Project Outline

Antimicrobial Resistance (AMR) is now widely understood to be a global healthcare emergency, exacerbated by many socio-economic factors. This includes the decline in new drug development due to disengagement from the sector by the major pharmaceutical companies resulting in a renewed emphasis on academic engagement in discovery and understanding of how resistance develops. Of all the targets for antibiotics and resistance development, the biosynthesis of the bacterial cell wall and its linkage to cell division is of particular significance it is the entry point for all antibiotics into bacteria as well as the target for the mainstay of antimicrobial chemotherapy: the b-lactams.

Outside the cytoplasmic membrane of all bacteria, there is a sugar-based polymer called peptidoglycan (PG) crosslinked by peptide bridges, which gives the cell wall strength, rigidity, cell shape characteristics and is a scaffold for a multitude of other molecular structures. Disruptions of the PG structure itself or its biosynthetic precursors by antibiotics, can result in cell lysis (bacteriolytic effect) or cessation of bacterial cell growth (bacteriostatic effect). Recently there has been a renascence in our understanding of the biosynthesis of the cell wall and how this is intimately linked to cell division¹. Whilst we have recognised this process for decades², the molecular interactions that are vital and underpin the biology of this process, are only just starting to be properly addressed.

In our laboratory we are focussed on several major aspects of this process including how the cell wall PG is made by the SEDs-Class B PBP complexes and how the process of cell division is coordinated with PG biosynthesis in both Gram-positive and Gram-negative pathogens.

We have recently solved the Cryo-EM structure of the cell elongation specific E.coli RodA-PBP2⁵ (see below) and are working in collaboration with research groups who have solved the structure of the divisome specific FtsW-PBP3 complex⁶ and have revealed mechanistic evolutionary relationship with other cell wall biosynthetic proteins that utilise a undecaprenyl phosphate carrier lipid substrates⁷. By understanding these factors at a molecular level we hope to bring new biological understanding of the processes involved and discover new route to future antimicrobials.

We use a variety of cutting-edge approaches to study this as well as collaboration with groups across the world in this research to allow a complete in-vivo to in-vitro approach. Importantly, our laboratory has two technological advantages over others including the ability to make the PG precursor lipid II allowing functional study of these proteins as enzymes and non-detergent methods to extract the membrane proteins involved in their native lipid environment⁸ allowing structural elucidation of their interactions at a molecular level.

The project will suit a student interested in microbiology, antibiotic resistance and biochemistry. Techniques used in this project can include basic microbiology, molecular biology including in-vivo mutant generation using CRISPR techniques, high resolution light and fluorescent microscopy of the resulting phenotypes to protein chemistry and structural biology including X-ray crystallography, Cryo-EM microscope or Mass spectrometry.

The ultimate PhD project will be developed with the student according to their interests and skill set with in a high collaborative research team environment. Further detail and project areas will be advertised on https://warwick.ac.uk/fac/sci/lifesci/research/droper/research/. We urge students interested in the project to get in contact at earliest possible opportunity and engage in mini project opportunities ( ).

References

1. Typas, A., Banzhaf, M., Gross, C. A. & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature reviews. Microbiology 10, 123-136, doi:10.1038/nrmicro2677 (2011).
2. Park, J. T. & Strominger, J. L. Mode of action of penicillin. Science 125, 99-101 (1957).
3. Galley, N. F., O'Reilly, A. M. & Roper, D. I. Prospects for novel inhibitors of peptidoglycan transglycosylases. Bioorg Chem 55, 16-26, doi:10.1016/j.bioorg.2014.05.007 (2014).
4. Zuegg, J. et al. Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity. Nature communications 6, 7719, doi:10.1038/ncomms8719 (2015).
5. Nygaard, R. et al. Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex. Nature communications 14, 5151, doi:10.1038/s41467-023-40483-8 (2023).
6. Kashammer, L. et al. Cryo-EM structure of the bacterial divisome core complex and antibiotic target FtsWIQBL. Nat Microbiol 8, 1149-1159, doi:10.1038/s41564-023-01368-0 (2023).
7. Ashraf, K. U. et al. Structural basis of lipopolysaccharide maturation by the O-antigen ligase. Nature 604, 371-376, doi:10.1038/s41586-022-04555-x (2022).
8. Teo, A. C. K. et al. Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein. Sci Rep 9, 1813, doi:10.1038/s41598-018-37962-0 (2019).

Techniques

• Basic microbiology and phenotyping of in-vivo mutants
• DNA cloning and site-directed mutagenesis.
• Protein expression and chemistry
• X-ray crystallography, CryoEM microscopy, Mass spectrometry
• High throughut and analytical assay design and development