AbstractThe gastrointestinal (GI) tract is a major source of nutrients for the microbial community which resides within it. Carbohydrates, specifically non-digestible polysaccharides, are key nutrients that profoundly affect the composition of the colonic microbiota which allows the maintenance of gut homeostasis and colonisation resistance. Perturbations in this gut microbiota structure can eliminate these processes allowing the colonisation of pathogens and disease.
Clostridium difficile is a Gram-positive, endospore-forming bacterium whose main virulence determinants are the production of the two exotoxins, TcdA and TcdB. Although much C. difficile research has mainly focused on characterising virulence traits in this pathogen, in the last decade a number of studies have begun to unravel its metabolic capabilities which contribute to its colonisation and proliferation within the gut. This thesis aims to further our knowledge of C. difficile metabolism by attempting to characterise the molecular mechanisms underpinning metabolism of the carbohydrate trehalose, a disaccharide proposed to have contributed towards the emergence of hypervirulence within this species.
Bioinformatic analyses of proteins to date implicated in trehalose metabolism within C. difficile, suggested that as expected each is predicted to carry out a function to allow the sensing, import and/or degradation of trehalose within the environment. An exception was TreX, whose functional role was thought inconclusive as the protein lacked a number of expected conserved trehalase motifs. Using representative strains from each of the five toxigenic clades, hypervirulent strains were then shown to be capable of growing on trehalose at low (10 mM) or high (50 mM) concentrations, whilst non-hypervirulent strains could not. Transcriptional profiling of the treR, treA, treA2, ptsT, treX and treR2 trehalose metabolism genes then showed further inter-strain differences at the gene expression level. Interestingly whilst an increase in expression levels was found in the non-hypervirulent strains at the low trehalose concentration this was evidently not sufficient to translate into observable growth. Finally, in the hypervirulent R20291 and M120 strains, the presence of trehalose was found to decrease expression levels of the tcdA and tcdB toxin genes and the early-sporulation sigma factor gene, sigE raising the possibility that trehalose may actually limit a number of key processes associated with pathogenesis.
For the first time it was then shown that the TreR regulator protein within the C. difficile genome displays DNA-binding activity to both the treR and treA genes. Additionally, it was confirmed experimentally for the first time that the presence of trehalose-6-phosphate acts as an effector leading to increased binding of the TreR protein from R20291 to its target DNA, thus suggesting a potential role as a transcriptional activator. Finally, RNA-sequencing analysis of the hypervirulent R20291 strain provided an insight into the metabolic rewiring that C. difficile carries out during growth on an alternative carbon source. Of importance and requiring further investigation, it revealed for the first time in C. difficile the induction of an alternative trehalose metabolism pathway, previously elucidated in Lactococcus lactis, whose trehalose metabolism relies on a similar pathway.
Thesis is embargoed until 31 July 2024.
|Date of Award||Jul 2023|
|Supervisor||Geoff McMullan (Supervisor) & John McGrath (Supervisor)|