Role of penicillin binding protein 1a in GBS virulence

青霉素结合蛋白 1a 在 GBS 毒力中的作用

• 批准号: 6611349
• 负责人: CRAIG E. RUBENS
• 金额: $ 33.53万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-15 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611349
• 关键词: Streptococcus agalactiae; bacteria infection mechanism; bacterial genetics; bacterial proteins; binding proteins; cell wall; clinical research; gene deletion mutation; gene expression; host organism interaction; human subject; laboratory rat; opsonin; penicillins; phagocytosis; phlebotomy; protein structure function; serum; site directed mutagenesis; virulence

DESCRIPTION (provided by applicant): Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection due to this organism. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. Recently, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. A significant proportion of the avirulent mutants had transposon insertions in genes involved in cell surface metabolism emphasizing the significance of these functions for in vivo survival of GBS. We characterized the most attenuated mutant from the cell-surface metabolism group, which had a transposon insertion in a putative penicillin-binding protein gene (ponA) homologue. Based on sequence homology, the disrupted GBS gene is predicted to code for a class A, high molecular weight penicillin-binding protein (PBP1a), possessing both transglycosylase and transpeptidase activity. These bifunctional enzymes catalyze both the polymerization and cross-linking of bacterial peptidoglycan. The PBP1a gene mutant was significantly attenuated in both competitive index and 50 percent lethal dose assays of GBS virulence in neonatal rats. Additionally, the PBP1a gene mutant displayed a significant defect in resistance to opsonophagocytic killing as measured by in vitro bactericidal assays. Complementation analysis in vivo confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. The PBP1a gene mutant had a normal growth rate in vitro, produced wild-type levels of capsular polysaccharide and was otherwise phenotypically identical to the parent strain. We hypothesize that the GBS ponA gene is required for resistance to opsonophagocytic killing in vitro and virulence in vivo. Our investigation seeks to define the role of PBP1a in interactions with the host and virulence of GBS in vivo. Aim 1 will complete analysis of the ponA gene and the prfA gene (penicillin binding protein related factor A), a gene cotranscribed with ponA. A nonpolar prfA deletion mutant will be constructed and subjected to phenotypic analysis. Aim 2 will use genetic and biochemical approaches to define the structure and function of the PBP1a protein. Site-directed mutations that disrupt enzymatic activity of the protein will be introduced into PBP1a. Analysis of these mutants will allow us to evaluate whether enzymatic activity of the protein is required for virulence and resistance to opsonophagocytic killing. Aim 3 will determine the role of the PBP1a protein in resistance of GBS to opsonophagocytic killing by investigating the interaction of aponA mutant with serum opsonins and phagocytic cells. While bacterial cell-wall associated enzymes, including PBPs, have been reported to be required for virulence in numerous animal models of infection, no mechanism has been proposed to explain these observations. These studies will be the first to systematically investigate the role of PBP1a in virulence, will further our understanding of the pathogenesis of GBS infections, and may identify targets for preventative or therapeutic modalities.

描述（由申请方提供）：B族链球菌（GBS）仍然是美国引起新生儿败血症、肺炎和脑膜炎的最重要细菌病原体，尽管CDC推荐了预防该微生物感染的化学预防策略。除了胶囊，GBS在宿主中存活所需的因素还没有很好的定义。最近，标记转座子诱变（STM）被用来确定所需的基因的生长和生存的新生大鼠败血症感染模型中的GBS。一个显着比例的无毒突变体转座子插入基因参与细胞表面代谢，强调这些功能在体内生存的GBS的意义。我们的特点是从细胞表面代谢组，其中有一个转座子插入在一个假定的青霉素结合蛋白基因（ponA）同源的最减毒突变体。基于序列同源性，预测破坏的GBS基因编码A类高分子量青霉素结合蛋白（PBP1a），具有转糖基酶和转肽酶活性。这些双功能酶催化细菌肽聚糖的聚合和交联。PBP1a基因突变体在新生大鼠GBS毒力的竞争指数和50%致死剂量测定中均显着减弱。此外，PBP1a基因突变体在体外杀菌试验中显示出对调理吞噬细胞杀伤的抗性的显著缺陷。体内互补分析证实，在突变体中观察到的改变的表型是由于转座子插入ponA。PBP1a基因突变体在体外具有正常的生长速率，产生野生型水平的荚膜多糖，并且在其他方面与亲本菌株表型相同。我们假设GBS ponA基因是抵抗体外调理吞噬杀伤和体内毒力所必需的。我们的研究旨在确定PBP1a在与宿主相互作用和体内GBS毒力中的作用。目的1完成ponA基因和与ponA共转录的prfA基因的分析。将构建非极性prfA缺失突变体并进行表型分析。目的2将利用遗传学和生物化学方法来确定PBP1a蛋白的结构和功能。破坏蛋白质酶活性的定点突变将被引入PBP1a。这些突变体的分析将使我们能够评估蛋白质的酶活性是否是毒力和抗调理吞噬杀伤所必需的。目的3通过研究aponA突变体与血清调理素和吞噬细胞的相互作用，确定PBP1a蛋白在GBS抵抗调理吞噬杀伤中的作用。虽然已报道细菌细胞壁相关酶（包括PBPs）是许多动物感染模型中毒力所需的，但尚未提出解释这些观察结果的机制。这些研究将首次系统地研究PBP1a在毒力中的作用，将进一步加深我们对GBS感染发病机制的理解，并可能确定预防或治疗方式的靶点。