Cersosimo, L. | Search bioRxiv

bioRxiv2023

The TcdE holin drives toxin secretion and virulence in Clostridioides difficile.

Clostridioides difficile is the leading cause of healthcare associated infections. The Pathogenicity Locus (PaLoc) toxins TcdA and TcdB promote host disease. These toxins lack canonical N-terminal signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release, which have included targeted secretion and passive release from cell lysis. While the holin TcdE has been implicated in TcdA and TcdB release, its role in vivo remains unknown. Here, we show profound reductions in toxin secretion in tcdE mutants in the highly virulent strains UK1 (epidemic ribotype 027, Clade 3) and VPI10463 (ribotype 087, Clade 1). Notably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection by reducing acute extracellular toxin to undetectable levels, limiting mucosal damage, and enabling long-term survival, in spite of continued toxin gene expression in tcdE mutants. Our findings confirm TcdEs critical functions in vivo for toxin secretion and C. difficile virulence.

microbiology↗

bioRxiv2023

Central in vivo mechanisms by which C. difficile's proline reductase drives efficient metabolism, growth, and toxin production

Clostridioides difficile (CD) is a sporulating and toxin-producing nosocomial pathogen that opportunistically infects the gut, particularly in patients with depleted microbiota after antibiotic exposure. Metabolically, CD rapidly generates energy and substrates for growth from Stickland fermentations of amino acids, with proline being a preferred reductive substrate. To investigate the in vivo effects of reductive proline metabolism on C. difficiles virulence in an enriched gut nutrient environment, we evaluated wild-type and isogenic{Delta} prdB strains of ATCC43255 on pathogen behaviors and host outcomes in highly susceptible gnotobiotic mice. Mice infected with the{Delta} prdB mutant demonstrated extended survival via delayed colonization, growth and toxin production but ultimately succumbed to disease. In vivo transcriptomic analyses demonstrated how the absence of proline reductase activity more broadly disrupted the pathogens metabolism including failure to recruit oxidative Stickland pathways, ornithine transformations to alanine, and additional pathways generating growth-promoting substrates, contributing to delayed growth, sporulation, and toxin production. Our findings illustrate the central role for proline reductase metabolism to support early stages of C. difficile colonization and subsequent impact on the pathogens ability to rapidly expand and cause disease.

microbiology↗