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Title: Biological containment of recombinant spores and dissemination of pathogenic spores
Author: Hosseini, Siamand
ISNI:       0000 0004 8500 6005
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2019
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Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores coupled with their desiccation resistance makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of spore GMOs by utilizing the concept of a 'thymine-less death', a phenomenon first reported six decades ago. Using Bacillus subtilis, we have inserted chimeric genes in the two thymidylate synthase genes, thyA, and thyB, using a two-step process. Insertion is made first at thyA followed by thyB where resistance to trimethoprim enables selection of recombinants. Importantly, this method requires the introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine) and in their absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes. Despite numerous attempts to reduce the risk of transmission of C. difficile still this nosocomial infection presents a considerable problem. BclA1 is a glycosylated protein expressed on the exosporium layer of C. difficile spores. So far two types of BclA1 have been identified, a full-length and truncated BclA1 encoding for a 693 and 48 amino acids protein respectively. Previously shown that BclA1 play a role in colonisation as a mutant strain, CD630, that had a deleted BclA1 required 2-logs higher spores to colonise in mice in comparison to the isogenic wild-type strain. In this work, the study on BclA1 has expanded by first Identify different types of bclA1 gene within different ribotypes and second test different ribotypes with different bclA1 for colonisation. The in vivo result shows that different non-isogenic strains of C. difficile, regardless the type of BclA1 they have, needed 100 spores to colonise in mice. Interestingly R176, a hypervirulent strain of C. difficile needed a higher number of spores to colonise. The hypervirulent strain also showed to produces more spores than other strains. The findings of this work are that, first, BclA1 may not play a role in colonisation within non-isogenic strains, and second, higher sporulation of hypervirulent C. difficile strains, possibly is a reason for faster dissemination and high incident despite reduced ability to colonise.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available