Prof David Roper
No more applications being accepted
Funded PhD Project (European/UK Students Only)
About the Project
The biosynthesis of the bacterial cell wall represents an area of bacterial metabolism, which is a validated target for numerous antimicrobials1. Outside the cytoplasmic membrane of all bacteria, a sugar-based polymer called peptidoglycan is synthesized which is crosslinked by peptide bridges, providing 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). All bacteria are dependent on a series of peptidoglycan biosynthesis enzymes to achieve this which work in complex with other proteins to bring about cell division and cell wall growth2. Recent advances in our understand of the basic and mechanistic biology of this process reveals that certain peptidoglycan biosynthesis enzymes work together as co-dependent complexes which are the preferential targets of old and next generation -lactam antibiotics3. This novel understanding has profound implication for existing combination antimicrobial chemotherapy and future drug discovery. Our laboratory is at the forefront of this new understanding and we are exploring the interaction and mechanism of these enzymes to drive this exploration.
In any particular bacterial species, there is a seemingly large number of penicillin binding proteins (PBPs) produced which are associated with various aspects of cell wall peptidoglycan biosynthesis. This apparent redundancy is brought into focus by the fact that analysis of the specificity of a range of -lactam antibiotics reveals that most clinically used drugs including the very well tolerated cephalosporins drugs, preferentially target the class B, monofunctional transpeptidase PBP enzymes. These PBP enzymes have recently been discovered to work in a co-dependent complex with SEDs (Sporulation, elongation and Division) glycosyltransferase enzymes3. With respect to the model Gram-negative rod shaped species E.coli, the SEDs-PBP complexes pairs are FtsW-PBP3 required for cell division, and RodA-PBP2 required for rod shape growth and cell morphology4,5. Insertion of the single transmembrane helix of the class B PBP into the multiple transmembrane helices of the SEDs protein causes a conformational change in the latter which activates its glycosyltransferase activity.
This knowledge profoundly changes our understanding of the basic biology of bacterial cell wall biosynthesis. Less than 5 years ago only class A , bifunctional PBPs (e.g. PBP1a, PBP1B) were known to produce the glycan strand and perform the peptide crosslinking reaction. We now know that the class A enzymes are more likely to “fill in” and “repair holes” in the peptidoglycan matrix which is primarily synthesised by the SEDs-class B PBP complexes pairs. This understanding explains the importance of class B PBPs as described above from a -lactam targeting perspective since their role is far more fundamental to the bacterial cell.
This project will focus on the a mechanistic understand of how class B Penicillin binding proteins work in complexes with SEDs (Sporulation, elongation and Division) glycosyltransferase enzymes. We will explore the way in which the glycan strand produced by the SEDs glycosyltransferase is channelled towards the class B Penicillin binding proteins active site and how the interaction of other proteins in the complex modulate this process. The project will involve a combination of structure based mechanistic biochemistry, substrate dependent assays, proteins biochemistry and phenotypic analysis of mutant proteins and their effect on the complex6,7.
Funding Notes
Studentship includes: fees, a tax free stipend of at least £15,009 p.a (to rise in line with UKRI recommendation); a travel allowance in year 1; a travel / conference budget; a generous consumables budget and use of a MacBook Pro for the duration of the programme.
References
1. Bugg, T.D., Braddick, D., Dowson, C.G. & Roper, D.I. Bacterial cell wall assembly: still an attractive antibacterial target. Trends Biotechnol 29, 167-73 (2011).
2. Typas, A., Banzhaf, M., Gross, C.A. & Vollmer, W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10, 123-36 (2011).
3. Meeske, A.J. et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature 537, 634-638 (2016).
4. Taguchi, A. et al. FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein. Nat Microbiol 4, 587-594 (2019).
5. Sjodt, M. et al. Structural coordination of polymerization and crosslinking by a SEDS-bPBP peptidoglycan synthase complex. Nat Microbiol (2020).
6. Catherwood, A.C. et al. Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B. J Am Chem Soc 142, 5034-5048 (2020).
7. Kuru, E. et al. Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis. ACS Chem Biol 14, 2745-2756 (2019).