Cell Wall Formation in Rod Shaped Bacteria

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences


Bacteria are surrounded by a mesh-like cell wall, made out of a polymer called peptidoglycan, which gives them their shape and protects them from bursting due to high internal turgor pressure. In many rod-shaped bacteria, including pathogens such as Escherichia coli, Salmonella species and Pseudomonas aeruginosa, cells grow by adding new material around the cell sidewall. This is principally performed by a multi-protein complex called the elongasome which maintains the cell's rod shape by precise insertion of new cell wall material. Inactivation of the elongasome leads to loss of rod-shape and severely impaired cell fitness.

Antimicrobial resistance now represents a major threat to modern medicine, motivating the urgent search for fundamentally new ways to attack the formation of the bacterial cell wall. Elongasome proteins are structurally very similar to the divisome cell wall synthesis proteins that divide the cell, and together, elongasome and divisome proteins represent the primary architects of the bacterial cell wall. Therefore, the research into the molecular basis of elongasome function proposed will not only advance our understanding of how the bacterial cell wall is built but may also identify new routes to next generation antibiotics that target elongasome and divisome cell wall synthesis proteins and are effective against a wide range of bacteria.

In this project we will determine the basis of elongasome function and regulation from an atomic to macromolecular scale. This will be achieved using synergy between advanced molecular imaging techniques, advanced biochemical and structural biology analysis, cutting-edge computational analysis and simulation methods and bespoke chemical probes, in a fundamentally integrative team-science research programme.

- We recently solved the structure of the E. coli RodA-PBP2 core of the elongasome, providing a foundational basis for understanding the molecular mechanism of elongasome function
- We have developed a repertoire of new approaches to realise this project including new chemical biology tools based on elongasome substrate mimetics, innovative molecular simulations of multi-protein complexes and new methods for single molecule biophysics of cell wall synthesis proteins
- This makes us uniquely placed to make major advances in our understanding of the elongasome and the molecular basis of bacterial cell wall synthesis.

The highly integrated team science work packages proposed here will enable major advances in bacterial cell wall biology that are simply not possible in an ordinary responsive model proposal.
Direct added value is provided by sustained strategic investment at Warwick to build a national centre of excellence in bacterial cell envelope biology, including:
- Recruitment of Stansfeld (2019) and Holden (2022)
- £54M investment in the new laboratory complex occupied by all Warwick applicants,
- A £1.7M charitable donation for a Howard Dalton research centre which provides underpinning support for cell wall research and funds a £120K smFRET instrument heavily used in this in this proposal.
Team science is greatly facilitated by location of Warwick applicants in the same new building and established collaborations between all Warwick and Belfast applicants.

-Fundamental knowledge gain: This proposal approach will reveal broad insights into the molecular function and regulation of the SEDS-PBP proteins that are the major architects of the bacterial cell wall.
-Potential applications in biomedicine and biotechnology: This will lay the foundations for future antimicrobial therapies targeting cell wall synthesis by SEDS proteins.
-Staff training: We will train a cohort of interdisciplinary pre and postdoctoral scientists orientated in an area of microbiology that desperately needs new talent and enable future generations of antimicrobial research in academia and industry.

Technical Summary

Bacterial cell shape is determined by the activity of a complex responsible for peptidoglycan (PG) biosynthesis called the elongasome. This evolutionarily ancient macromolecular machine coordinates with inner and outer cell membrane biogenesis to permit cellular growth. The biosynthetic core of the elongasome is a complex between the SEDS protein, RodA, and a monofunctional penicillin binding protein, PBP2. This mirrors a similar SEDS-PBP complex required for bacterial cell division. The homology between the two complexes, emphasises the critical nature of these functional protein complexes in bacterial cell biology. Additional elongasome proteins; MreB, MreC, MreD and RodZ associate with the central core and modulate function ensuring correct assembly of the peptidoglycan sacculus and ensuring correct cell shape. These additional components of the elongasome are essential to function and in their absence the cell loses control of its shape, leading to cell death.

In previous work we have determined the cryo-EM structure of the RodA-PBP2 complex from E. coli, devised synthetic routes to create lipid-linked substrates and mimetics, modelled the dynamic interactions of substrates and proteins and recorded the single-molecule motions of the active elongasome at a microscopic level of detail. The challenge is now to use these collective skills to understand the mechanistic role of RodA-PBP2 in the context of the native substrates and the other elongasome components. Our project therefore directly addresses an area of fundamental microbiology within the fronter bioscience goal of understanding the rules of life. We will focus this sLoLa project around three key biological questions relating to Activation, Elongation and Regulation.
This heavily integrated proposal will combine microbial biochemistry, single-molecule microscopy, computational biophysics, and chemical biology to unpack the molecular mechanisms of cell wall biogenesis in bacteria.


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