Towards phage therapy against Clostridioides difficile
Publication Date
July 29, 2022
Creator
Abstract
Clostridioides difficile is a serious public health threat and the rise of CDI epidemics over the last few years has triggered an increased interest in using phages as a form of therapy. Natural phage therapies rely on the lytic activity of phages to eradicate infection. To this date, no strictly lytic phage infecting C. difficile has been isolated. To address this issue, we propose to test the concept of using temperate phage to deliver lethal cargo to this pathogen.
We have isolated phages ΦCD3801, ΦCD8601 and ΦCD9301. Several locations in the ΦCD9301 genome have been identified and experimentally proven suitable for cargo sequence knock-in using an antibiotic marker. Initially, the construction of recombinant temperate phage carrying Small Acid Soluble Protein (SASP) as an antimicrobial against C. difficile was investigated. In vegetative cells, SASP binds to the DNA and interfere with DNA replication and gene expression leading to cell death. Two SASPs cargos were individually integrated into the ΦCD9301 genome, however no change in the phage activity was observed in vitro. Next, we proposed to test the concept of re-directing endogenous CRISPR-Cas systems towards toxic genes specific to this pathogen using recombinant phage ΦCD9301. Although, endogenous CRISPR-Cas was successfully re-directed to autoimmunity with the use of a plasmid, this result was not detected using recombinant phage carrying enCRISPR miniarrays.
Overall, this work shed some light on the generation of recombinant phages infecting C. difficile. Moreover, an endogenous CRISPR system of this pathogen was characterised and successfully redirected to autoimmunity. Further work is required to determine the optimal choice of promotor systems to achieve strong enough expression of cargo without the interference with the manufacturing strain.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
68471
UoN Repository URI
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PhD thesis_Zuzana Grmanova.pdf
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Full-text
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Examined
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