Characterising regulation of virulence factors affecting the interaction between Clostridioides difficile and the intestinal microbiome.

Clostridioides difficile is the leading cause of antibiotic associated diarrhoea, causing significant morbidity and mortality worldwide. Treatment of C. difficile infection (CDI) with antibiotics can be effective, however, up to 30% of patients suffer from infection recurrence, often multiple episodes, either through infection relapse or reinfection. Even treatment with narrow spectrum antibiotics such as fidaxomicin, which is considered microbiome sparing, is still associated with a significant proportion of patients experiencing recurrence. Therefore, treatments targeting C. difficile’s specific virulence factors, including their expression and regulation, offer a promising route to manage CDI and reduce recurrence, however, a greater understanding of their underlying biology is required. A major C. difficile virulence factor that provides it with a competitive advantage over certain species of the gut microbiota is the production of the antibacterial compound p-cresol. p-cresol is produced by the fermentation of tyrosine to give the precursor p-HPA which is converted to p-cresol by the actions of HpdBCA decarboxylase, encoded on the hpdBCA operon. Three different transcriptional reporter systems were used, and evaluated, to characterise the hpdBCA promoter and found that supplementation of growth media with p-HPA drives expression of hpdBCA leading to high level pcresol production. This response was found across strains representing all five C. difficile clades showing that this is an extremely well conserved pathway. Additionally, exogenous p-HPA was found to be inhibitory to C. difficile growth, an effect enhanced by inactivation of HpdBCA, suggesting conversion of p-HPA to p-cresol provides C. difficile with a growth advantage. Similarly to p-cresol, p-HPA was also found to inhibit commensal species commonly found in the gut, with Gram-negative strains more susceptible than Gram-positive strains. Confocal microscopy revealed that HpdB is expressed ubiquitously by all cells within a C. difficile culture highlighting the importance of this response to C. difficile. These findings validate the hypothesis that targeting p-cresol production by inhibition of HpdBCA would be effective across all C. difficile infections regardless of causative strain. An alternative approach to targeting C. difficile virulence factors directly is to target the regulators that control their expression and understand how they influence C. difficile’s lifestyle. The sinRCD2215 locus is formed of two transcriptional regulators and is involved in the control of C. difficile lifestyle choice with a major role in sporulation regulation. The work in this study describes key regulatory controls of the sinR-CD2215 locus including autoregulation by SinR itself, together with identification of its binding site, and control of the operon’s expression by major regulators Spo0A, CodY and CcpA. This study has added significant insight into the regulatory controls of key virulence factors by C. difficile and highlights the importance of these controls to C. difficile infection.