The most widely used antibiotics ever discovered (beta-lactams) are targeting the enzymes that polymerize peptidoglycan (PG), the essential component of the bacterial cell wall. Resistance to beta-lactams is largely associated with modifications of the PG synthetic pathway, leading to changes in PG structure. In collaboration with Protein Metrics, a US-based company, we showed that a high throughput and automated data analysis of PG can be performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS; [1,2]).
We propose to build on our preliminary work to develop a novel pipeline for the structural analysis of bacterial PG, combining ultra-high resolution LC-MS/MS with the Byos software platform from Protein Metrics. We will then use this novel strategy to analyse structural diversity of C. difficile PG in relation to beta-lactam resistance.
The specific objectives of the project are the following:
1) Optimisation of the LC-MS/MS strategy for PG structural analysis using model PGs. PGs with distinct compositions, sugar modifications and crosslinks will be analysed by LC-MS/MS. Chromatography conditions, fragmentation and quantification strategies will be optimized. During this part of the project, the student will become familiar with PG structural analysis.
2) Software development and customization of the interface dedicated to PG structural analysis. A 5-month industrial placement will take place at Protein Metrics (12 employees) in Cupertino, California, USA. The student will work with members of the software development team to customize several aspects of the MS analysis software to identify specific PG properties. During the placement, the student will be immersed in the translation of research into commercial products, being exposed to marketing efforts, sales cycles, and customer support. Protein Metrics participates in multiple conferences and also runs bespoke user conferences, and everyone, including students, interact with potential customers and demo some software. The student will have opportunities to network with representatives from major pharmaceutical companies (Genentech, Janssen) and academic partners from the area (Stanford, UCSF, Berkeley).
3) Application. We will explore the natural variation in PG structure in Clostridium difficile. This opportunistic pathogen is the leading cause of antibiotic-associated diarrhoea worldwide, causing significant morbidity and mortality, with a consequent healthcare cost burden of over €3B in the EU alone . C. difficile natural resistance to beta-lactams underpins the capacity of this organism to cause infections but the molecular basis for resistance is unknown. PG structure in circulating clinical strains will be analysed to determine how this relates to the spectrum of resistance to beta-lactam antibiotics. The strains will be provided by our collaborator Pr M. Wilcox, (Leeds teaching Hospitals NHS Trusts). Identifying PG properties associated with resistance will provide critical information to gain mechanistic insights into antimicrobial resistance of this pathogen.
This project will involve a multidisciplinary approach encompassing microbiology, cell wall biochemistry, mass spectrometry and computer sciences for software development. The candidate will be part of a team with a highly collaborative ethos and will therefore be trained to acquire a unique set of skills that will open up a wide range of job opportunities after the PhD. http://www.sheffield.ac.uk/mbb/staff/stephanemesnage/stephanemesnage https://www.proteinmetrics.com/ https://www.sheffield.ac.uk/bms/research/collins https://medicinehealth.leeds.ac.uk/medicine/staff/3541/professor-mark-wilcox
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/