Rob FaganDr Robert Fagan

Lecturer

Room: F25a
Tel: 0114 222 4182
Email: r.fagan@sheffield.ac.uk

Research

Research Precis

precisClostridium 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.

Research Keywords

Microbiology, Clostridium difficile, spore formation, S-layer

Research In Depth

fig11. Making an S-layer
S-layer biogenesis is a complex multi-step process, involving a dedicated secretion ATPase (SecA2) and cysteine protease (Cwp84). To maintain an intact layer of nearly 600,000 subunits, each cell must produce, secrete, process and assemble 164 S-layer precursors per second, making this the most metabolically expensive structure in the cell. We study all aspects of S-layer biogenesis using protein biochemistry, bacterial genetics, super-resolution light microcoscopy and X-ray crystallography.

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
The S-layer is an essential structure, which has long hampered functional analysis. We can now genetically manipulate the S-layer at will and are studying the role this critical surface structure plays in interactions with the host and in the life cycle of the organism. We also collaborate with AvidBiotics on the development of novel phage tail-like bacteriocins that target the S-layer to kill C. difficile (Kirk et al. Sci Transl Med 2017).

fig2Figure 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.
Image credit: Ella Maru Studio, Inc.

3. S-layer structure
We combine cutting edge cryo-electron microscopy and tomography with X-ray crystallography to understand the structural and molecular basis of S-layer function and biogenesis.

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

fig33. High-throughput genetics
The first effective methods for genetic manipulation of the Clostridia were only developed within the last decade. Progress has been rapid however. We have previously developed tools for inducible expression in C. difficile (Fagan and Fairweather JBC 2011), transposon mutagenesis (Dembek et al MBio 2015) and fluorescence microscopy (Buckley et al Sci Rep 2016). We continue to develop new genetic tools and apply these to study interactions between C. difficile and its host.

4. n-Butanol production
We are using our innovative genetic tools to understand the genetic basis of sporulation and response to membrane stress in C. saccharoperbutylacetonicum in collaboration with Green Biologics Ltd.

Postdoctoral fellowships
Talented postdoctoral scientists interested in applying for an independent fellowship to join the Fagan lab should contact Rob directly to discuss possible projects and funding opportunities.

Graduate Student Applications
We always want to hear from students interested in our research. Well qualified and motivated graduates should contact Rob directly to discuss potential projects and options for funding. Fully funded PhD studentships in the Fagan lab will be advertised on this page as they become available.

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.

Fagan Group Members:

Dr Peter Oatley
Dr Joseph Kirk
Dr Jason Wilson
Nadia Fernandes
Shauna O’Beirne
Laurence de Lussy-Kubisa

Former group members:

Dr Ashley Davies
Dr Oishik Banerji
Dr Yasir Alabdali

Collaborators:

Prof Per Bullough
Dr Paula Salgado
Dr Gilian Douce
Dr Roy Chaudhuri
Prof Mike Brockhurst
AvidBiotics Corp., South San Francisco, USA
Green Biologics Ltd., Abingdon


PhD Opportunities



A crystal shell: function of the critical Clostridium difficile surface layer - FULLY FUNDED

Clostridium difficile is the most frequent cause of hospital acquired infection across Europe. This highly antibiotic resistant pathogen relies on antibiotic-mediated disruption of the gut microbiota in order to cause disease. We urgently need to develop species-specific antimicrobials that kill C. difficile without further disruption to the gut microbiota. The C. difficile cell is coated by a crystalline protein surface layer (S-layer) that mediates contact between the bacterium and its host and environment. The S-layer is a promising, albeit unexploited, target for new therapeutics but until recently we have not had the tools necessary to study this critical surface structure. Our work focusses on the structure, function and biogenesis of this crystal shell.

The successful candidate will join our multi-disciplinary Wellcome Trust-funded project in collaboration with Dr Paula Salgado in Newcastle University and Dr Gillian Douce at the University of Glasgow. The PhD student will work alongside a postdoctoral scientist at the University of Sheffield, with regular collaborative meetings in Newcastle and Glasgow and the opportunity of a placement in a partner’s laboratory.

Reference
Robert P. Fagan and Neil F. Fairweather (2014) Biogenesis and functions of bacterial S-layers. Nature Reviews Microbiology 12:211-222.

Apply online here. For further details contact Dr Rob Fagan at r.fagan@sheffield.ac.uk

Biogenesis of the Clostridium difficile cell surface

Clostridium difficile infection (CDI) is a major cause of morbidity and mortality in our hospitals. Infection normally follows disruption of the gut normal flora by broad-spectrum antibiotic treatment. Following disruption of the microbiota C. difficile colonises the gut and begins uncontrolled proliferation. The C. difficile cell surface is completely covered by a proteinaceous surface layer (S-layer) that mediates interactions between the pathogen and its host (Fagan and Fairweather, 2014). Biogenesis of this S-layer is a complex multistep process that begins with secretion of protein precursors via a dedicated accessory Sec system (Fagan and Fairweather, 2011), and is a promising target for the development of novel antimicrobials. The purpose of this project is to characterise key proteins in the S-layer secretion system. The project will involve the use of classic molecular microbiology and biochemical techniques, including genetic manipulation of an anaerobic pathogen, and analysis of protein secretion.

Fagan, R. P. and N. F. Fairweather (2014) Biogenesis and functions of bacterial S-layers. Nature Rev. Micro. 12:211-222

Fagan, R. P. and N. F. Fairweather (2011) Clostridium difficile has two parallel and essential Sec secretion systems. J. Biol. Chem. 286(31):27483-27493

Dissecting the mechanisms of antibiotic resistance in Clostridium difficile

Antibiotic resistance in pathogenic bacteria is one of the greatest health challenges facing humanity today. Without the development of novel antimicrobial therapies, inhibiting novel targets or restoring the efficacy of existing drugs, we are facing a reversal of the dramatic improvements to both human health and lifespan seen over the last 60 years. Clostridium diffiicle is the leading cause of antibiotic-associated diarrhoea in the UK. C. difficile infection is a direct consequence of our use of antibiotics; the organism is highly resistant to a broad range of antibiotics and requires antibiotic disruption of the normal flora to give it a competitive advantage in the gut.

Despite the importance of C. difficile in the context of human health we know surprisingly little about C. difficile pathogenesis and even less about the molecular mechanisms underpinning its remarkably broad range antibiotic resistance. The bacterial cell wall is the target of several classes of antibiotic, including the β-lactams (e.g. penicillin) and glycopeptide antibiotics (e.g. vancomycin). C. difficile is resistant to most commonly used β-lactam antibiotics, however, the basis of this resistance is completely uncharacterised.

This project aims to dissect the mechanisms of resistance to several antibiotics including broad-spectrum β-lactams. We will use a combination of classical microbiology and genetics to identify the genes responsible for conferring resistance and will then use protein biochemistry approaches to determine the mechanisms of resistance. As a starting point we will employ both random transposon mutagenesis and targeted mutagenesis to identify antibiotic sensitive mutants. For the targeted experiments we will seek to examine the contribution of genes with homology to previously identified resistance mechanisms (e.g. the penicillin-binding proteins and β-lactamases).

Teaching

Level 3 Modules

Career History

Career History

  • 2013 - present: Lecturer, Dept. of Molecular Biology and Biotechnology, University of Sheffield, UK
  • 2008 - 2012: Senior Research Associate, Centre for Molecular Microbiology and Infection, Imperial College London, UK
  • 2005 - 2008: Research Associate, Centre for Molecular Microbiology and Infection, Imperial College London, UK
  • 2001 - 2005: PhD, Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Ireland








































Publications

Journal articles