Carter, Z. A. | Search bioRxiv

bioRxiv2025

Proline Stickland fermentation supports C. difficile spore maturation

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. Further, we discovered that the end product of proline reduction, 5-aminovalerate, can support the growth of C. difficile through an unidentified, PrdR-dependent mechanism. IMPORTANCEC. 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.

physiology↗

bioRxiv2023

The pH-responsive SmrR-SmrT system modulates C. difficile antimicrobial resistance, spore formation, and toxin production

Clostridioides difficile is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine, C. difficile must adapt to a variety of conditions in its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect C. difficile toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in C. difficile cell outcomes. In this study, we investigated two genes, CD2505 and CD2506, that are differentially regulated by pH to determine if they impact C. difficile growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein smrR and smrT) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of smrRT that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance. IMPORTANCEC. difficile is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease. C. difficile is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes, smrR and smrT on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses smrT expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.

microbiology↗