Dr Robert Fagan
Tel: 0114 222 4182
Clostridium difficile is the most common cause of antibiotic-associated diarrhea. It’s a highly antibiotic resistant pathogen that can cause severe disease following antibiotic-mediated disruption of the protective gut microbiota. The aim of our research is to understand the molecular basis of interactions between the bacterium and its host. We study the outermost layer of the C. difficile cell envelope, the surface- or S-layer, a 2-dimensional proteinaceous crystal that completely coats the surface of the bacterium. The S-layer has been implicated in adhesion and induction of innate immunity. Our work combines molecular microbiology and structural biology to study S-layer biogenesis and function.
Microbiology, Clostridium difficile, spore formation, S-layer
Research In Depth
1. Making an S-layer
Figure 1: The S-layer precursor, SlpA, is translocated through SecA2/SecYEG membrane channels (Fagan and Fairweather JBC 2011). 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 (Dang et al ACS Chem Biol 2010; Dang et al Bioorg Med Chem). These two subunits form a high-affinity heterodimer that assembles to form the crystalline S-layer (Fagan et al Mol Micro 2009). 3 cell wall binding mofits within the HMW SLP interact with the secondary cell wall polysaccharide PS-II to anchor the whole structure onto the cell surface (Willing et al Mol Micro 2015).
2. S-layer function and exploitation
Figure 2: Artist's impression showing an engineered antimicrobial bacteriocin killing a C. difficile cell. The Avidocin-CD nanomachine has bound to the S-layer (green) on the cell surface and contracted to drive the harpoon-like nanotube core through the cell envelope, killing the bacterium.
3. S-layer structure
Figure 3: A. Crystal structure of the outermost two domains of the LMW S-layer protein (Fagan et al. Mol Micro 2009). These are the most highly variable portion of the S-layer protein complex and represent the majority of the cell surface. Although the primary sequence of the LMW SLP varies greatly between strains the structural fold appears to be conserved. B. 20 Å structure of the native C. difficile S-layer generated using 2D electron crystallography in collaboration with Prof Per Bullough
3. High-throughput genetics
4. n-Butanol production
Graduate Student Applications
Graduate students usually join the department via the excellent MBB PhD program, you can apply here. Further details on the funding opportunities available at the university are available here.
Level 3 Modules
- McHale TM, Garciarena CD, Fagan RP, Smith SGJ, Martin-Loches I, Curley GF, Fitzpatrick F & Kerrigan SW (2018) Inhibition of Vascular Endothelial Cell Leak Following Escherichia coli Attachment in an Experimental Model of Sepsis.. Crit Care Med.
- Kirk JA, Gebhart D, Buckley AM, Lok S, Scholl D, Douce GR, Govoni GR & Fagan RP (2017) New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. Science Translational Medicine, 9(406). View this article in WRRO
- Kirk JA, Banerji O & Fagan RP (2017) Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics. Microbial Biotechnology, 10(1), 76-90. View this article in WRRO
- Kirk JA & Fagan RP (2016) Heat shock increases conjugation efficiency in Clostridium difficile. Anaerobe, 42, 1-5. View this article in WRRO
- Janganan TK, Mullin N, Tzokov SB, Stringer S, Fagan RP, Hobbs JK, Moir A & Bullough PA (2016) Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains. Food Microbiology, 59, 205-212. View this article in WRRO
- Buckley AM, Jukes C, Candlish D, Irvine JJ, Spencer J, Fagan RP, Roe AJ, Christie JM, Fairweather NF & Douce GR (2016) Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains. Scientific Reports, 6. View this article in WRRO
- Willing SE, Candela T, Shaw HA, Seager Z, Mesnage S, Fagan RP & Fairweather NF (2015) Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII. Molecular Microbiology, 96(3), 596-608. View this article in WRRO
- Dembek M, Barquist L, Boinett CJ, Cain AK, Mayho M, Lawley TD, Fairweather NF & Fagan RP (2015) High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile. MBIO, 6(2). View this article in WRRO
- Pettit LJ, Browne HP, Yu L, Smits WK, Fagan RP, Barquist L, Martin MJ, Goulding D, Duncan SH, Flint HJ, Dougan G, Choudhary JS & Lawley TD (2014) Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism. BMC Genomics, 15(1). View this article in WRRO
- Fagan RP & Fairweather NF (2014) Biogenesis and functions of bacterial S-layers. Nature Reviews Microbiology, 12(3), 211-222. View this article in WRRO
- Mahon V, Fagan RP & Smith SGJ (2012) Snap denaturation reveals dimerization by AraC-like protein Rns. Biochimie, 94(9), 2058-2061. View this article in WRRO
- Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G & Lawley TD (2012) The Clostridium difficile spo0A gene is a persistence and transmission factor.. Infect Immun, 80(8), 2704-2711. View this article in WRRO
- Tam Dang TH, Fagan RP, Fairweather NF & Tate EW (2012) Novel inhibitors of surface layer processing in Clostridium difficile.. Bioorg Med Chem, 20(2), 614-621.
- Fagan RP & Fairweather NF (2011) Clostridium difficile has two parallel and essential Sec secretion systems.. J Biol Chem, 286(31), 27483-27493. View this article in WRRO
- Fagan RP, Janoir C, Collignon A, Mastrantonio P, Poxton IR & Fairweather NF (2011) A proposed nomenclature for cell wall proteins of Clostridium difficile.. J Med Microbiol, 60(Pt 8), 1225-1228.
- Reynolds CB, Emerson JE, de la Riva L, Fagan RP & Fairweather NF (2011) The Clostridium difficile cell wall protein CwpV is antigenically variable between strains, but exhibits conserved aggregation-promoting function.. PLoS Pathog, 7(4), e1002024. View this article in WRRO
- Dang THT, de la Riva L, Fagan RP, Storck EM, Heal WP, Janoir C, Fairweather NF & Tate EW (2010) Chemical probes of surface layer biogenesis in Clostridium difficile.. ACS Chem Biol, 5(3), 279-285.
- Fagan R & Fairweather N (2010) Dissecting the cell surface.. Methods Mol Biol, 646, 117-134.
- Emerson JE, Reynolds CB, Fagan RP, Shaw HA, Goulding D & Fairweather NF (2009) A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein.. Mol Microbiol, 74(3), 541-556. View this article in WRRO
- Fagan RP, Albesa-Jové D, Qazi O, Svergun DI, Brown KA & Fairweather NF (2009) Structural insights into the molecular organization of the S-layer from Clostridium difficile.. Mol Microbiol, 71(5), 1308-1322.
- Fagan RP, Lambert MA & Smith SGJ (2008) 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, 76(3), 1135-1142. View this article in WRRO
- Smith SGJ, Mahon V, Lambert MA & Fagan RP (2007) A molecular Swiss army knife: OmpA structure, function and expression.. FEMS Microbiol Lett, 273(1), 1-11.
- Fagan RP & Smith SGJ (2007) The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin.. FEMS Microbiol Lett, 269(2), 248-255.