of methicillin resistance. Resistance is determined by several proteins that interact with penicillin. The interactions among these proteins are critical, but poorly understood. Knowledge of these interrelationships may lead to new drug discovery and new and more effective approaches to therapy./ Resistance is mainly due to production a novel low affinity penicillin bind protein, PBP 2a, a well wall synthetic enzyme. PBP 2a seems to substitute for all other PBPs. mecA, the gene encoding PBP 2a, is regulated by the same regulatory genes that control production of inducible beta-lactamase. Another type of penicillin interactive protein, a penicillin sensory signal transducer BlaR1, signals the cell to express PBP 2a and beta-lactamase, which together mediate all beta-lactam resistance in staphylococci. BlaR1 appears to be a PBP fused to an intracellular Zn++ metalloprotease, and as such may represent a completely new type of transmembrane signaling system. There are three aims. Aim1. To determine the intracellular pathway by which penicillin binding to BlaR1 signals induction of beta- lactamase and PBP 2a. The effect of specific mutations in BlaR1 on signaling will be determined to prove whether or not Blar1 is a metalloprotease. Putative consensus motifs of this superfamily of proteins will be targeted. The relationship between BlaR1 activation and proteolysis of BlaI, the repressor of the beta-lactamase regulon, will be defined. Aim 2. To identify PBPs, structural determinants, and other elements that interfere with PBP 2a mediated resistance. Effects of PBP deletion and mutations on PBP 2a mediated resistance will test whether PBP 2a can substitute for other PBPs and where essential functions reside within the molecule. The curious phenomenon of negative selection for expression of PBP 2a that we observed in mec naive cells also will be examined. Aim 3. To determine when during the cell cycle PBPs are expressed and where they are localized. An electron microscopic method for immunolocalization of specific myc-targeted PBPs in the cell will be developed. To augment information about where PBPs localize, when they are expressed during the cell cycle will be determined by Northern blotting.

金黄色葡萄球菌的甲氧西林耐药菌株是一个主要的临床问题。它们具有多重耐药性，但青霉素和 β-内酰胺抗生素的无效才是真正的问题，因为这些是治疗葡萄球菌感染的首选药物。本研究的目的是进一步了解甲氧西林耐药机制。耐药性是由与青霉素相互作用的几种蛋白质决定的。这些蛋白质之间的相互作用至关重要，但人们对其知之甚少。了解这些相互关系可能会导致新药的发现和新的更有效的治疗方法。/耐药性主要是由于产生了一种新型低亲和力青霉素结合蛋白 PBP 2a，一种孔壁合成酶。 PBP 2a 似乎可以替代所有其他 PBP。 mecA 是编码 PBP 2a 的基因，受到控制诱导型 β-内酰胺酶产生的相同调控基因的调控。另一种类型的青霉素相互作用蛋白，青霉素感觉信号转导器 BlaR1，向细胞发出信号以表达 PBP 2a 和 β-内酰胺酶，它们共同介导葡萄球菌中的所有 β-内酰胺耐药性。 BlaR1 似乎是与细胞内 Zn++ 金属蛋白酶融合的 PBP，因此可能代表一种全新类型的跨膜信号系统。有三个目标。目标1。确定青霉素与 BlaR1 结合发出诱导 β-内酰胺酶和 PBP 2a 信号的细胞内途径。将确定 BlaR1 中特定突变对信号传导的影响，以证明 Blar1 是否是金属蛋白酶。该蛋白质超家族的假定共有基序将成为目标。 BlaR1 激活与 BlaI（β-内酰胺酶调节子阻遏物）的蛋白水解之间的关系将被定义。目标 2. 鉴定 PBP、结构决定因素和其他干扰 PBP 2a 介导的耐药性的元件。 PBP 缺失和突变对 PBP 2a 介导的耐药性的影响将测试 PBP 2a 是否可以替代其他 PBP 以及分子内的基本功能。我们在 mec 幼稚细胞中观察到的 PBP 2a 表达负选择的奇怪现象也将得到检验。目标 3. 确定 PBP 在细胞周期中的表达时间及其定位位置。将开发一种电子显微镜方法，用于细胞中特定 myc 靶向 PBP 的免疫定位。为了增强有关 PBP 定位位置的信息，将通过 Northern 印迹确定它们在细胞周期中何时表达。