Role of penicillin binding protein 1a in GBS virulence

描述（由申请人提供）：在美国，B族链球菌（GBS）仍然是导致新生儿败血症、肺炎和脑膜炎的最重要的细菌病原体，尽管cdc推荐了预防这种微生物感染的化学预防策略。除了荚膜外，GBS在宿主体内存活所需的因素尚未明确。最近，在新生儿大鼠脓毒症感染模型中，使用标记转座子诱变（STM）来鉴定GBS生长和存活所需的基因。相当大比例的无毒突变体在参与细胞表面代谢的基因中插入转座子，强调了这些功能对GBS体内存活的重要性。我们鉴定了细胞表面代谢组中最弱的突变体，该突变体在假定的青霉素结合蛋白基因（ponA）同源物中插入转座子。基于序列同源性，预测被破坏的GBS基因编码a类，高分子量青霉素结合蛋白（PBP1a），具有转糖基化酶和转肽酶活性。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%致死剂量试验中均显著减弱。此外，PBP1a基因突变体在体外杀菌试验中显示出对调理自噬细胞杀伤的抗性显著缺陷。体内互补分析证实，突变体的表型改变是由于转座子插入ponA。PBP1a基因突变体在体外具有正常的生长速度，产生野生型水平的荚膜多糖，其他表型与亲本菌株相同。我们假设GBS ponA基因是体外抗调理自噬细胞杀伤和体内毒力所必需的。我们的研究旨在确定PBP1a在体内与宿主相互作用和GBS毒力中的作用。目的1将完成ponA基因和prfA基因（青霉素结合蛋白相关因子A）的分析，prfA基因是与ponA共转录的基因。构建非极性prfA缺失突变体并进行表型分析。目的2将使用遗传和生化方法来定义PBP1a蛋白的结构和功能。位点定向突变破坏蛋白质的酶活性将被引入PBP1a。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需要对调理自噬细胞杀伤的毒力和抗性。目的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抗调理吞噬细胞杀伤中的作用。虽然包括PBPs在内的细菌细胞壁相关酶已被报道为许多感染动物模型的毒力所必需，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，将进一步了解GBS感染的发病机制，并可能确定预防或治疗方式的靶点。

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.