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oai:www.repository.cam.ac.uk:1810/3631962025-12-19T20:07:35Zcom_1810_195217com_1810_256065col_1810_219484

Functionalization of phage endolysins to treat infections of spore-forming Clostridia bacteria in the era of antibiotic resistance https://doi.org/10.17863/CAM.105377 Mills, Gabrielle Christie, Graham Certain *Clostridia* species are notable pathogens, not only because they form highly resistant endospores but because they produce toxins and exhibit antibiotic resistance. *Clostridioides difficile* is a nosocomial pathogen responsible for thousands of deaths every year, whereas *Clostridium perfringens* is a common cause of food poisoning and a notable veterinary pathogen. Research on *Clostridia* spores and phage therapy has been limited to a selection of species, partially because of their anaerobic nature. While recent work has endeavoured to bridge this gap, there is still a pressing need to understand the resistance mechanisms of *Clostridia* spore-formers—especially *Clostridioides difficile*—and develop improved treatments for these difficult pathogens. The aim of this thesis was to augment current knowledge of *Clostridia* and their phage-based therapies, specifically through analysis of *Clostridium perfringens* SM101 as well as *Clostridioides difficile* strains 630, R20291, and SH1. The first portion of the thesis specifically looks at the *Clostridia* spore coat: the composition through proteomics and genetic engineering and the structure via phase contrast and TEM imaging. Over 3,000 coat and exosporium proteins were identified in *Clostridia* species, and TEM showed that *Clostridioides difficile* 630 and *Clostridium perfringens* SM101 both possess an exosporium like that of *Clostridium sporogenes*. Then, phage were induced and isolated to assess their specificity and efficacy against *Clostridia*, yet endolysin domains were the ultimate focus for host range analyses and subsequent experimentation. Finally, the feasibility of combining endolysin binding domains and antibacterial proteins was investigated for improvements in lytic and binding activity. To do so, a novel methodology was formulated to synthesize these proteins in susceptible *Escherichia coli*. Fusions of hen-egg-white- lysozyme and a phage endolysin domain of *Clostridium perfringens* show enhanced lytic activity against *Clostridia* bacteria, including outgrown vegetative cells. University of Cambridge 2023-10-17 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/363196 https://www.repository.cam.ac.uk/bitstreams/567a5352-28b4-459c-903c-39c1b7f1fdd4/download dc29eb66839f47fd4d9d0d5748c77fd5 https://www.repository.cam.ac.uk/bitstreams/68516cc1-1b9f-430a-ace1-d47cb245f4cd/download 87eda9de84448d1f82354d60eee3eb5f https://creativecommons.org/licenses/by-nd/4.0/ antibiotics Clostridium endolysins phage