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.

金黄色葡萄球菌的耐甲氧西林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-内酰胺类抗生素的无效是真实的问题，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的机制。耐药性由几种与青霉素相互作用的蛋白质决定。这些蛋白质之间的相互作用是至关重要的，但了解甚少。这些相互关系的知识可能会导致新的药物发现和新的和更有效的治疗方法。耐药主要是由于产生了一种新的低亲和力青霉素结合蛋白PBP 2a，这是一种井壁合成酶。PBP 2a似乎可以替代所有其他PBP。编码PBP 2a的基因mecA受控制诱导型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BlaR 1，向细胞发出信号以表达PBP 2a和β-内酰胺酶，它们一起介导葡萄球菌中的所有β-内酰胺抗性。BlaR 1似乎是融合到细胞内Zn++金属蛋白酶的PBP，因此可能代表一种全新类型的跨膜信号传导系统。有三个目标。目标1.确定青霉素与BlaR 1结合信号诱导β-内酰胺酶和PBP 2a的细胞内途径。将确定BlaR 1中的特定突变对信号传导的影响，以证明Blar 1是否是金属蛋白酶。该蛋白质超家族的推定共识基序将成为目标。将确定BlaR 1激活和BlaI（β-内酰胺酶调节子的阻遏物）蛋白水解之间的关系。目标2.鉴定干扰PBP 2a介导的抗性的PBP、结构决定簇和其他元件。PBP缺失和突变对PBP 2a介导的抗性的影响将测试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表达的负选择的奇怪现象也将被检查。目标3.为了确定在细胞周期期间PBPs何时表达以及它们定位在何处。将开发用于细胞中特定myc靶向PBPs的免疫定位的电子显微镜方法。为了增加关于PBP定位于何处的信息，将通过北方印迹法确定它们在细胞周期期间何时表达。