title: Clostridium difficile spore-macrophage interactions: spore survival.
abstract: BACKGROUND
  Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment.
  
  
  RESULTS
  In this work, we provide evidence that C. difficile spores are well suited to survive the host's innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells' ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1.
  
  
  CONCLUSIONS
  These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.
authors.affiliation: Laboratorio de Mecanismos de Patogénesis Bacteriana, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chile. daniel.paredes.sabja@gmail.com
authors.forename: Daniel
authors.initials: D
authors.lastname: Paredes-Sabja
authors.forename: Glenda
authors.initials: G
authors.lastname: Cofre-Araneda
authors.forename: Christian
authors.initials: C
authors.lastname: Brito-Silva
authors.forename: Marjorie
authors.initials: M
authors.lastname: Pizarro-Guajardo
authors.forename: Mahfuzur R
authors.initials: MR
authors.lastname: Sarker
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D016360:Clostridioides difficile / Q000502:physiology*; D054884:Host-Pathogen Interactions; D008264:Macrophages / Q000166:cytology / Q000382:microbiology*; D051379:Mice; D010587:Phagocytosis; D010588:Phagosomes / Q000382:microbiology; D013010:Sonication; D013171:Spores, Bacterial / Q000502:physiology