The Roper group's research interests are in focused in several main areas:
- Peptidoglycan Biosynthesis: The majority of my work is concentrated on understanding the molecular mechanisms that underpin the biosynthesis of peptidoglycan pathogens with particular emphasis on the way in which cell shape is determined and controlled by RodA-PBP and the wider elongasome. This involved coordination of processes from inside to outside the cell membrane
- Bacteria Cell division: Cell division is the largest morphological change that any cell undergoes and has common elements in all forms of life. In bacteria this is controlled by a dynamic complex of proteins, many of which are located at or in the cell membrane making their study and analysis challenging. FtsZ and the proteins which interact are of key interest in that respect.
- New antibacterial drug discovery: Our studies on cell division and their linkage to peptidoglycan synthesis has an intimate connection to the targets of existing and potentially future antimicrobial drugs. We are investigating a number of these including the potential of the lipid II polymerisation process as a target for such drugs.
- Molecular Mechanisms of Antibiotic resistance: We are interested in the molecular mechanisms of antibiotic resistance, in relation to the biosynthesis of the cell wall including the mode of action of the lipid II sequestering antibiotic, vancomycin and our discovery of the way in which D-cycloserine targets three different aspects of peptidoglycan biosynthesis.
We have a number of possible Mbio, Mini projects and full PhD project opportunities as per the below:
1. Utilizing nanodisc technology to understand the biology of late stage bacterial cell division proteins
2. Exploring protein-protein interactions in bacterial cell division: targets for next generation antibiotic discovery?
3. Investigating the role of bacteria Serine-Threonine kinases in antibiotic resistance
4. Protein complexes required for Methicillin resistance in S. aureus
5. Fuelling the bacterial cell wall biosynthetic pipeline, PPIs required for cell wall biosynthesis
6. Next generation Inhibitor discovery in bacterial peptidoglycan biosynthesis: glycosyltransferases
1. Project Title: Utilizing nanodisc technology to understand the biology of late stage bacterial cell division proteins
Mini Project: Yes
PhD Project: Possibly
There are around 10 proteins in E. coli that are required for form a complex called the divisiome that brings about cell division. The bacterial homologue of tubulin. FtsZ is at the hub of this wheel and we have used a new, detergent free extraction method (SMALP) to get an negative stain EM structure of FtsZ bound to its membrane anchor ZipA. Much later in the cell division process FtsW and FtsI (also called PBP3) form another sub complex of the divisome that we would also like to extract and study. The project would be to use Gibson cloning to make a construct for this, explore purification using the extraction method and go on to prelim EM studies as well as biochemical characterization. This project is related to an existing PhD project in the lab in which we have successfully applied the same approach to an early stage divisomal complex called FtsEX that is involved earlier in the formation of the divisiome
2. Project Title: Exploring protein-protein interactions in bacterial cell division: targets for next generation antibiotic discovery?
Mini Project: Yes
PhD Project: yes, new area with Dr Josef Lawandowski (Chemistry)
Bacterial cell division is orchestrated by a group of membrane bound and cytoplasmic proteins, which form a complex known as the Divisome. In order for the cell to divide correctly, the division process must be coordinated with biosynthesis of new cell wall material and separation of daughter chromosomes mediated by specific divisomal protein sub-complexes. Arrest of these events leads to the cessation of normal growth or cell lysis and is of interest therefore from both a basic scientific and translational perspective. At the core of the divisome is the bacterial homologue of tubulin; FtsZ, which polymerises and exerts a cytokinetic force on the cell membrane that leads to cell division. Whilst X-ray crystallography has provided high-resolution “snap shot” information on FtsZ and FtsZ modulating proteins, there is relatively little information about how the protein-protein interactions with FtsZ are mediated and how that interaction effects FtsZ polymerisation and function and thus cell division overall. A number of proteins are known to bind to the carboxyl terminal domain of FtsZ, anchoring it to the membrane (ZipA, FtsA) and are known to be important in the overall cell division process. In addition, the FtsZ-associated proteins ZapA to ZapE interact at an early stage of FtsZ-ring assembly. Our previous work has shown that the cell division protein ZapA needs to tetramerise into a “dog bone” shape in order to achieve ordered assembly of FtsZ fibres, but a mutant dimer form of ZapA will still bind to FtsZ. The interaction of ZapA decreases the overall GTPase activity of FtsZ stabilising the formation of FtsZ fibres as seen by light microscopy and measurements of GTP turnover. These experiments in combination with existing X-ray structures strongly implicate the conserved structure of the globular head group of ZapA as the site of interaction with FtsZ. The ZapA protein is widely found in bacterial species and is a relatively small protein which can be expressed in high yield making it ideal for specialised NMR labelling and biochemical studies. When combined with the library of established biochemical assays for FtsZ function as well as in-vivo and in-vitro assay of function and structure, we have a powerful arsenal of experimental techniques assembled (including solution and solid state NMR, EM and other biophysical and biochemical techniques) to address important biological questions including:
- What is the interaction site between FtsZ and ZapA?
- How does the binding of ZapA to FtsZ mediate its effect on ftsZ polymerisation.
- How is this interaction effected by other binding partners including ZipA, ZapB and FtsA?
