Ingle, Patrick S.
(2018)
Expanding the genome editing repertoire in Clostridium difficile for improved studies of sporulation and germination.
PhD thesis, University of Nottingham.
Abstract
Clostridium difficile is an anaerobic, Gram-positive, endospore-forming, pathogenic bacterium which is the leading cause of antibiotic-associated diarrhoea, and causes a significant burden to healthcare facilities and communities, worldwide. Bacterial endospores are one of the most resilient forms of life, able to withstand exposures to wet-heat, desiccation, UV radiation, oxygen, and some disinfectants, which would otherwise kill the vegetative cell form. Thus, endospores of C. difficile are able to persist in the environment and contaminate surfaces within healthcare settings. Once ingested, these spores pass into the anaerobic lower intestines and in susceptible individuals find favourable conditions in which to germinate, generating the toxin-producing vegetative cells responsible for C. difficile associated disease. Consequently, spores are the infectious agent of this disease and both sporulation and germination processes are essential for disease. Whilst these processes have been well studied in Bacillus subtilis, it is only recently, with the development of appropriate reverse genetics tools for clostridia, that the mechanisms of sporulation and germination have begun to be described for C. difficile. This study uses the currently available mutagenesis tools of ClosTron and allelic exchange to generate mutant spores lacking spore-specific proteins, and through a range of assays characterises the sporulation, germination and resistance properties of these mutants, to understand the roles of these proteins in C. difficile endospores. Furthermore, these genetics tools are established in a novel C. difficile strain with beneficial properties for studying the processes of sporulation and germination in vitro. Finally, this study establishes CRISPR/Cas9 genome editing in C. difficile for the first time to overcome the major pitfalls associated with the previously available reverse genetics tools. This mutagenesis method was found to offer fast, highly efficient genome editing of two different C. difficile strains and will be the method of choice for future studies in C. difficile.
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