Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis

Area of Science:

• Microbiology and Infectious Diseases
• Gastroenterology
• Cell Biology

Background:

• Clostridioides difficile is a major cause of nosocomial infections, leading to severe diarrhea and colitis via toxin production.
• Current models using cancer cell lines lack biological relevance for studying C. difficile-induced enteritis.
• A need exists for a relevant model to investigate C. difficile pathogenesis in the small intestine.

Purpose of the Study:

• To establish and validate human intestinal enteroids (HIEs) as a novel model for studying C. difficile toxin effects in the small intestine.
• To investigate the differential sensitivity of HIEs to C. difficile toxins A (TcdA) and B (TcdB).
• To explore the role of mucus in modulating C. difficile toxin interactions with intestinal epithelium.

Main Methods:

• Generation of biopsy-derived jejunal HIEs and Vero cells expressing fluorescently labeled F-actin (LifeAct-Ruby).
• Live-cell microscopy to monitor actin cytoskeleton dynamics in response to C. difficile toxins.
• Quantitative PCR to assess toxin receptor mRNA expression.
• Addition of mucin 2 (MUC2) to cell cultures to evaluate mucus barrier function.

Main Results:

• Pathogenic C. difficile toxins induced cell rounding in HIEs in a strain-dependent manner.
• HIEs exhibited tenfold greater sensitivity to TcdA compared to TcdB.
• Despite higher toxin receptor mRNA levels, HIEs showed decreased sensitivity to toxins compared to traditional cell lines, attributed to mucus secretion.
• Human MUC2 addition to Vero cells delayed toxin-induced cell rounding, confirming mucus as a protective barrier.

Conclusions:

• Human intestinal enteroids provide a biologically relevant in vitro model for studying C. difficile cytotoxicity in the small intestine.
• Mucus secreted by HIEs acts as a significant barrier, influencing the interaction between C. difficile toxins and the intestinal epithelium.
• This model facilitates deeper insights into C. difficile infection mechanisms and host-pathogen interactions in the gut.