3. Project Title: Investigating the role of bacteria Serine-Threonine kinases in antibiotic resistance
Mini Project: Yes, also existing Mbio
PhD project: Yes
Mbio Project: Yes
The second concerns the fundamentals of antibiotic resistance in Enterococcus. An existing student in the lab, Chris Thoroughgood, has found the kinase domain of the only ST kinase in the bacterium is responsible for phosphorylation control of a number of other proteins required for various responses to antibiotics. The first job would be to try and crystallise the ST kinase domain with a variety of ligands including staurosporin which is a known inhibitor and then go on to study the likely targets of the kinase including DdcY and VanY which are responsible for turning the peptide stem of the peptidoglycan precursors into tetra peptides. For DdcY and VanY we would like to know if the enzyme activity (i.e. Penta to tetra) is effected by phosphorylation and then perhaps to go on to structural studies with those as well.
4. Project Title: Protein complexes required for Methicillin resistance in S. aureus
Mini project: Yes
PhD project: Yes
Mbio Project: Yes
S. aureus is a prominent human and animal pathogen that can cause a diverse range of diseases ranging from relatively minor skin infections to serious and life-threatening infections such as endocarditis, pneumonia, and sepsis. Its impact is enhanced by the development of antibiotic resistance, most notably methicillin-resistant S. aureus (MRSA) that is resistant to virtually all b-lactam antibiotics.
Although methicillin is no longer in use MRSA are regarded as referring to resistance to virtually all b-lactam antibiotics including oxacillin and/or cefoxitin in common use. beta-Lactams bind to the penicillin-binding proteins (PBP) transpeptidase active site which is essential for cell wall biosynthesis and inhibit peptidoglycan crosslink formation which can lead to direct bacterial cell lysis or cessation of cell growth.
MRSA strains are characterized by the acquisition of a mobile genetic element, (SCCmec) carrying the mecA gene, which encodes an altered enzyme: PBP2a (MecA), with reduced affinity for beta-lactam antibiotics. As a result, cell wall biosynthesis in MRSA strains continues even in the presence of otherwise inhibitory levels of beta-lactams, which inhibit the activity of other PBPs.
This resistance mechanism requires the formation of a complex between bifunctional (glycosyltransferase & transpeptidease) S. aureus PBP2 and monofunctional (transpeptidease) PBP2a and certain antimicrobials work (e.g. found in green tea!) by delocalising the two proteins. PBP2 is a class A bifunctional enzyme in S. aureus strains and uses its glycosyltransferase activity to assemble lipid II (peptidoglycan building block) molecules into a long glycan polymer. This glycan polymer is the substrate for transpeptidase reactions catalysed by its own transpeptidase domain or that of other PBPs. In the presence of beta lactams, the inhibited TP activity of PBP2 is complimented by the TP activity of PBP2a thus allowing cell wall biosynthesis in the presence of these drugs.
Recently, a novel MRSA isolate, designated MecC has been discovered in agricultural settings, which has a significantly different sequence signature to MecA. Moreover, genetic experiments suggest that MecC does not partner with PBP2 for the required glycosyltransferase activity implicating either the monofunctional peptidoglycan associated glycosyltransferase or newly discovered SEDs family glycosyltransferases FtsW as the essential partner(s).
Essential question in this project will be:
- What is the partnering glycosyltransferase for MecC?
- What factors are important for complex formation (Protein-protein interactions? Lipid environment?)
5. Title: Fueling the bacterial cell wall biosynthetic pipeline: Protein complexes required for cell wall biosynthesis
Mini Project: Yes
PhD Project: Yes
Mbio Project: Yes
Biosynthesis of the bacterial cell wall represents an area of bacterial metabolism, which is a validated target for antimicrobial drugs and natural products (e.g. vancomycin, B-lactams, D-cycloserine etc). Outside the cytoplasmic membrane of all bacteria, there is a sugar-based polymer called 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). Biosynthesis of this polymer begins in the cytoplasm with a pathway of enzymes wherein UDP-GlcNAc is converted by MurA and MurB to UDP-MurNAc and then to the final cytoplasmic PG precursor by the sequential action of MurC-F ligases which add a pentaptide to the MurNAc sugar. This soluble precursor is linked to C55 carrier lipid via the integral membrane protein MraY to form lipid I 4-6 on the intracellular face of the cell membrane. MurG then adds UDP-GlcNAc to lipid I, to yield lipid II.7,8 prior to translocated through the cytoplasmic membrane9, where it becomes a substrate for the peptidoglycan synthease enzymes (PBPs).
In rod shaped bacteria, the actin homologue protein MreB which is located on the cytoplasmic side of the membrane has been shown to interact with several proteins that are proven to be involved in length growth, including the extracellular PBPs. Current thinking in the field suggest that MreB may have a central role in linking peptidoglycan precursor biosynthesis in the cytoplasm with peptidoglycan polymer formation on the outside of the cell. Our hypothesis is that MreB forms a dynamic filament on which the Mur enzymes assemble to form an “assembly line” for the peptidoglycan intermediates which channels the product to the membrane of the cell for lipid II biosynthesis. Recent elegant microscopy studies show a direct linkage between these activities inside and outside the bacterial cell.