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The identification and characterization of novel haemolysin genes from Clostridium difficile

Alkudmani, Zeina Subhi B.; (2019) The identification and characterization of novel haemolysin genes from Clostridium difficile. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Clostridium difficile is a Gram positive spore forming bacteria that is the leading cause of antibiotic associated diarrhoea and pseudomembranous colitis. C. difficile infection is a significant health care burden in hospital settings and in the community. One of the main factors that lead to C. difficile infection is the prolonged use of antimicrobial agents that disturb the microflora of the gut. This allows it to colonize and produce toxins which disrupt the integrity of the intestinal epithelium. Other than toxin production, C. difficile has a number of virulence factors including surface proteins and enzymes that aid in bacterial attachment and spread of infection. Another possible virulence factor that was described in clostridia and other bacteria is the production of haemolysins. These are proteins (sometimes lipids) produced by the bacterium and cause cytolysis of red blood and eukaryotic cells for example epithelial cells, leukocytes, lymphocytes, and macrophages. This work is the first report of C. difficile showing haemolytic activity and the aim of this thesis is to identify genes responsible for haemolysis in C. difficile. A CD630 genomic library was constructed in E. coli and six genes were found to confer haemolysis. One clone contained a gene (designated atlf) encoding a peptide homologous to an anthrax toxin lethal factor which exhibited large zones of haemolysis. Clostron and CRISPR-Cas9 based CD630 mutants were constructed with insertion and deletion in atlf respectively. The haemolysis of the mutants was tested on agar and by a quantitative assay, which revealed no change in the haemolytic phenotype compared to the wild type. This work suggests that haemolysis is a multifactorial phenomenon in C. difficile and may involve several genes. The CRISPR-Cas9 mutagenesis system constructed in this work will aid in many mutagenic studies to understand haemolysis and other virulence mechanisms in C. difficile.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The identification and characterization of novel haemolysin genes from Clostridium difficile
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. - Some third party copyright material has been removed from this e-thesis.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10078163
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