Sarah Lynn Martz1, Mabel Guzman-Rodriguez1, Shu-Mei He1, Curtis Noordhof1, David John Hurlbut2, Gregory Brian Gloor3, Christian Carlucci4, Scott Weese4, Emma Allen-Vercoe4, Jun Sun5, Erika Chiong Claud6, Elaine Olga Petrof7. 1. Division of Infectious Diseases/GI Diseases Research Unit Wing, Department of Medicine, Kingston General Hospital, Queen's University, 76 Stuart Street, Kingston, ON, K7L 2V7, Canada. 2. Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, K7L 2V7, Canada. 3. Department of Biochemistry, University of Western Ontario, London, ON, N6A 5C1, Canada. 4. Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada. 5. Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA. 6. Departments of Pediatrics and Medicine, University of Chicago, Chicago, IL, 60637, USA. 7. Division of Infectious Diseases/GI Diseases Research Unit Wing, Department of Medicine, Kingston General Hospital, Queen's University, 76 Stuart Street, Kingston, ON, K7L 2V7, Canada. eop@queensu.ca.
Abstract
BACKGROUND: A defined Microbial Ecosystem Therapeutic (MET-1, or "RePOOPulate") derived from the feces of a healthy volunteer can cure recurrent C. difficile infection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host. METHODS: A combination of in vivo (antibiotic-associated mouse model of C. difficile colitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed. RESULTS: MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis. CONCLUSIONS: MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host.
BACKGROUND: A defined Microbial Ecosystem Therapeutic (MET-1, or "RePOOPulate") derived from the feces of a healthy volunteer can cure recurrent C. difficileinfection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host. METHODS: A combination of in vivo (antibiotic-associated mouse model of C. difficilecolitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed. RESULTS:MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis. CONCLUSIONS:MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host.
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