P150 Ulcerative Colitis Patients’ Luminal Zinc Induces Clostridioides difficile Virulence and Exacerbates Inflammatory Responses

Abstract

Abstract Background The refractory acute severe UC patients are often complicated by C. difficile infection (CDI), resulting in higher surgery and mortality rates. However, few studies have observed C. difficile’s pathogenicity in real UC patients’ gastrointestinal microenvironments, and the actual role of C. difficile in UC development remains unknown. We found zinc concentrations among UC patients’ feces were significantly higher than those among Crohn’s disease patients and healthy subjects. Additionally, we previously reported that excessive zinc would promote the growth and virulence of C. difficile. Therefore, we hypothesized that the increased UC patients’ luminal zinc may up-regulate C. difficile virulence and promote intestinal inflammatory responses, leading to an exacerbated UC progression. Methods UC patients’ stool samples (n=40) were collected and analyzed by qPCR and ICP-MS to determine the presence of C. difficile and the fecal zinc concentrations. C. difficile was cultured in BHIs with or without zinc (ZnSO4) in an anaerobic chamber. Expression of virulence genes was analyzed using RT-qPCR, transcriptome sequencing, and western blot. Human colonic organoids and Caco-2 cells were co-cultured with C. difficile or zinc-treated C. difficile, and cytotoxicity, permeability, and inflammatory cytokine release were assessed. TLR5 knock-out Caco-2 cells were constructed using sgRNA-guided Cas9 nuclease and co-cultured with C. difficile or zinc-treated C. difficile to verify whether zinc-induced C. difficile’s pathogenicity was mainly through flagellar formation. Results C. difficile was detected in ~35% of UC patients’ fecal samples. Luminal zinc concentrations (~915μM, p<0.01) were significantly higher among UC patients than CD patients or healthy subjects. The UC patients’ luminal concentration of zinc promoted C. difficile growth, suppressed its exotoxins release, and induced higher expression of its flagella-related genes, resulting in higher cytotoxicity and inflammatory responses in human colonic organoids and Caco-2 cells. However, zinc-treated C. difficile did not induce higher inflammation and pathogenicity in TLR5 knock-out Caco-2 cells. Conclusion The association between C. difficile and UC aggravation may partially stem from UC patients’ excess luminal zinc promoting C. difficile flagellin expression, inducing inflammatory responses, and worsening UC progression.