Defining PcsB: Essential Murein Biosynthesis Protein in Pathogenic Pneumococci

项目概述：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，利用一组保守和定义的晚期细胞壁生物合成蛋白，这些蛋白是特定茎肽物种交联所必需的，创造了一系列微妙的特定交联。这些交联使细胞壁充当网状包裹，使细胞耐受渗透裂解，从而使细胞存活。特别令人感兴趣的是革兰氏阳性致病菌（如肺炎链球菌）中小鼠生物合成所需的新型必需蛋白质，因为为了生产更有效的抗生素，需要新的抗菌靶点。其中一种蛋白质是部分表征和必要的细胞壁转肽酶PcsB。本工作的重点是确定PcsB在肺炎链球菌中的功能，这将有两个具体目标。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，利用单一和共定位策略，以及免疫印迹技术来完成，并将用于设定PcsB的作用时间，并建议可能的相互作用蛋白。第二个目的是通过反相高压液相色谱和质谱法检查体外气氛中交联的程度，从生物化学角度定义PcsB的功能。了解PcsB在肺炎球菌细胞周期中的定位模式，以及确定这种必需蛋白的生化功能，将有助于确定我们对革兰氏阳性人类病原体肺炎链球菌细胞壁生物合成的认识。相关性：肺炎链球菌是一种主要存在于人类呼吸道的革兰氏阳性细菌病原体，感染后可导致鼻窦炎、脑膜炎、肺炎、败血症、中耳炎等多种疾病，是生物医学研究的重要细菌。在过去四十年中，肺炎球菌疾病呈上升趋势，对内酰胺类抗生素和其他常用的抗微生物药物产生耐药性，因此始终需要找到对抗这种人类病原体的新方法。确定抗菌药物新蛋白靶点的一种方法是研究肺炎链球菌细胞壁的生物合成，特别是研究对生物体生存能力至关重要的蛋白质，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.