C. difficile Spores and Macrophage Interactions Figure 3. Complement decreases internalization of C. difficile spores by Raw 264.7 cells. Alexa- biotin-labeled C. difficile spores of strains 630 and Pitt177 were incubated for 30 min with culture medium (white bars), fetal bovine serum (FBS) (light grey bars), heat inactivated FBS (dark grey bars), and heat inactivated FBS supplemented with rabbit complement (black bars) prior to infection of monolayers of Raw 264.7 cells as described figure legend of Figure 2. The relative percentage of Raw 264.7 cells with at least one spore (A), relative number of spores per Raw-spore complex (B), and relative percentage of internalization (C) were quantified and calculated as described in Methods section and legend of Figure 2. 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.g003 Factors Affecting the Survival of C. difficile Spores During Infection of Raw 264.7 Cells The ability of bacterial spores to survive inside macrophages depends on their germination capabilities inside the macrophage environment [13,22]. In addition, host factors with muramidase activity have been reported to trigger germination of C. perfringens and B. anthracis spores lacking the PG cortex hydrolysis machinery [24,36,37]. In this context, we evaluated the survival of spores after infecting Raw 264.7 cells with germinant- and/or human serum- treated C. difficile spores. Initial experiments demonstrated that when Raw 264.7 cells were incubated with DMEM in absence of FBS for 24 h under either aerobic or anaerobic conditions, there was no decrease in viability of Raw 264.7 cells as determined by trypan blue viability assay (data not shown). As expected, during infection of Raw 264.7 cells with untreated Figure 4. Sonication does not affect binding and internalization of C. difficile spores by Raw 264.7 cells. Monolayers of Raw 264.7 cells were infected with untreated (white bars) and sonicated (grey bars) C. difficile spores of strains 630 and Pitt177 at an MOI of 10 for 30 min, and analyzed by fluorescence microscopy for: relative percentage of Raw 264.7 cells with at least one spore (A), relative number of spores per Raw-spore complex (B), and relative percentage of internalization (C) as described in Methods section and in the legend of figure 2. Results are the average of at least three independent experiments and error bars are standard error of the mean. doi:10.1371/journal.pone.0043635.g004 PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43635