Bollard, Niall
(2018)
Characterisation of phosphotransferase systems (PTS) in Clostridium difficile.
PhD thesis, University of Nottingham.
Abstract
Phosphotransferase systems (PTS) represent an important method of sugar uptake in bacteria and have been well described in the past. However, research into PTS within the genus Clostridium has been mainly restricted to the non-pathogens. Analysis of the genome of Clostridium difficile 630 revealed over 40 intact PTS; this is over three times as many as in other pathogenic Clostridia, such as Clostridium perfringens and Clostridium botulinum. Previously, carbon catabolite repression has been shown to affect toxin production in C. difficile. Being capable of utilising different carbohydrates efficiently could be important for C. difficile to adapt to, grow, and survive in the human gut. So far, little work has been done to corroborate the role of individual PTS in carbohydrate uptake, sensing of environmental stimuli and regulation of virulence, i.e. toxin expression. A deeper understanding of the PTS in C. difficile, and their importance in virulence, could lead to the development of new drug targets.
The aim of this study is to characterise the main PTS of C. difficile, determine their role in carbohydrate uptake, and their effect on regulation of virulence. To date, we have chosen the main candidates thought to be involved in mannitol and sorbitol uptake, and have inactivated these PTS using the ClosTron and in-frame deletion methods. Phenotypic characterisation of these mutants was undertaken to prove their role in uptake of the relevant sugar and to determine their role in virulence regulation.
This study has demonstrated, by growth assays and HPLC, that the operons at CD630_0762-8 and CD630_2331-4 respectively encode PTS specific for sorbitol and mannitol uptake. In the case of the mannitol operon, it has been proved (through the use of cytotoxicity assays, which showed reduced bacterial toxicity in the presence of the sugar) that the suppression of toxin synthesis in the presence of mannitol is dependent upon uptake of the substrate via this operon. With sorbitol, toxin levels are, seemingly, not directly reliant upon uptake of the sugar, resulting in, mainly, an increase of toxin in sorbitol. Presently, it is not possible to say whether these systems have a distinct role or not in the motility of the organism.
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