Molecular and structural analysis of Streptococcus pneumoniae teichoic acid biosynthesis and implications of teichoic acid alterations on the bacterial pathophysiology

肺炎链球菌磷壁酸生物合成的分子和结构分析以及磷壁酸改变对细菌病理生理学的影响

• 批准号: 258667519
• 负责人: Dr. Nicolas Gisch
• 金额: --
• 依托单位: Forschungsgruppe Bioanalytische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/258667519?language=en
• 关键词: Molecular; structural; analysis; Streptococcus; pneumoniae

Teichoic acids (TAs) are important components of the Streptococcus pneumoniae cell wall. Wall teichoic acids (WTA) and lipoteichoic acids (LTA) of S. pneumoniae are structurally identical and contain phosphorylcholine (P-Cho) substituents. The P-Cho anchors a special class of pneumococcal surface proteins, the so-called choline-binding proteins (CBPs) non-covalently to the cell surface. In the last funding period, we gained detailed insights into the genetic and structural requirements of pneumococcal teichoic acid, especially LTA, biosynthesis and modification, as well as on the impact of presence/absence of LTAs on the pathophysiology of S. pneumoniae. Most importantly, we demonstrated that SPD_1672 (strain D39) / SP_1893 (strain TIGR4), now renamed by us to TacL, is the lipoteichoic acid ligase in S. pneumoniae. Pneumococcal mutants deficient in TacL showed a total loss of LTA and were significantly impaired in virulence in murine models of acute pneumonia and systemic infection, although they grew normally in culture and did not exhibit changes in cell morphology or division compared to parental strains.The first major aim of the proposed project is to understand the consequences of LTA absence in pathophysiological processes in detail. Therefore, we will use a broad range of approaches like cell-based adhesion assays, analysis of biofilm formation capacity and determine the lipid mediator profile in e.g. epithelial cells after stimulation with pneumococcal wild-type and mutant strains to analyze this. As a perspective, we aim to reconstitute the TacL-mediated transfer of the Und-PP bound TA-precursor chain onto the glycolipid anchor in an in vitro assay. The second major focus of this project is to gain insight into the mode of action of the LytR-Cps2A-Psr (LCP) protein family. These proteins/enzymes possess most likely semi-redundant roles in connecting the WTA and/or the capsule to the peptidoglycan. Based on methodologies, basically set up during the first funding period, we aim to decipher the impact of LytR, Cps2A, or Psr on the amount of WTA and capsule attachment and we will investigate the virulence potential of single, double or triple LCP-mutants in comparison to the isogenic wild-type. The mutants will be phenotypically characterized in S. pneumoniae TIGR4 and D39. The TAs of these strains will be isolated, structurally analyzed and quantified. For this, we again combine the two major expertises of two successfully collaborating groups working in the research area of infection biology and chemical structural analysis, respectively. The characterization of the binding of CBPs to P-Cho of TAs and the identification of minimal TA part structures required for these interactions will be continued. Finally, we will investigate the role of pneumococcal TAs in the immune recognition, with a special focus on the complement system, in a defined lipopeptide and capsule deficient background to figure out the specific contribution of pnTAs.

磷壁酸是肺炎链球菌细胞壁的重要组成部分。S.肺炎链球菌和肺炎链球菌在结构上相同，并且含有磷酸胆碱（P-Cho）取代基。P-Cho将一类特殊的肺炎球菌表面蛋白，即所谓的胆碱结合蛋白（CBP）非共价地锚定在细胞表面。在上一个资助期，我们详细了解了肺炎球菌磷壁酸的遗传和结构要求，特别是LTA，生物合成和修饰，以及LTA的存在/不存在对S.肺炎。最重要的是，我们证明了SPD_1672（菌株D39）/ SP_1893（菌株TIGR 4），现在我们重新命名为TacL，是S.肺炎。TacL缺陷的肺炎球菌突变体表现出LTA的总损失，并在急性肺炎和全身性感染的小鼠模型中的毒力显着受损，虽然他们在培养中正常生长，并没有表现出细胞形态或分裂的变化相比，亲本strain.The第一个主要目的的拟议项目是详细了解LTA的缺乏在病理生理过程中的后果。因此，我们将使用广泛的方法，如基于细胞的粘附测定，生物膜形成能力的分析，并确定例如用肺炎球菌野生型和突变株刺激后上皮细胞中的脂质介质谱，以分析这一点。作为一个角度来看，我们的目标是重建TacL介导的Und-PP结合TA-前体链转移到糖脂锚在体外测定。该项目的第二个主要重点是深入了解LytR-Cps 2A-Psr（LCP）蛋白家族的作用模式。这些蛋白质/酶在将WTA和/或胶囊连接至肽聚糖中具有最可能的半冗余作用。基于基本上在第一个资助期内建立的方法，我们的目标是破译LytR，Cps 2A或Psr对WTA和胶囊附着量的影响，我们将研究单，双或三重LCP突变体与同基因野生型相比的毒力潜力。突变体将在S. pneumoniae TIGR 4和D39。这些菌株的TA将被分离、结构分析和定量。为此，我们再次联合收割机的两个主要专业知识的两个成功的合作小组工作在感染生物学和化学结构分析的研究领域，分别。将继续对CBP与TA的P-Cho结合进行表征，并鉴定这些相互作用所需的最小TA部分结构。最后，我们将研究肺炎球菌TA在免疫识别中的作用，特别关注补体系统，在一个定义的脂肽和胶囊缺陷的背景下，找出具体的贡献pnTA。