Impact of vancomycin and Clostridioides difficile on the secretome and pathogenicity of Clostridium innocuum

Clostridium innocuum, a member of the human gut microbiome with intrinsic resistance to vancomycin, has been increasingly associated with inflammatory bowel diseases (IBD). Clinical observations indicate that co-infection with Clostridioides difficile and C. innocuum could lead to poorer clinical remission in ulcerative colitis; however, the pathogenic mechanism of C. innocuum remains unclear. Here, we investigated the effects of vancomycin and C. difficile on C. innocuum secretomes and the functions of the modified secretomes on C. innocuum pathogenicity. The results indicated that, compared to co-culturing with C. difficile, vancomycin was more effective in stimulating the secretion of proteins without a signal peptide, whereas C. difficile was better at promoting the secretion of classical secretory proteins. Based on these results, we further analyzed the effects of three abundant classical secretory proteins on C. innocuum virulence utilizing recombinant proteins. The results demonstrated that the NlpC/P60-containing protein (NlpC/P60) can enhance C. innocuum biofilm formation and adherence to HT-29 cells. Additionally, NlpC/P60, D-Ala-D-Ala carboxypeptidase, and a polysaccharide deacetylase were able to stimulate IL-8 production of HT-29 cells and TNF-α production of Raw264.7 macrophages. Additionally, recombinant NlpC/P60 and polysaccharide deacetylase exhibited cytotoxicity on Raw264.7 cells at 48 h. As the production of IL-8 and TNF-α is closely associated with IBD development, it is suggested that C. innocuum secretomes, under the influence of vancomycin or C. difficile, could contribute to IBD progression by enhancing inflammation and host-pathogen interactions.