C. difficile Spores and Macrophage Interactions Figure 2. Adherence and internalization of C. difficile spores by Raw 264.7 cells. Monolayers of Raw 264.7 cells were infected at an MOI of 10 with Alexa- and biotoin-labeled C. difficile spores of strains 630 and Pitt177, unbound spores washed, and samples prepared for fluorescence microscopy. Percentage of raw macrophages with at least one spore (A), number of spores per macrophage complex (B), and percentage of intracellular spores (C) were quantified as described in Methods section. D, E, F) The effect of cytochalasin D on the relative binding of spores to Raw 264.7 cells (D), relative number of spores per Raw-spore complex (E), and relative percentage of internalization (F) was evaluated without (white bars) and with 1 mM cytochalasin D (grey bars). Relative values refer to the relative percentage of Raw 264.7 cells with at least one spore (D), relative number of spores per Raw-spore complex (E), and relative percentage of internalization (F) in presence of cytochalasin D normalized to the culture medium control. Results are combined from at least three independent experiments and error bars are standard error of the mean. Asterisks (*) denote statistical difference at p,0.05, and double asterisks (**) denote statistical difference at p,0.01 compared to culture medium control. doi:10.1371/journal.pone.0043635.g002 suggesting that mature phagolysosome were formed and that the spores were attacked by the antimicrobial machinery of Raw 264.7 cells. Although it seems as if some surface layers might be detaching from C. difficile spores (Fig. 7B), it was most striking that the ultrastructure of spores remained intact even after 24 h of infection with Raw 264.7 cells (Fig. 7C), indicating that C. difficile spores were able to survive inside macrophages during infection. Most notably, from a total of 30 spores analyzed after 0.5 and 24 h of infection with Raw 264.7 cells, no germinated spore was detected (data not shown), suggesting that C. difficile spores remained dormant inside macrophages. Since C. difficile strain 630 spores are able to survive inside the macrophage’s phagolysosome, we hypothesized that these spores might be modulating the phagolysosome’s activity through direct interaction with spore’s surface proteins. Indeed, once phagocy- tosed, the fate of pathogenic bacteria varies depending on their ability to escape the phagosome. For example, B. anthracis spores localize to the late phagolysosome [21], whereas C. perfringens vegetative cells escape from the phagosome by degrading the phagosome membrane [35]. Therefore, to gain more insight into the fate of C. diffcile spores during macrophages infection, we analyzed TEM images of phagosytosed C. difficile strain 630 spores after 30 min of infection. As a control, we infected Raw 264.7 cells with B. subtilis PS832 wild-type spores, and observed that B. subtilis spores remained inside the phagosome with no disruption of the phagosome’s membrane (Fig. 7D). In contrast, in Raw 264.7 cells infected with C. difficile spores, several phagosomes containing C. difficile spores had a disrupted membrane (Fig. 7E and data not shown). A closer examination revealed that the surface of C. difficile spores is closely interacting with the phagosome’s membrane (Fig. 7F). Collectively, these results clearly indicate that at least some C. difficile spore-containing phagosome loss their membrane integrity and that C. difficile spore surface interacts with the phagosome’s membrane. PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43635