title: A human gut ecosystem protects against C. difficile disease by targeting TcdA.
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.
authors.affiliation: 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.
authors.forename: Sarah Lynn
authors.initials: SL
authors.lastname: Martz
authors.affiliation: 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.
authors.forename: Mabel
authors.initials: M
authors.lastname: Guzman-Rodriguez
authors.affiliation: 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.
authors.forename: Shu-Mei
authors.initials: SM
authors.lastname: He
authors.affiliation: 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.
authors.forename: Curtis
authors.initials: C
authors.lastname: Noordhof
authors.affiliation: Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, K7L 2V7, Canada.
authors.forename: David John
authors.initials: DJ
authors.lastname: Hurlbut
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, ON, N6A 5C1, Canada.
authors.forename: Gregory Brian
authors.initials: GB
authors.lastname: Gloor
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Carlucci
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors.forename: Scott
authors.initials: S
authors.lastname: Weese
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors.forename: Emma
authors.initials: E
authors.lastname: Allen-Vercoe
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Sun
authors.affiliation: Departments of Pediatrics and Medicine, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Erika Chiong
authors.initials: EC
authors.lastname: Claud
authors.affiliation: 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.
authors.forename: Elaine Olga
authors.initials: EO
authors.lastname: Petrof
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001691:Biological Therapy / Q000379:methods*; D018938:Caco-2 Cells; D016360:Clostridioides difficile / Q000254:growth & development* / Q000302:isolation & purification; D003599:Cytoskeleton / Q000473:pathology; D004195:Disease Models, Animal; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology / Q000473:pathology / Q000517:prevention & control*; D004768:Enterotoxins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005243:Feces / Q000737:chemistry / Q000166:cytology / Q000382:microbiology; D005347:Fibroblasts / Q000473:pathology; D000069196:Gastrointestinal Microbiome; D006801:Humans; D008810:Mice, Inbred C57BL
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile
keywords: C. difficile; Microbiota; TcdA