C. difficile Spores and Macrophage Interactions Figure 1. Representative fluorescence micrographs of internalization of C. difficile spores by Raw 264.7 cells. C. difficile spores were labeled with biotoin and Alexa Fluor 488 (green) prior to infection of monolayers of Raw 264.7 cells (red). Infected Raw 264.7 cells were washed; fixed and extracellular spores were stained with Streptavidin-Alexa Fluor 350 conjugate (blue), stained for F-actin and analyzed by fluorescence microscopy as described in Methods section. Representative micrographs of phagocytosis of C. difficile spores are shown: A) Extracellular C. difficile spores (blue); B) Total C. difficile spores (green); C) Merged images. Bars represent 5 mm. White arrows highlight internalized spores. doi:10.1371/journal.pone.0043635.g001 membrane ruffling (Fig. 5B, C). In addition, some spores are being entrapped by Raw 264.7 cells’ membrane through a mechanism similar to coiling phagocytosis (Fig. 5D,E) [32]. Transmission electron microscopy (TEM) of spore-infected Raw 264.7 cells showed that the site of adherence of C. difficile spores to Raw 264.7 cells can also occur at the end of protrusions from the surface of Raw 264.7 cytoplasmic membrane (Fig. 5F). Collectively, these results suggest that phagocytosis of C. difficile spores by Raw 264.7 cells might be mediated through various phagocytic pathways [33]. C. difficile Spores Survive Inside Raw 264.7 Cells Since C. difficile spores were efficiently recognized and in- ternalized by Raw 264.7 cells, we evaluated if these spores could survive inside Raw 264.7 cells under aerobic conditions. Results demonstrate that no significant reduction in spore viability, measured by spores’ colony forming ability, was observed after 5 h of infection with Raw 264.7 cells at an MOI of 10 (Fig. 6A). Extension of infection periods to 24 h showed a slight but significant (p,0.05) reduction in colony forming ability (Fig. 6A). Strikingly, extending the length of infection up to 48–72 h at an MOI of 10 did not produce a further decrease in spore viability (Fig. 6A). Similar results were observed when Raw 264.7 cells were infected with C. difficile spores at an MOI of 1 (data not shown). These results suggest that C. difficile spores are well suited to survive inside phagocytic cells. Next, we evaluated the survival of C. difficile vegetative cells during macrophage infection for comparative purposes. More than three decimal reductions of vegetative cells of C. difficile strain 630 were observed during the first 30 min of infection with Raw 264.7 cells under aerobic conditions and no viable counts were detected after 5 h of infection. This killing was due to presence of oxygen rather than Raw 264.7 cells, since in absence of Raw 264.7 cells similar degree of killing of vegetative cells of C. difficile strain 630 was observed (data not shown). Since C. difficile is a strictly anaerobic bacterium that lacks most defense mechanisms against reactive oxygen species [34], infection experiments were repeated under anaerobic conditions. Macrophages have been previously reported to be able to survive up to 48 h under anaerobic conditions [35]. Prior to infection, Raw 264.7 cells and DMEM medium were prereduced for 1 and 72 h, respectively. After 24 h of incubation under anaerobic condition, more than 95% of Raw 264.7 cells remained viable as determined by trypan blue viability assay (data not shown). Interestingly, a significant (p,0.01) decrease on viability of C. difficile strain 630 vegetative cells was observed after 30 min of infection (Fig. 6B). After 5 h of incubation under anaerobic conditions, there was a slight increase in viable cell counts (Fig. 6B). Extending the infection time to 24 h demonstrated that C. difficile cells were able to out-grow Raw 264.7 cells (Fig. 6B). These results suggest that: i) under aerobic conditions, C. difficile cells are easily killed by Raw 264.7 cells; ii) under anaerobic conditions, although there is significant killing of C. difficile cells, the surviving cells are able to grow. C. difficile Spores Remain Intact Inside the Phagosome of Raw 264.7 Cells To understand better the fate of C. difficile spores once inside the macrophage, TEM images were obtained after infection of Raw 264.7 cells with C. difficile spores. Raw 264.7 cells were able to efficiently phagocytose several C. difficile strain 630 spores and keep them inside the phagosomes (Fig. 7A). Furthermore, lysosomes fused to the phagosomes containing C. difficile spores (Fig. 7B), PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43635