Dr Robert Fagan

email: r.fagan@sheffield.ac.uk

Tel: 0114 2224182

Career History

Research Interests

Clostridium difficile is a spore forming, anaerobic, Gram positive bacterium which causes severe disease in patients following antibiotic treatment. My work focuses on the interaction between C. difficile and its host. My interests fall into three broad areas:

1 Characterisation of surface protein secretion

Protein translocation into and across the cytoplasmic membrane is an essential process in all bacteria. The majority of translocated proteins utilise the multi-component Sec system, comprising, at its core, a membrane pore (SecYEG) and an energising ATPase (SecA). Recently, several Gram positive species have been found to possess a second accessory Sec system, characterised by the possession of a second SecA ATPase, SecA2. In species which possess an accessory Sec system, it is responsible for the translocation of a subset of proteins, often virulence factors. Compared to many model bacterial pathogens, the mechanisms of protein secretion in the Clostridia are poorly understood. I recently identified and characterised the essential C. difficile accessory Sec secretion system and identified its major substrates as the S-layer precursor, SlpA, and the major phase variable cell wall protein, CwpV.

Figure legend: The S-layer precursor, SlpA, and the major phase variable cell wall protein, CwpV, are exported through accessory SecA2/SecYEG membrane channels. Following translocation and signal peptide cleavage, SlpA undergoes a second cleavage event, mediated by a cell wall cysteine protease, Cwp84, yielding the high and low molecular weight (HMW and LMW) SLPs which form the assembled S-layer. CwpV also undergoes a post-translocation cleavage event (non-enzymatic autoproteolysis) and inserts into the S-layer, comprising up to 15 % of the completed layer.

2 Structural characterisation of surface proteins

For many years I have had an interest in the structural characterisation of Clostridial surface proteins. Current projects aim to determine the high-resolution structures of several surface proteins using X-ray crystallography, in collaboration with Paula Salgado at the University of Newcastle (http://www.ncl.ac.uk/camb/staff/profile/paula.salgado#tab_profile), in combination with analysis of the assembled macromolecular structures using EM and other microscopic techniques.

Figure legend: Crystal structure of the outermost two domains of the LMW S-layer protein. These are the most highly variable portion of the S-layer protein complex and represent the majority of the bacterial surface. Although the primary sequence of the LMW SLP varies greatly between strains the structural fold appears to be conserved.

3 Characterisation of novel virulence factors which contribute to the colonisation of the human host

I apply novel genetic tools in combination with biochemistry, chemical biology and structural biology to the study of Clostridial virulence factors.

Joining the lab

Prospective PhD students and Postdoctoral fellows are encouraged to contact me directly by email r.fagan@sheffield.ac.uk

PhD studentships: Funded PhD studentships will be advertised here as they become available but well qualified graduates, including those intending to self fund, should contact me directly to discuss possible projects.

Postdoctoral fellowships: I welcome postdocs who wish to apply for a fellowship to join the group. Please contact me directly to discuss potential projects.

Publications

1. Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G, Lawley TD. Clostridium difficile spo0A gene is a persistence and transmission factor. Infect. Immun. 2012, 80(8):2704-2711 http://www.ncbi.nlm.nih.gov/pubmed/22615253

2. Mahon V, Fagan RP, Smith SGJ. Snap denaturation reveals dimerization by AraC-like protein Rns. Biochimie 2012, 94:2058-2061 http://www.ncbi.nlm.nih.gov/pubmed/22627379

3. Fagan RP, Fairweather NF. Clostridium difficile has two parallel and essential Sec secretion systems. J. Biol. Chem. 2011, 286(31):27483-27493 http://www.ncbi.nlm.nih.gov/pubmed/21659510

4. Reynolds CB, Emerson JE, de la Riva L, Fagan RP, Fairweather NF. The Clostridium difficile cell wall protein CwpV is antigenically variable between strains, but exhibits conserved aggregation-promoting function. PLoS Pathogens 2011, 7(4):e1002024 http://www.ncbi.nlm.nih.gov/pubmed/21533071

5. Fagan RP, Janoir C, Collignon A, Mastrantonio P, Poxton IR, Fairweather NF. A proposed nomenclature for cell wall proteins of Clostridium difficile. J. Med. Micro. 2011, 60(8):1225-1228 http://www.ncbi.nlm.nih.gov/pubmed/21252271

6. Dang TH, Fagan RP, Fairweather NF, Tate EW. Novel inhibitors of surface layer processing in Clostridium difficile. Bioorg. Med. Chem. 2011, 20(2):614-621 http://www.ncbi.nlm.nih.gov/pubmed/21752656

7. Fagan R, Fairweather N. Dissecting the cell surface. Methods Mol. Biol. 2010, 646:117-134 http://www.ncbi.nlm.nih.gov/pubmed/20597006

8. Dang TH, de la Riva L, Fagan RP, Storck EM, Heal WP, Janoir C, Fairweather NF, Tate EW. Chemical probes of surface layer biogenesis in Clostridium difficile. ACS Chem. Biol. 2010, 5(3):279-285 http://www.ncbi.nlm.nih.gov/pubmed/20067320

9. Emerson JE, Reynolds CB, Fagan RP, Goulding D, Fairweather NF. A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein. Mol. Micro. 2009, 74:541-556 http://www.ncbi.nlm.nih.gov/pubmed/19656296

10. Fagan RP, Albesa-Jove D, Qazi O, Svergun DI, Brown KA, Fairweather NF. Structural insights into the molecular organisation of the S-layer from Clostridium difficile. Mol. Micro. 2009, 71:1308-1322 http://www.ncbi.nlm.nih.gov/pubmed/19183279

11. Fagan RP, Lambert MA, Smith SGJ. The Hek outer membrane protein of Escherichia coli strain RS218 binds to proteoglycan and utilizes a single extracellular loop for adherence, invasion and autoaggregation. Infect. Immun. 2008, 76:1135-42 http://www.ncbi.nlm.nih.gov/pubmed/18160475

12. Smith SGJ, Mahon V, Lambert MA, Fagan RP. A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol. Lett. 2007, 273:1-11 http://www.ncbi.nlm.nih.gov/pubmed/17559395

13. Fagan RP, Smith SGJ. The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin. FEMS Microbiol. Lett. 2007, 269:248-55 http://www.ncbi.nlm.nih.gov/pubmed/17241243