C. difficile Spores and Macrophage Interactions Figure 5. Raw 264.7 cells bind and phagocytose C. difficile spores. A,B,C,D,E,F) SEM of Raw 264.7 cells infected with C. difficile spores under aerobic conditions. Note the active phagocytosis of the spores by Raw 264.7 cells in both panels. White arrows denote coiling phagocytosis. F) Transmission electron microscopy (TEM) of Raw 264.7 cells infected with C. difficile 630 spores at an MOI of 10 for 30 min under aerobic conditions. The area of adherence of C. difficile spores occurred at patchy regions at the end of protrusions from the surface of Raw 264.7 cells. Bar: 1 mm. doi:10.1371/journal.pone.0043635.g005 C. difficile spores, no significant inactivation of spores was observed (Fig. 8A). However, when Raw 264.7 cells were infected with ST treated spores, a significant (p,0.05) increase in spore killing was observed under aerobic and anaerobic conditions (Fig. 8A), indicating that germinated spores are easily killed by Raw 264.7 cells. The amount of spore killing in anaerobic condition was lower than under aerobic conditions but higher than in absence of ST (Fig. 8A), supporting the fact that the killing of ST-treated spores is due to Raw 264.7 cells and that absence of oxygen might allow some extracellular germinated C. difficile spores to survive during macrophage infection and increase number of viable cells. Addition of the co-germinant L-glycine increased the amount of spore killing during infection of Raw 264.7 cells under anaerobic conditions (Fig. 8A). Fluorescence microscopy of infected Raw 264.7 cells with ST-treated C. difficile spores demonstrated significant spore outgrowth only during anaerobic incubation (Fig. 8C,D), supporting the fact that at least some ST-treated C. difficile spores are able to outgrow during macrophage infection under anaerobic conditions. Collectively, these results suggest that C. difficile spores treated with ST are easily killed by Raw 264.7 cells. Next, experiments were carried out with reduced concentration of ST (0.1%) in presence of 5 mM of L-glycine (STG) to unmask the effect of other potential factors such as human serum (HS). Significant killing was observed when Raw 264.7 cells were infected with STG treated spores (Fig. 8B). Strikingly, when C. difficile spores were pre-incubated with HS prior to infection of Raw 264.7 cells, there was also a significant (p,0.05) increase in spore killing (Fig. 8B). Pretreatment of spores with HS and STG produced a slight but not significant increase in spore killing by Raw 264.7 cells (Fig. 8B). Heat treatment activates bacterial spore’s germinant receptors to initiate the nonreversible germina- tion process [38]. Therefore, experiments were repeated with heat- activated spores incubated with HS and STG prior infection. There was a slight difference (p = 0.06) in the amount of spore killing between heat-activated versus non heat activated spores incubated with STG and HS. However, this difference became significant (p,0.01) when killing of heat-activated spores in- cubated with STG and HS was compared to that of non-heat activated spores incubated with HS (Fig. 8B). These results indicate that germination factors sensitize C. difficile spores to macrophage mediated killing. C. difficile Spores are Cytotoxic to Raw 264.7 Cells Since above results suggest that C. difficile spores are able to survive and also interact with the phagosome’s membrane, we hypothesized that C. difficile spores might be cytotoxic to Raw 264.7 cells. When monolayers of Raw 264.7 cells were infected with C. difficile spores at an MOI of 1, some macrophage cell death was observed after 24 h of infection; however, cell death did not PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43635