ATOMICROBIOLOGY · Elucidation of the architecture and dynamics of the bacterial cell wall by an interdisciplinary approach

Elucidation of the architecture and dynamics of the bacterial cell wall by an interdisciplinary approach

Project context and objectives The major component of the bacterial cell wall, peptidoglycan (PG), is essential for shape determination and cellular integrity. PG is unique to the bacterial kingdom and its biosynthesis is the site of action of some of the most clinically important antibiotics, such as the beta-lactams (penicillin) and glycopeptides (vancomycin). However, the recent alarming and rapid spread of antibiotic resistance reinforces the necessity to find new drugs and drug targets. Understanding the basic essential principles of PG assembly is therefore a prerequisite for the discovery of new approaches toward the control of pathogens. PG is a complex polymer made up of glycan chains of repeating disaccharide residues, cross-linked via peptide side chains. Despite a wealth of knowledge concerning the components required for PG synthesis and remodelling, our knowledge of the architecture of the polymer and its dynamics during cell growth and division has remained largely elusive. Project results Taking a clearly interdisciplinary approach across biology, chemistry and physics, the host institution has begun to elucidate the architecture of the cell-wall PG of the rod-shaped organism Bacillus subtilis. This has led to the discovery of a totally novel macro-architecture and the development of new models of PG and its dynamics during growth and division, overturning the classical textbook images of cell-wall structure. Crucial to these developments has been the application of high-resolution imaging using atomic force microscopy (AFM). AFM has also been developed to enable the study of living bacteria at high resolution, allowing for the determination of the dynamics of growth and division at the nanometre scale. Thus, we are at the leading edge of both the technology and its application in the study of such important fundamental aspects of bacteria, coupled with the potential for novel drug development. The aim of this project was to combine the cutting-edge expertise of the fellow on wall biochemistry, with that of the host institution in AFM, other biophysical methods and modelling to study bacterial cell-wall architecture, properties and dynamics. The first part of the work has revealed the relationship between cell-wall architecture and the dynamics of peptidoglycan synthesis for a group of organisms with ovoid cell shape ('ovococci') which includes a number of important pathogens. We combined biochemical analyses with super-resolution microscopy to dissect the cell-wall peptidoglycan architecture and dynamics of three species of ovococcus: Streptococcus pneumoniae, Enterococcus faecalis and Lactococcus lactis. Chain length analysis revealed the existence of surprisingly long glycan strands similar to B. subtilis. Extensive AFM analysis showed a preferential orientation of the peptidoglycan network parallel to the short axis of the cells. Finally, we applied for the first time in bacteria, super-resolution structured illumination fluorescence microscopy to unravel the dynamics of peptidoglycan assembly in ovococci. Based on our results, we have shown that ovococci have a unique peptidoglycan architecture not observed previously in other model organisms. The second part of the project was aimed at understanding the topology of the bacterial cell surface. We explored the possibility of using cell-wall binding domains as functional probes to identify surface nanodomains. As a model system, we focused on the LysM peptidoglycan binding domain from E. faecalis peptidoglycan hydrolase AtlA. Using deconvolution microscopy, we showed that this enzyme is specifically targeted to the septum during the cell cycle, and that its C-terminal LysM domain is responsible for this subcellular localisation. Using a multidisciplinary approach encompassing biochemistry, biophysics and structural biology, we have identified the peptidoglycan motif recognised by LysM and elucidated the molecular basis of this interaction. Our work represents a first step towards the development of peptidoglycan binding domains as novel functional fluorescent probes to provide a nanoscale topological map of the bacterial cell surface. As a long-term aim, we propose to develop the study of peptidoglycan binding domains to generate an arsenal of probes targeting various peptidoglycan structures and to use them as a tool to explore peptidoglycan architecture and dynamics during cell division. From a translational research perspective, we can envisage the use of peptidoglycan binding domains as a means to target therapeutic molecules to pathogens.

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