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

• Applicants: Dr. Nicolas Gisch; Professor Dr. Sven Hammerschmidt
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Analytical Chemistry ; Biological and Biomimetic Chemistry
• Term: from 2014 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258667519

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.

• DFG Programme: Research Grants