Background: Clostridium difficile infection (CDI) is a leading cause of hospital-associated and acquired diarrhoea, leading to high morbidity and mortality. The disease is mostly caused by disrupting the normal gut microflora through administration of broad-spectrum antibiotics, creating a niche for C. difficile spores to colonise, then germinate and grow out into the vegetative, toxin producing cell form. The spore is the infective form and is 1-2 μm in size, consisting of a central core surrounded by layers of different thickness and density. Despite its importance in spreading disease, there are several key questions surrounding the exact mechanism of germination. In contrast to other Clostridia and Bacillus, C. difficile does not have any classical germination receptor homologues. The pseudo-protease CspC has been suggested as the primary receptor for taurocholate . However, there is no direct biochemical evidence for its interaction with bile acids. CspC’s amino acid sequence shows no homology with that of known bile salt-binding proteins in bacteria or mammalian cells. This project will employ life and physical sciences approaches together with synthetic chemistry to enable further understanding of the germination mechanism of C. difficile which is key to prevention and treatment of infection.
Hypotheses: 1. The alkyl sulfonate binding lipoprotein SsuB/CD2365 and the equivalent binding proteins CD0999 and CD1979 are potential bile salt receptors. They are present in the spore proteome  and their genes are upregulated during germination . 2. A correlative light and electron microscopy (CLEM) imaging pipeline using fluorescent and gold-labelled germinant probes, respectively can be employed to show the pathways these take through the spore.
Methodology: 1. Genomic and phenotypic characterisation of putative germination receptors Three putative taurocholate receptors in C. difficile 630 spores have been identified through proteomic, transcriptomic and genetic screens [2, 3]. Using newly established CRISPR-Cas methodology , the three target genes will be inactivated in C. difficile 630 and effects upon sporulation and germination will be assayed. This will be done following a previously published strategy aimed at determining which part of the germination/sporulation process is affected by a given mutation .
2. Microscopy to elucidate spatial and temporal binding of germinants The putative structure of C. difficile spores has been described previously , but the pathway of the germinant through the outer layers and the location of the target receptors are not known. We will perform multimodal 4D (3D+time) imaging of the germination process to reveal the changing structure of the spore during germination and gain functional information of germinant binding receptors and their locations. To achieve this, we will use gold-labelled and fluorescently labelled  germinant analogues (synthesising of these probes will be supervised at UoAston) and image using facilities at UoB and additionally apply for beamtime at Harwell Campus (Central Laser Facility). Having established optimum imaging conditions, we will investigate the interaction of the probes with selected mutants.
Scientific outcomes: Representing a multi-disciplinary research project incorporating basic bioscience (microbiology), molecular biology, synthetic chemistry and advanced microscopy, the proposed PhD project will lead to enhanced understanding of the mechanisms underlying C. difficile spore germination. Ultimately this can inform novel preventative and therapeutic approaches for a disease affecting, in particular, an ageing population. The PhD student will acquire expertise in state-of-the-art methods in anaerobic microbiology and molecular biology, specifically in the fast-moving area of the human gut microbiome. The student will also work in synthetic chemistry. In addition, the student will be trained in advanced imaging technologies.
References  Francis MB et al. PLoS Pathog. (2013) 9(5), e1003356  Lawley TD et al. J Bacteriol. (2009) 191(7), 5377-86  Dembek M et al. PLoS One. (2013) 8(5), e64011  Hong W et al. ACS Synth Biol. (2018). 7:1588-1600  Burns DA & Minton NP. J. Microbiol. Methods (2011) 87, 133-8  Ulrich G et al. Angewandte Chemie Int. (2008) 47: 1184–1201
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