Soutenance de thèse : The interplay of Teichoic Acids and Peptidoglycan synthesis in Streptococcus pneumoniae cell envelope

Par Mai NGUYEN (IBS/Groupe Pneumocoque)

All bacteria are surrounded by a multilayered envelope that protects the cell, defines cell morphology, and acts as a semi-permeable barrier that regulates the entry and efflux of various molecules. In the Gram-positive opportunistic human pathogen Streptococcus pneumoniae, the envelope contains a thick cell wall made up of peptidoglycan (PG), a 3D network of glycan strands cross-linked by peptide chains, which confers the cell shape, and mechanical resistance against the cytoplasmic turgor pressure. Also residing in the Gram-positive cell envelope are teichoic acids (TA), consisting of wall teichoic acids (WTA) covalently attached to the PG layer, and lipo-teichoic acids (LTA) anchored in the cytoplasmic membrane. In S. pneumoniae, TA are especially decorated with phosphocholine residues that serve as docking sites for Choline-Binding Proteins (CBPs), which play various important functions, such as PG cleavage (including autolysis) and virulence.

While PG has been intensively studied over the years, TA remain poorly understood in terms of functions and assembly, despite being a major cell envelope component. To examine the synthesis of TA and their relationship with PG metabolism, we employed complementary cellular and molecular approaches. We used in particular a click-chemistry labelling method, based on the metabolic incorporation of modified metabolites into TA and PG. In this method, compatible clickable fluorophores can be conjugated to the incorporated metabolite analogues, permitting the observation of the polymers by fluorescence imaging (SDS-PAGE analysis of isolated TA, and optical microscopy visualization of S. pneumoniae cells). Due to the sub-diffraction spatial scale ( 200 nm) of the pneumococcal PG and TA synthesis region at midcell, we used direct STochastic Optical Reconstruction Microscopy (dSTORM), which can achieve spatial resolution of 20 nm, to visualize newly inserted cell wall components along cell growth.

In order to differentiate the roles of WTA and LTA in the architecture of the cell envelope, we examined mutant strains lacking lytR or tacL, the two genes encoding the enzymes respectively responsible for the last step of WTA and LTA assembly. Cryo-electron microscopy of vitreous sections (CEMOVIS) showed a markedly reduced periplasmic space upon deletion of lytR or tacL, highlighting the importance of TA in maintaining the architecture of this still poorly understood cell envelope region. Intriguingly, the PG layer was also thinner in the absence of LytR or TacL, suggesting a molecular interplay between TA and PG synthesis processes. Phase-contrast microscopy further showed that ∆tacL cells harbor drastic divisional and morphological defects compared to ∆lytR cells. By combining fluorescent TA labelling with SDS-PAGE to titrate the amount of TA per cell and determine the ratio of WTA to LTA, we discovered a significant accumulation of membrane-bound polymerized TA precursors in the ∆tacL strain. This indicates that production of WTA and LTA are competing pathways for TA precursors. In support of this, dSTORM analysis provided evidence that the excess of TA precursors in ∆tacL cells can be used for WTA synthesis. In addition, analysis using atomic force microscopy (AFM) alluded to a weakened cell wall in ∆tacL cells compared to ∆lytR and wild-type strains. Altogether, these findings suggest that in S.pneumoniae, LTA have an important function in PG assembly that cannot be compensated by WTA or membrane-bound TA precursors. Curiously, our preliminary data showed that the absence of LTA is associated with impaired incorporation of fluorescent D-amino acids by PG synthases, as well as enhanced sensitivity to PG-cleaving enzymes. Based on these observations, we propose a model in which LTA would be one of the regulatory factors of PG synthesis and hydrolysis in the periplasmic space of S. pneumoniae.