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

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
 
Final Report Year 2025

Final Report Abstract

Teichoic acids (TAs) belong to the important components of the Streptococcus pneumoniae (Spn) cell wall. We here gained new insights into the TA biosynthesis and modification as well as on the impact of LTAs on the pathophysiology of Spn. We demonstrated that SPD_1672 (strain D39)/SP_1893 (strain TIGR4), named now TacL, is the lipoteichoic acid ligase in Spn. Mutants deficient in TacL showed a total loss of LTA and were significantly impaired in virulence in murine models, although phenotypically inconspicuous. To understand the consequences of LTA absence, TacL-deficient mutants and respective complemented strains were constructed. We showed that LTA is indispensable for Spn adherence to human nasopharyngeal cells and colonization. Further a decreased membrane fluidity and higher surface abundance of lipoproteins (LPs) in TacL mutants, accompanied by an increased susceptibility to antimicrobial peptides, is indicated. Thus, LTA is crucial for colonization and required for effectively protecting pneumococci from host immune defence mechanisms. Till date, a quantitative description of the membrane lipidome of Spn remained elusive. We developed a standardized pipeline for in-depth lipidomic analysis of Spn and applied it to serotype 2 strain D39 as well as three isogenic mutant strains (D39∆tacL, D39∆lgt, D39∆cps). All mutants are in the biosynthesis of a major cell wall component (LTA, LPs, or capsular polysaccharide (CPS), respectively), thus a change in the lipidome was theoretically conceivable. Lipid profiles of the ΔtacL and Δlgt mutant showed no significant alterations, but the Δcps strain exhibited substantial lipidome remodeling. This data is the basis for future lipidomic investigations e.g., of non-encapsulated, therefore non-typeable clinical isolates. Further, we gained insight into the mode of action of the LytR-Cps2A-Psr (LCP) protein family in Spn. These enzymes possess semi-redundant roles in connecting the WTA and the CPS to the peptidoglycan. We aimed to decipher the impact of LytR, Cps2A, or Psr on the amount of WTA and CPS attachment as well as on virulence. We generated lcp single- or double-mutants in Spn D39. Mutant phenotypes indicated an increased susceptibility to various antibiotics and oxidative stress as well as a decreased virulence in in vivo infection models. While the lytR-mutant showed an impaired cell division, the psr-mutants showed an overall reduced amount CPS, but also striking CPS-expression heterogeneity in the double mutant ∆cps2A∆psr. Even after isolating the subpopulations, the heterogeneity remained stable. Interestingly, a genome-wide CRISPRi-seq screen showed that genes in TA-biosynthesis are crucial during competence. Data suggested that LytR is important for WTA formation and together with ComM essential for immunity against CbpD. The development of a GC/MS-based method for pneumococcal teichoic acid quantification was promoted in this project and first experiments underlined the importance of LytR for WTA formation.

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