Defining PcsB: Essential Murein Biosynthesis Protein in Pathogenic Pneumococci

描述(由申请人提供)：项目摘要：革兰氏阳性细菌的肽聚糖细胞壁仍然是细菌细胞的活性成分，利用一组保守和明确的晚期细胞壁生物合成蛋白质，这是特定干肽物种交联所必需的，创造了一系列微妙的特定交联物。这些交叉连接使细胞壁起到网状包裹的作用，使细胞对渗透溶解具有耐受性，从而允许细胞存活。尤其令人感兴趣的是革兰氏阳性病原菌(如肺炎链球菌)中毛霉素素生物合成所需的新的和必要的蛋白质，因为为了生产更有效的抗生素，需要新的抗菌靶。其中一种蛋白质是部分特征的和必要的细胞壁转肽酶，PCSB。这项工作的重点是确定PCSB在肺炎链球菌中的功能，这将通过两个特定的目标来实现。第一个目标是在细胞周期中定位PCSB。这将通过免疫荧光方法实现，利用单一和共定位策略，以及免疫印迹技术，并将用于设定PCSB的作用时间，以及建议可能的相互作用蛋白。第二个目标是通过反相高压液相色谱和质谱学方法检测体外环境中PCSB的交联度，从而从生化角度确定PCSB的功能。了解肺炎球菌PCSB在细胞周期中的定位模式，以及确定这一必需蛋白的生化功能，将有助于我们确定革兰氏阳性人类病原体肺炎链球菌细胞壁生物合成的知识。肺炎链球菌是一种革兰氏阳性细菌，主要发生在人体呼吸道，其感染可导致鼻窦炎、脑膜炎、肺炎、败血症和中耳炎等多种疾病，使肺炎链球菌成为生物医学研究的重要细菌。在过去的40年里，肺炎球菌疾病一直在增加，对内酰胺类抗生素和其他常用的抗微生物药物产生了耐药性，因此始终需要找到新的方法来对抗这种人类病原体。确定新的抗菌药物蛋白质靶点的一种方法是研究肺炎链球菌的细胞壁生物合成，特别是对生物体生存所必需的蛋白质的研究，如PCSB。

DESCRIPTION (provided by applicant): Project Summary: The peptidoglycan cell wall of gram-positive bacteria remains an active element of the bacterial cell, utilizing a conserved and defined set of late cell wall biosynthesis proteins that are required for the cross-linking of specific stem peptide species, creating a delicate array of specific cross-links. These cross-links allow the cell wall to act as a mesh encasement, making the cell tolerant to osmotic lysis, and therefore permitting cell survival. Of particular interest are novel and essential proteins required for murein biosynthesis in gram- positive pathogenic bacteria, such as Streptococcus pneumoniae, as new antimicrobial targets are needed in order to produce more effective antibiotics. One such protein is the partially characterized and essential putative cell wall transpeptidase, PcsB. The focus of this work is to determine the function of PcsB in S. pneumoniae, which will be accomplished with two specific aims. The first aim is to localize PcsB during the cell cycle. This will be accomplished with immunofluorescence methods, utilizing single and co-localization tactics, as well as immunoblot techniques, and will serve to set a time of action for PcsB as well as suggest possible interacting proteins. The second aim is to biochemically define the function of PcsB by examining the extent of cross-linking in an in vitro atmosphere by means of reverse-phase high pressure liquid chromatography and mass spectrometry methods. Understanding both the localization patterns of PcsB in pneumococcus during the cell cycle, as well as determining the biochemical function of this essential protein will help to define our knowledge of cell wall biosynthesis in the gram-positive human pathogen, S. pneumoniae. Relevance: S. pneumoniae is a gram-positive bacterial pathogen mainly of the human respiratory tract, infection of which can result in several diseases, including sinusitis, meningitis, pneumonia, septicemia, and otitis media, making S. pneumoniae an important bacteria for biomedical research. In the past four decades, pneumococcal diseases have been on the rise with resistance to ¿-lactam antibiotics and other commonly used anti-microbial drugs, therefore creating an ever-present need to find new ways to combat this human pathogen. One way to determine new protein targets for antimicrobial drugs is the study of cell wall biosynthesis in S. pneumoniae, particularly the study of proteins essential to the viability of the organism, such as PcsB.