C. difficile Spores and Macrophage Interactions Figure 7. C. difficile spores remain intact inside the phagosome of Raw 264.7 cells. TEM images of Raw 264.7 cells infected with C. difficile spores under aerobic conditions (A–C, E, F) and with B. subtilis spores (D). TEM micrographs were taken after 30 min (A,B and D–F) and 24 h of infection. A) TEM shows that C. difficile spores are efficiently phagosytosed by Raw 264.7 cells. B), Phagosome containing C. difficile spores fuses with lysosomes, white arrows denotes fusion of lysosomes with the phagosome. C), C. difficile spores remain intact after 24 h of infection with Raw 264.7 macrophages. D) TEM micrograph of phagosytosed B. subtilis spores by Raw 264.7 cells. Phagosome’s membrane remains intact. E) TEM image showing phagocytosed C. difficile spore in a phagosome with membrane damage, white arrows denote disrupted phagosome membrane. F) TEM image rendering direct interactions between the surface of C. difficile spores and the phagosome’s membrane. White scale bar is 500 nm for panels A–C, and 100 m, for panels D-F. doi:10.1371/journal.pone.0043635.g007 because remnants of the exosporium-like structure hold sufficient motifs recognized by Raw 264.7 cells to phagocytose C. difficile spores. Therefore, in an in vivo situation in the absence of some exosporium material, activated primary macrophages localized in the lamina propria, might efficiently recognize and phagocytose C. difficile spores. In addition, it was quite surprising to note that the percentage of C. difficile spore adherence of and phagocytosis by Raw 264.7 cells were lower for a clinical isolate Pitt177 than for the domesticated strain 630, suggesting that some sort of antigenic variation might have occurred on the surface of Pitt177 spores allowing it to evade attacks from the innate immune system. Unfortunately, our knowledge of the spore surface proteins is limited, and further studies on the identification of spore surface antigens will help uncover the reason for these differences. Complement also plays a role in recognition and opsonization of bacterial pathogens [40]. Our current results showed that in presence of untreated or heat inactivated fetal bovine serum supplemented with complement there was a significant decrease of spore phagocytosis, suggesting that C. difficile spores might be able to modulate components of complement to evade the innate immune system. This phenomenon has been previously reported in numerous bacterial pathogens including B. anthracis [41–45]. Notably, B. antharcis spores’ surface a-enolase and Elongation factor Tu can bind plasminogen which in turn degrades deposited C3b resulting in a decrease in phagocytosis [41]. Although not yet proven, this raises the possibility that a similar mechanism might be applicable for C. difficile spores’ phagocytosis. Once phagocytosed, survival of pathogenic bacteria is de- pendent on their ability to survive or escape the phagosome and/ or inhibit the maturation of phagolysosome. Our results clearly suggest that C. difficile spores are unable to germinate during macrophage infection in absence of germinants. It was most PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43635