Discovery of a Widespread Polyamine-Low-Molecular-Weight Thiol Hybrid Pathway in Clostridioides difficile

Clostridioides difficile infection can cause severe inflammation in the gastrointestinal (GI) tract, leading to diarrhea, colitis, and an increased risk of colorectal cancer. Colonization of C. difficile is associated with microbial community-level changes in the expression of polyamine and polyamine precursor biosynthesis genes. Polyamines are abundant cationic metabolites that serve indispensable functions for all kingdoms, particularly in gut homeostasis. Catabolism of the polyamine precursors arginine and ornithine offers C. difficile supplemental nutrition while subverting host immunity, yet existing models of C. difficile metabolism are incomplete regarding polyamines with comparable importance in the gut (e.g., spermidine). In this study, we conducted feeding studies with isotope-labeled polyamines and discovered a network of low-molecular-weight thiol (LMWT) molecules termed clostridithiols (CSHs) constructed from polyamines conjugated with N-acetylcysteine (NAC) moieties. NAC is clinically used as a mucolytic agent and is a well-established redox molecule. Through the analysis of a human microbiota diversity collection, we established that these previously uncharacterized hybrid metabolites are widely detected in Firmicutes and Bacteroidetes. A genetic screen using DNA from an alternative CSH producerBacteroides uniformis enabled the identification and validation of a two-gene operon, including a gene encoding a domain of unknown function, that was conserved in both producing organisms and other members of the microbiome. CSH abundance in GI mucosal biopsies positively correlated with colorectal cancer compared with matched healthy control samples. These studies indicate that human microbial metabolism broadly unites polyamine and LMWT functionalities to generate metabolites that may be associated with disease.