Cytoplasmic membrane vesicles from Clostridioides difficile R20291 are remodeled by osmotic stress

Introduction – Bacterial extracellular vesicles are important mediators of host–microbe interactions, facilitating the transport of proteins, toxins, and other bioactive molecules across biological barriers. Although vesicle production has been reported in several Gram-positive pathogens, the environmental factors influencing vesicle characteristics in Clostridioides difficile remain poorly understood. This study investigated the impact of osmotic stress on the physicochemical properties and epithelial cell association of cytoplasmic membrane vesicles (CMVs) produced by the hypervirulent strain R20291. Methods – C. difficile R20291 was cultured under standard conditions or in medium supplemented with 350 mM NaCl. CMVs were isolated from culture supernatants by ultrafiltration and ultracentrifugation. Vesicle morphology, size distribution, and abundance were evaluated using transmission electron microscopy and nanoparticle tracking analysis. Protein and lipid contents were quantified using biochemical assays. CMV association with HT-29 epithelial cells was assessed by confocal microscopy and flow cytometry. Glucosyltransferase-associated enzymatic activity was evaluated using a colorimetric substrate assay before and after vesicle disruption. Results – CMVs were produced under both culture conditions and displayed the typical spherical morphology observed for bacterial membrane vesicles. Osmotic stress was associated with significant changes in vesicle physicochemical properties. NaCl-derived CMVs exhibited smaller particle diameters and a narrower size distribution than control CMVs. Biochemical analyses showed increased protein content and reduced lipid levels in vesicles produced under high-salinity conditions. Confocal microscopy and flow cytometry demonstrated increased CMV-associated fluorescence in HT-29 cells exposed to NaCl-derived vesicles compared with control vesicles. Vesicle preparations displayed measurable glucosyltransferase-associated enzymatic activity, which increased following vesicle disruption, suggesting the presence of enzyme-associated cargo within the vesicle structure. Discussion – These findings demonstrate that osmotic stress is associated with alterations in the size, composition, and epithelial cell association of CMVs produced by C. difficile. The observed changes suggest that environmental conditions may influence vesicle-mediated interactions between C. difficile and host cells, potentially affecting processes relevant to bacterial colonization and pathogenesis.