# Proline Stickland fermentation supports C. difficile spore maturation

## TL;DR

The study shows that proline fermentation influences spore maturation in Clostridioides difficile, a gut pathogen.

## Contribution

The novel finding is that proline fermentation and its byproducts regulate spore development and quality in C. difficile.

## Key findings

- Proline reductase is important for the development of mature C. difficile spores.
- Excess proline can repress sporulation through regulation by PrdR.
- 5-aminovalerate, a product of proline reduction, supports C. difficile growth via a PrdR-dependent mechanism.

## Abstract

Clostridioides difficile is an anaerobic pathogen that thrives in the metabolically diverse intestinal environment. C. difficile is readily transmitted due to its transformation into a dormant spore form that is highly resistant to heat and disinfectants. Nutrient limitation is a key driver of spore formation; however, few metabolites have been directly shown to influence the regulation of C. difficile sporulation. A distinct aspect of C. difficile biology is the fermentation of amino acids through Stickland metabolism pathways, which are critical sources of energy for this pathogen. We hypothesized that as a preferred energy source, the amino acid proline may serve as a signal that regulates the initiation of sporulation or the development of spores. Using mutants in the proline reductase gene, prdA, and the proline-dependent regulator, prdR, we examined the impact of proline on C. difficile physiology and differentiation. Our results demonstrate that proline reductase is important for the development of mature spores and that excess proline can repress C. difficile sporulation through PrdR regulation. Furthermore, we discovered that the end product of proline reduction, 5-aminovalerate, can support the growth of C. difficile through an unidentified, PrdR-dependent mechanism.

C. difficile is an anaerobic intestinal pathogen that disseminates in the environment as dormant, resilient spores. Nutrient limitation is known to stimulate spore production, but the contribution of specific nutrients to sporulation is poorly understood. In this study, we examined the contribution of proline and proline fermentation to spore formation. Our results demonstrate the effect of proline fermentation on spore quality and the importance of the proline reductase pathway on spore maturation.