Mechanisms by which proline reductase of Clostridioides difficile promotes efficient metabolism and disease progression in vivo

Clostridioides difficile is a toxin-producing pathogen that opportunistically infects those with a depleted gut microbiome, often triggered by antibiotic use. C. difficile preferentially utilizes Stickland amino acids, including proline, to promote energy generation and growth for colonization. We evaluated host outcomes from infection with wild-type and Δ prdB mouse-infective C. difficile ATCC 43255 strains to investigate how proline metabolism modulates C. difficile’s pathogenesis and interactions with commensals in an enriched gut nutrient environment. While gnotobiotic mice infected with the Δ prdB mutant succumbed to infection, they showed delayed colonization and toxin production, thereby extending their survival. In vivo C. difficile transcriptomic analyses demonstrated a shift from Stickland fermentation to carbohydrate metabolism in the Δ prdB mutant. To investigate functions of C. difficile’s proline reductase in interactions with the commensal microbiota, we evaluated infection outcomes in mice co-colonized with the disease-promoting commensal Clostridium sardiniense (CSAR). One-third of CSAR and Δ prdB- infected mice survived, while co-colonized mice with the wild-type strain rapidly succumbed within 48 h. In vivo transcriptomic analyses from co-colonized mice identified additional metabolic defects in the Δ prdB mutant, including failure to express oxidative Stickland pathways and ornithine fermentations to harness CSAR-produced ornithine, suggesting conversion of the mutant into a glycolytic strain that was now in direct competition with CSAR for carbohydrate substrates. CSAR remained metabolically active during the Δ prdB infection through its mucin degradation and polysaccharide and disaccharide metabolism. Our findings illustrate critical functions of C. difficile’s proline reductase in coordinating early metabolism to facilitate gut colonization, including interactions with a cross-feeding commensal species.

IMPORTANCE

Clostridioides difficile is a spore-forming bacterium that commonly causes pseudomembranous colitis in patients exposed to antibiotics, with infections leading to 30,000 deaths annually in the United States. We conducted further in vitro and in vivo analyses to determine how proline reductase modulates C. difficile colonization, growth, and metabolism, and showed that this pathway is critical to Clostridium sardiniense’ s ability to cross-feed with the pathogen to cause toxic megacolon. We demonstrated that sporulation and toxin production are delayed when C. difficile’ s proline reductase pathway is interrupted through the deletion of the prdB gene. Survival was enhanced in mice co-colonized with C. sardiniense and the C. difficile Δ prdB mutant, as C. difficile relies on glycolytic pathways over Stickland fermentations. The present findings support the central role of proline reductase metabolism in early pathogen growth, metabolism, and toxin, and as a potential therapeutic target against C. difficile infection .