Unraveling the dynamics of bacterial cell wall synthesis and maturation at the nanoscale – NanoWall

The cell wall plays a major role in bacterial proliferation, survival and virulence. However, the molecular processes responsible for its architecture and final composition are still largely misunderstood. Peptidoglycan (PG) and teichoic acids (TA) are the two major components of the wall of Gram-positive bacteria. The cell wall is assembled in cell regions of nanometric dimensions, requiring the implementation of high-resolution investigation methods. To this end, the NANOWALL project brings together three partners with complementary expertise in synthetic chemistry, super-resolution cell imaging, biochemistry, NMR (nuclear magnetic resonance) spectroscopy and microbial genetics. Thanks to this synergy of expertise, the project aims to elucidate the dynamics of synthesis and maturation of TA and PG, and to discover molecular mechanisms that coordinate their metabolism. It focuses on the human pathogen Streptococcus pneumoniae, whose wall assembly processes are similar to those of many pathogenic species (streptococci and enterococci). The NANOWALL project will provide a better understanding of how the cell surface of Gram-positive bacteria is shaped during different phases of growth, which is an essential aspect of their physiology. As PG and TA metabolism are associated with S. pneumoniae virulence and antibiotic susceptibility, the project will also generate important information for the understanding of host-pathogen interactions and the discovery of new antibacterial strategies.
The strengths of NANOWALL lie in original strategies developed in the participants' laboratories for metabolic wall labeling by click chemistry, coupled with in vivo and in vitro analyses.
In 2021, a first study using the super-resolution fluorescence microscopy technique called dSTORM (direct STochastic Optical Reconstruction Microscopy, about 25 nm resolution) allowed us to correlate the elongation and cell division processes with the dynamics (localization, temporal sequence, quantification) of two modes of PG assembly. The study showed the progressive uncoupling of these two modes of assembly in time and space, and revealed the kinetic parameters of the associated synthesis and maturation events. It has thus provided a better understanding of how S. pneumoniae acquires its ovoid shape. By extending this study to TA, whose metabolism is linked to that of PG, the NANOWALL project will reveal the contribution of these complex polysaccharides to the constitution and maintenance of the shape and integrity of the cell wall. The implementation of co-labeling and dSTORM experiments in two colors will also allow the visualization of neo-synthesized PG and TA within the same cell in order to better characterize the coordination of their assembly processes.
Intriguingly, the proteins that bind to TA show different localizations from each other, suggesting that TA composition varies across the cell surface. Another objective of the NANOWALL project will therefore be to correlate the size and composition of TA with their spatial distribution. For this purpose, TA will be analyzed by biochemistry, NMR and mass spectrometry.
Cellular and molecular analyses will be performed on wild-type and mutant strains of S. pneumoniae (whose phenotype will also be characterized by more classical approaches in cellular electron microscopy and light microscopy) to dissect the contribution of key genes related to the assembly of TA and PG, and to identify molecular interactions between these processes. Finally, the comparison of data obtained during the exponential, stationary and lytic growth phases will allow characterizing the variations associated with bacterial physiology.