PENICILLIN INTERACTIVE PROTEINS OF STAPHYLOCOCCUS AUREUS

耐甲氧西林金黄色葡萄球菌是一个主要的临床问题。它们具有多重耐药，但青霉素和β -内酰胺类抗生素的无效是真正的问题，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。耐药性是由几种与青霉素相互作用的蛋白质决定的。这些蛋白质之间的相互作用至关重要，但人们对其了解甚少。了解这些相互关系可能会导致新的药物发现和新的更有效的治疗方法。耐药主要是由于产生一种新的低亲和力青霉素结合蛋白PBP 2a，一种井壁合成酶。PBP 2a似乎可以替代所有其他PBP。mecA，编码PBP 2a的基因，受控制诱导型β -内酰胺酶产生的相同调控基因的调控。另一种青霉素相互作用蛋白是青霉素感觉信号传感器BlaR1，它向细胞发出信号，表达PBP 2a和β -内酰胺酶，它们共同介导葡萄球菌的所有β -内酰胺抗性。BlaR1似乎是细胞内锌金属蛋白酶融合的PBP，因此可能代表了一种全新的跨膜信号系统。有三个目标。Aim1。确定青霉素结合BlaR1信号诱导β -内酰胺酶和PBP 2a的胞内途径。BlaR1特异性突变对信号传导的影响将被确定，以证明BlaR1是否是金属蛋白酶。这个蛋白质超家族的假定共识基序将被靶向。BlaR1的激活与β -内酰胺酶调控的抑制因子BlaI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介导的抗性的因素。PBP缺失和突变对PBP 2a介导的耐药的影响将测试PBP 2a是否可以替代其他PBP，以及基本功能在分子内的位置。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被检查。目标3。确定pbp在细胞周期中的表达时间和定位位置。将开发一种在细胞中免疫定位特异性myc靶向PBPs的电镜方法。为了增加关于pbp定位的信息，它们在细胞周期中何时表达将通过Northern印迹法确定。

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.