One size does not fit all – Trehalose metabolism by Clostridioides difficile is variable across the five phylogenetic lineages

Clostridioides difficile , the leading cause of antibiotic-associated diarrhoea worldwide, is a genetically diverse species which can metabolise a number of nutrient sources upon colonising a dysbiotic gut environment. Trehalose, a disaccharide sugar consisting of two glucose molecules bonded by an α 1,1-glycosidic bond, has been hypothesised to be involved in the emergence of C. difficile hypervirulence due to its increased utilisation by the RT027 and RT078 strains. Here, growth in trehalose as the sole carbon source was shown to be non-uniform across representative C. difficile strains, even though the genes for its metabolism were induced. Growth in trehalose reduced the expression of genes associated with toxin production and sporulation in the C. difficile R20291 (RT027) and M120 (RT078) strains in vitro , suggesting an inhibitory effect on virulence factors. Interestingly, the R20291 TreR transcriptional regulatory protein appeared to possess an activator function as its DNA-binding ability was increased in the presence of its effector, trehalose-6-phosphate. Using RNA-sequencing analysis, we report the identification of a putative trehalose metabolism pathway which is induced during growth in trehalose: this has not been previously described within the C. difficile species. These data demonstrate the metabolic diversity exhibited by C. difficile which warrants further investigation to elucidate the molecular basis of trehalose metabolism within this important gut pathogen.