PENICILLIN INTERACTIVE PROTEINS OF STAPHYLOCOCCUS AUREUS

耐甲氧西林金黄色葡萄球菌是临床面临的主要问题。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正的问题，因为它们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是至关重要的，但人们对此知之甚少。对这些相互关系的了解可能导致新的药物发现和新的更有效的治疗方法。/耐药性主要是由于产生一种新的低亲和力青霉素结合蛋白，PBP 2a，一种良好的壁合酶。PBP 2a似乎取代了所有其他PbP。编码PBP 2a的基因MecA受控制诱导性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蛋白Blar1，向细胞发出信号，使其表达PBP 2a和β-内酰胺酶，这两种酶共同介导葡萄球菌对所有β-内酰胺类抗生素的耐药性。BlaR1似乎是一种与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。目的：1.目的：确定青霉素与BlaR1结合的细胞内途径，以诱导β-内酰胺酶和PBP2a的产生。BlaR1中特定突变对信号的影响将被确定，以证明Blar1是否是一种金属蛋白酶。这个蛋白质超家族的假定共识基序将成为靶点。BLAI是β-内酰胺酶调节子的抑制子，BLAI的蛋白分解与BLAR1的激活之间的关系将被定义。目的2.鉴定影响PBP2a介导的抗性的PBPs、结构决定因素和其他因素。PBP缺失和突变对PBP 2a介导的抗性的影响将检验PBP 2a是否可以替代其他PBP，以及必要的功能驻留在分子中的哪里。我们还将研究我们在原始细胞中观察到的对PBP2a表达进行负选择的奇怪现象。目的3.确定PBPs在细胞周期中的表达时间和定位。将开发一种电子显微镜方法，用于在细胞中免疫定位特定的myc靶向PBPs。为了增加关于PBPs定位的信息，它们在细胞周期中何时表达将由Northern blotting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.