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

壁苔酸（TAs）是肺炎链球菌细胞壁的重要成分。肺炎链球菌壁磷壁酸（WTA）和脂磷壁酸（LTA）结构相同，均含有磷酰胆碱（P-Cho）取代基。P-Cho锚定了一类特殊的肺炎球菌表面蛋白，即所谓的胆碱结合蛋白（CBPs）非共价地附着在细胞表面。在上一个资助期，我们深入了解了肺炎球菌壁藻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/缺失对肺炎链球菌病理生理的影响。最重要的是，我们证明了SPD_1672（菌株D39） / SP_1893（菌株TIGR4）（现在被我们重新命名为TacL）是肺炎链球菌的脂质胆酸连接酶。缺乏TacL的肺炎球菌突变体在急性肺炎和全身感染的小鼠模型中表现出LTA的完全丧失，并且毒力明显受损，尽管它们在培养中正常生长，并且与亲本菌株相比没有表现出细胞形态或分裂的变化。该项目的第一个主要目的是详细了解LTA缺失在病理生理过程中的后果。因此，我们将使用广泛的方法，如基于细胞的粘附试验，分析生物膜形成能力，并确定在受到肺炎球菌野生型和突变菌株刺激后上皮细胞中的脂质介质谱来分析这一点。从这个角度来看，我们的目标是在体外实验中重建tacl介导的将Und-PP结合的ta前体链转移到糖脂锚上的过程。该项目的第二个重点是深入了解LytR-Cps2A-Psr （LCP）蛋白家族的作用模式。这些蛋白/酶在将WTA和/或胶囊连接到肽聚糖方面很可能具有半冗余作用。基于第一个资助期基本建立的方法，我们的目标是破译LytR， Cps2A或Psr对WTA和胶囊附着量的影响，我们将研究单，双或三重lcp突变体与等基因野生型相比的毒力潜力。突变体将在肺炎链球菌TIGR4和D39中表现出表型特征。这些菌株的TAs将被分离、结构分析和定量。为此，我们再次结合了两个成功合作小组在感染生物学和化学结构分析研究领域的两个主要专业知识。将继续研究CBPs与TA的P-Cho结合的特征以及这些相互作用所需的最小TA部分结构的鉴定。最后，我们将研究肺炎球菌TAs在免疫识别中的作用，特别关注补体系统，在确定脂肽和胶囊缺乏的背景下，找出pnt TAs的具体贡献。

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.