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Title: The function and biogenesis of the Clostridium difficile S-layer
Author: Kirk, Joseph
ISNI:       0000 0004 6424 053X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Clostridium difficile is the most common cause of antibiotic-associated diarrhoea and the severity and incidence of C. difficile infections (CDIs) have risen dramatically in the last decade. C. difficile is only able to cause disease once the microbiota of the gut has been damaged, usually through the use of broad spectrum antibiotics. CDIs are treated using antibiotics which further damage the microbiota, resulting in high rates of recurrence. This highlights the need for more specific CDI therapies that do not damage the natural flora. Avidocin-CDs are a potential alternative therapy for CDI that resemble the R-type Pyocins of Pseudomonas aeruginosa, and kill C. difficile by inserting a needle like core through the bacterial cell envelope. We have demonstrated that these complexes bind SlpA, the major component of the S-layer. Spontaneous mutants resistant to Avidocin-CDs, that lacked a functional S-layer, were isolated. These S-layer mutants, and a strain in which functional slpA was returned to the chromosome of these mutants, provided a unique opportunity to study the function of the S-layer. We have now demonstrated that the S-layer affects the growth and morphology of cells, sporulation efficiency, resistance to innate immune effectors and CwpV expression. Interestingly, we also demonstrated that the S-layer mutants are avirulent in the hamster model of infection, although maintain the ability to persist in the gut. Sequencing of the S-layer mutant genome also allowed the identification of a new phase variable promoter. In addition to studying S-layer function we also investigated S-layer biogenesis and the role of SecA2. Using pull-down experiments, we have identified potential SecA2 binding partners including members of the Cell Wall Protein family and the cell wall synthesis machinery components MreB and FtsZ. We provide evidence that S-layer biogenesis is localised to areas of cell wall growth and suggest a model in which SecA2 interacts with MreB or FtsZ to localise the secretion of SlpA to these areas.
Supervisor: Fagan, Robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available