Membrane vesicles from selected Clostridioides difficile strains induce epithelial-mesenchymal transition in colonic epithelial cells: insights from in vitro and in vivo studies

Clostridioides difficile, as the leading cause of antibiotic-associated diarrhea, contributes to nosocomial and community-acquired infections. Chronic inflammation is a well-known stimulator of epithelial-mesenchymal transition (EMT) pathway, which can subsequently induce carcinogenesis in host tissues. Here, we examined the ability of membrane vesicles (MVs) derived from two clinical C. difficile ribotypes (RT001 and RT084), and the non-toxigenic strain ATCC 700057 to induce EMT process using in vitro and in vivo experiments. The proliferation and viability of HT-29 and SW-480 cells exposed to different concentrations of C. difficile MVs were assessed by trypan blue exclusion test and MTT assay, respectively. The expression level of EMT markers was determined using RT-qPCR and Western blotting. EMT induction was further investigated in 8-week-old male wild-type C57BL/6 mice orally gavaged with MVs for up to 6 weeks. C. difficile MVs did not significantly affect the viability and proliferation of both cell lines. We found upregulation of β-catenin, vimentin, snail, and reduction of E-cadherin genes in colon cancer cells and tissues of mice treated with MVs. Our results demonstrated that RT084 MVs had a stronger impact on EMT induction. Additionally, Western blot analysis of proteins of SW-480 cells corroborated the enhancement of EMT by RT084 MVs. Our findings suggest that C. difficile MVs, especially RT084 MVs, can induce EMT in mice colonic tissues. These findings provide novel mechanistic insights into how C. difficile MVs may promote pro-tumorigenic changes and highlight their potential as platforms for vaccine design, host-pathogen interaction studies, and targeted delivery systems. Further studies are needed to precisely corroborate the role of C. difficile MVs and their content on EMT induction and progression in CDI.