d-Proline Reductase Underlies Proline-Dependent Growth of Clostridioides difficile

Area of Science:

• Microbiology
• Molecular Biology
• Host-Pathogen Interactions

Background:

• Clostridioides difficile is a major nosocomial pathogen causing gastrointestinal illness.
• Collagen degradation by C. difficile releases amino acids essential for its anaerobic Stickland metabolism.
• The roles of collagen-derived amino acids (proline, glycine, hydroxyproline) and specific enzymes in C. difficile physiology are not fully understood.

Purpose of the Study:

• To investigate the impact of proline, glycine, and hydroxyproline on C. difficile growth and physiology.
• To elucidate the specific roles of d-proline reductase and glycine reductase in Stickland metabolism.
• To determine the effect of these amino acids and related enzymes on C. difficile toxin production.

Main Methods:

• Culturing C. difficile strains (wild-type and mutant) in various media (BHIS, TY, CDMM) supplemented with proline, glycine, or hydroxyproline.
• Assessing growth yields and analyzing the necessity of d-proline reductase and glycine reductase activities.
• Measuring toxin production in response to amino acid supplementation in wild-type and mutant strains.

Main Results:

• Proline and hydroxyproline, but not glycine, enhanced growth yields in rich media; proline-stimulated growth required d-proline reductase activity.
• C. difficile could grow in a defined medium without proline only when d-proline reductase was absent, indicating enzyme-induced proline auxotrophy.
• Proline and hydroxyproline supplementation reduced toxin production, a mechanism dependent on selenoproteins.

Conclusions:

• The presence of d-proline reductase imposes a strict proline dependence on C. difficile, influencing its host colonization strategies.
• Stickland metabolism, particularly selenium-dependent reactions involving proline and hydroxyproline, is crucial for C. difficile colonization.
• Proline and hydroxyproline can suppress C. difficile toxin production via a selenoprotein-dependent pathway, offering potential therapeutic targets.