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: 6521686
• 负责人: CRAIG E. RUBENS
• 金额: $ 29.73万
• 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/6521686
• 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（由申请人提供）：尽管 CDC 推荐了化学预防策略来预防该微生物引起的感染，但 B 族链球菌 (GBS) 仍然是导致美国新生儿败血症、肺炎和脑膜炎的最重要的细菌病原体。除荚膜外，GBS 在宿主体内存活所需的因素尚不明确。最近，使用特征标签转座子诱变 (STM) 来鉴定新生大鼠脓毒症感染模型中 GBS 生长和存活所需的基因。很大一部分无毒突变体在涉及细胞表面代谢的基因中插入了转座子，强调了这些功能对于 GBS 体内存活的重要性。我们鉴定了细胞表面代谢组中减毒程度最高的突变体，该突变体在假定的青霉素结合蛋白基因（ponA）同源物中插入了转座子。基于序列同源性，被破坏的 GBS 基因预计会编码 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，根据体外杀菌测定的结果，PBP1a 基因突变体在抵抗调理吞噬杀伤方面表现出显着的缺陷。体内互补分析证实，在突变体中观察到的表型改变是由于 ponA 中转座子的插入所致。 PBP1a基因突变体在体外具有正常生长速率，产生野生型水平的荚膜多糖，并且在其他表型上与亲本菌株相同。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 在与宿主相互作用以及 GBS 体内毒力中的作用。目标 1 将完成对 ponA 基因和 prfA 基因（青霉素结合蛋白相关因子 A）（与 ponA 共转录的基因）的分析。将构建非极性 prfA 缺失突变体并进行表型分析。目标 2 将使用遗传和生化方法来定义 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 中。对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对调理吞噬杀伤的抵抗力所必需的。目标 3 将通过研究 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。虽然据报道，在许多感染动物模型中，细菌细胞壁相关酶（包括 PBP）是毒力所必需的，但尚未提出任何机制来解释这些观察结果。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。