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
MBB328 The Organisation of Bacterial Cells (Module Coordinator)
- Architecture and self-assembly of clostridium sporogenes and clostridium botulinum spore surfaces illustrate a general protective strategy across spore formers. mSphere, 5(4).
- Applying transposon-directed insertion site sequencing to industrially important, solventogenic species Clostridium saccharoperbutylacetonicum. Access Microbiology, 1(1A).
- Unraveling the role of C. difficile S-layer in infection and disease. Access Microbiology, 1(1A).
- Clostridium difficile: cell surface biogenesis. Access Microbiology, 1(1A).
- Editorial. Anaerobe, 53, 1-1. View this article in WRRO
- Inhibition of Vascular Endothelial Cell Leak Following Escherichia coli Attachment in an Experimental Model of Sepsis.. Crit Care Med, 46(8), e805-e810.
- 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
- Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics. Microbial Biotechnology, 10(1), 76-90. View this article in WRRO
- Heat shock increases conjugation efficiency in Clostridium difficile. Anaerobe, 42, 1-5. View this article in WRRO
- 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
- Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains. Scientific Reports, 6. View this article in WRRO
- 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
- High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile. MBIO, 6(2). View this article in WRRO
- Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism. BMC Genomics, 15(1). View this article in WRRO
- Biogenesis and functions of bacterial S-layers. Nature Reviews Microbiology, 12(3), 211-222. View this article in WRRO
- Snap denaturation reveals dimerization by AraC-like protein Rns. Biochimie, 94(9), 2058-2061. View this article in WRRO
- The Clostridium difficile spo0A gene is a persistence and transmission factor.. Infect Immun, 80(8), 2704-2711. View this article in WRRO
- Novel inhibitors of surface layer processing in Clostridium difficile.. Bioorg Med Chem, 20(2), 614-621.
- Clostridium difficile has two parallel and essential Sec secretion systems.. J Biol Chem, 286(31), 27483-27493. View this article in WRRO
- A proposed nomenclature for cell wall proteins of Clostridium difficile.. J Med Microbiol, 60(Pt 8), 1225-1228.
- 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
- Chemical probes of surface layer biogenesis in Clostridium difficile.. ACS Chem Biol, 5(3), 279-285.
- Dissecting the cell surface.. Methods Mol Biol, 646, 117-134.
- 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
- Structural insights into the molecular organization of the S-layer from Clostridium difficile.. Mol Microbiol, 71(5), 1308-1322.
- 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
- A molecular Swiss army knife: OmpA structure, function and expression.. FEMS Microbiol Lett, 273(1), 1-11.
- The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin.. FEMS Microbiol Lett, 269(2), 248-255.
- Architecture and self-assembly of the Clostridium sporogenes/botulinum spore surface illustrate a general protective strategy across spore formers.