Clostridium difficile Spore-Macrophage Interactions: Spore Survival Daniel Paredes-Sabja1,2*, Glenda Cofre-Araneda1, Christian Brito-Silva1, Marjorie Pizarro-Guajardo 1 , Mahfuzur R. Sarker2,3* 1 Laboratorio de Mecanismos de Patoge´nesis Bacteriana, Departamento de Ciencias Biolo´ gicas, Facultad de Ciencias Biolo´ gicas, Universidad Andres Bello, Santiago, Chile, 2 Department of Biomedical Sciences, Oregon State University, Corvallis, Oregon, United States of America, 3 Department of Microbiology, Oregon State University, Corvallis, Oregon, United States of America 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. Methodology/Principal Findings: 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/Significance: 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. Citation: Paredes-Sabja D, Cofre-Araneda G, Brito-Silva C, Pizarro-Guajardo M, Sarker MR (2012) Clostridium difficile Spore-Macrophage Interactions: Spore Survival. PLoS ONE 7(8): e43635. doi:10.1371/journal.pone.0043635 Editor: Dipshikha Chakravortty, Indian Institute of Science, India Received February 23, 2012; Accepted July 24, 2012; Published August 27, 2012 Copyright: 2012 Paredes-Sabja et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MECESUP UAB0802, Fondo Nacional de Ciencia y Tecnolog´ıa de Chile (FONDECYT REGULAR 1110569), by a grant from the Research Office of Universidad Andres Bello (DI-35-11/R) (to D.P.-S); grant from the Agricultural Research Foundation of Oregon State University and by a Department of Defense Multi-disciplinary University Research Initiative (MURI) award through the U.S. Army Research Laboratory and the U.S. Army Research Office under contract number W911NF-09-1-0286 (to MRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: daniel.paredes.sabja@gmail.com (DPS); sarkerm@oregonstate.edu (MRS) Introduction Clostridium difficile is a Gram-positive, anaerobic bacterial pathogen, responsible for ,20% of antibiotic-associated diarrheas, pseudomembranous colitis and toxic megacolon [1,2]. Onset of Clostridium difficile infections (CDI) typically occur during or after antibiotic treatment of hospitalized patients depending on whether the infected C. difficile isolate exhibits resistance to the antibiotic being administered [2]. Antibiotics disrupt the normal colonic flora, which normally suppresses C. difficile growth, therefore allowing C. difficile to colonize empty niches and secrete two major toxins, TcdA and TcdB producing massive intestinal epithelium damage [2]. In addition, both toxins trigger the release of various cytokines and chemokines that lead to an intensive immune response resulting in the recruitment of neutrophils and macro- phages from the systemic system [2]. During the onset of CDI, C. difficile begins a sporulation cycle in the colon [1,3] leading to persistence of spores in the colonic tract which can be shedded to the environment for up to 1 to 4 weeks after CDI treatment [4]. Indeed, in vitro work has demonstrated that C. difficile spores adhere particularly well to intestinal epithelial cells in culture [5]. Dormant C. difficile spores are impermeable to all known antibiotic treatments [6,7]. These persistent spores then germinate, colonize and produce recurrent CDI episodes [7]. C. difficile spores germinate in presence of cholates and its derivatives [8,9] but germination is more efficient in the presence of certain amino acids that act as co-germinants [10]. The massive macrophage and neutrophil recruitment during the course of CDI suggests that there must be some sort of interaction between C. difficile and the innate immune system. Indeed, recent studies have demonstrated that Toll-like receptor 4 and the nucleotide-binding oligomeriza- tion domain 1 (Nod1) recognizes the C. difficile vegetative cells and mediate protection against CDI [11,12]. Once phagocytosed, the fate of bacterial spores will vary depending on their specific virulence traits that will enable them to either escape of or to modulate the host innate immune system. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43635