Modeling Clostridium difficile infections

Clostridium difficile (Cd) is a gram-positive, spore-forming bacterium and the primary cause of nosocomial diarrhea and pseudomembranous colitis. The pathogenicity of Cd has been linked to its production of TcdA and TcdB. While they cause fluid secretion, inflammation, and colonic damage, their respective and synergistic roles have been difficult to ascertain. In infection animal model, TcdB has been demonstrated to be a key virulence factor, and TcdB causes obvious damage in human and porcine colonic explants. Using the colonic epithelia derived from cloned colonic stem cells, we have developed a model to test the response to TcdB. Epithelia generated in air-liquid interface cultures from cloned transverse colon stem cells were challenged with TcdB at different concentrations and durations. Colonic stem cells were differentiated in air-liquid interface cultures and treated with C. difficile toxin B at different doses (100 pM, 250 pM, and 500 pM) and times (8 hrs, 16 hrs and 24 hrs). At appropriate time points, membranes with ALI structure were collected for histology and microarray analysis.