Increased sporulation underpins adaptation of Clostridium difficile strain 630 to a biologically–relevant faecal environment, with implications for pathogenicity

TLDR

Clostridium difficile virulence is driven primarily by toxinogenesis and sporulation, yet many in vitro systems lack relevance to the human colonic environment. We aimed to develop a physiologically relevant colonic model by incorporating human faecal water into growth media to study strain 630. The model involved adding faecal water to media and assessing its physiological effects on strain 630, including metabolite profiling and transcriptomic analysis. In faecal‑water media, strain 630 produced novel hexanoyl‑ and pentanoyl‑amino acid derivatives, grew longer without changing rate, and upregulated up to 300‑fold sporulation genes while downregulating motility and toxin genes, with no stress‑gene changes, indicating enhanced transmissibility.

Abstract

Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically-relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile-derived metabolites in culture supernatants, including hexanoyl- and pentanoyl-amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300-fold increases in the expression of sporulation-associated genes were observed in FW media-grown cells, along with reductions in motility and toxin genes' expression. Moreover, the expression of classical stress-response genes did not change, showing that C. difficile is well-adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility.