faganr

Dr Robert Fagan

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

General

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 student, Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Ireland

Research Keywords

Microbiology, Clostridium difficile, spore formation, S-layer

Research

I welcome post-docs who wish to apply for a fellowship to join our research group.
Please contact me directly to discuss potential projects.


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 S-layer biogenesis and function

C. difficile has a paracrystalline surface layer (S-layer) that is largely comprised of a single protein, SlpA, and is interspersed with an additional 28 surface proteins which functionalise the cell surface. The majority of surface proteins are secreted by the multi-component Sec system, comprising, at its core, a membrane pore (SecYEG) and an energising ATPase (SecA). The S-layer is an essential structure and has its own dedicated secretion ATPase, SecA2. We have previously characterised the C. difficile SecA2 secretion system and carried out the first structural characterisation of individual S-layer subunits. We have ongoing projects focusing on the secretion of S-layer precursors, the structure of individual S-layer subunits and the assembled layer, and the function of S-layer.

fig1

Figure 1: 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 bacterial virulence factors

For many years we 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 and components of the secretion system using X-ray crystallography, in collaboration with Paula Salgado at the University of Newcastle (http://sbl.ncl.ac.uk/people/paula_research.shtml), in combination with analysis of the assembled macromolecular structures using EM and other microscopic techniques.

fig2

Figure 2: 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

We apply novel genetic tools in combination with biochemistry, chemical biology and structural biology to the study of Clostridial virulence factors. Recent projects include the development of the first efficient tool for comprehensive transposon mutagenesis of C. difficile.

Teaching

Module Coordinator: MBB328 The Organisation of Bacterial Cells, MBB335 Bacterial Pathogenicity

Level 3 Modules

Level 1 Modules

PhD Opportunities

I welcome applications from self-funded prospective home and international PhD students; see examples of possible projects below. I also have a FULLY FUNDED project available for UK/EU applicants.

You can apply for a PhD position in MBB here.

Contact me at r.fagan@sheffield.ac.uk for further information.



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).









































Publications

Journal articles