appropriate filters for ethidium homodimer-1 (filter with excitation at 535 nm and emission at 620 nm) and calcein (filter with excitation at 485 nm and emission at 535 nm). The percentage of dead cells was calculated using the following formula: % Dead Cells = [[F(620 nm)sample–F(620 nm)minimum]/ [F(620 nm)maximum–F(620 nm)minimum ]]6100%; where F(620 nm) is fluorescence intensity at a wavelength of 620 nm, F(620 nm)sample is fluorescence of Raw 264.7 cells infected with spores, F(620 nm)minimum is fluorescence of Raw 264.7 cells with no spores, F(620 nm)maximum is fluorescence of Raw 264.7 cells treated with 0.06% Triton 100X. The percentage of live cells was calculated using the following formula: % Live Cells = [[F(535 nm)sample–F(535 nm)minimum]/[F(535 nm)maximum– F(535 nm)minimum ]]6100%; where F(535 nm) is fluorescence intensity at a wavelength of 535 nm, F(535 nm)sample is fluorescence of Raw 264.7 cells infected with spores, F(535 nm)minimum is fluorescence of Raw 264.7 cells treated with 0.06% Triton 100 X, F(535 nm)maximum is fluorescence of Raw 264.7 cells with no spores. All experiment were done in triplicate and repeated at least three times. Statistical Analysis All experiments were repeated at least three times. Error bars represent standard error from the mean. Statistical analysis in some experiments involved Student’s t test, used to identify significant difference between groups, or pairwise comparison with Least Significant Difference. Statistical difference was considered with a p,0.05. Statistical analysis was done with Statgraphics Centurion XVI (StatPoint Technologies, Inc). Results Characterization of Internalization of C. difficile Spores by Raw 264.7 Cells To investigate binding and phagocytosis of C. difficile spores by Raw 264.7 cells, C. difficile spores were labeled with Alexa Fluor 488 and biotin and then used to infect Raw 264.7 cells. The spore interaction to and spore internalization by Raw 264.7 cells was evaluated by fluorescence microscopy (Fig. 1), with green C. difficile spores stained blue corresponding to extracellular spores. Our preliminary results demonstrated that Alexa-488 and biotin labeling did not affect Raw 264.7 cells to recognize C. difficile spores, and Raw 264.7 cells were able to adhere and phagocytose C. difficile spores better than THP-1 and J937 macrophage-like cell lines (data not shown). Therefore, Raw 264.7 cells were used in all subsequent experiments. Infection of Raw 264.7 cells with C. difficile strain 630 spores in DMEM and absence of serum showed that 83% of macrophages attached to at least one spores (Fig. 2A), with each Raw 264.7 cell binding to an average of 2.5 spores (Fig. 2B). Interestingly, 59% of all C. difficile 630 spores attached to Raw 264.7 cells were efficiently internalized after 30 min of incubation (Fig. 2C). Incubation for up to 3 h did not increase adherence and internalization of C. difficile spores by Raw 264.7 cells (data not shown). When Raw 264.7 cells were infected with spores of C. difficile strain Pitt177, only 40% of Raw 264.7 cells (Fig. 2A) were attached to at least one spore, with an average ,1.5 spores per Raw 264.7 cell (Fig. 2B), results that were significantly (p,0.01) lower than those observed with C. difficile strain 630 spores (Fig. 2A,B). Similar to C. difficile strain 630 spores, 50% of adhered C. difficile strain Pitt177 spores had been internalized by Raw 264.7 cells (Fig. 2C). Interestingly, no effects of cytochalasin D, an actin polymeri- zation inhibitor [28], on adherence of spores of C. difficile strain 630 to Raw 264.7 cell was observed (Fig. 2D, E). Although there C. difficile Spores and Macrophage Interactions was no significant decrease in the percentage of Raw 264.7 cells with at least one spore of C. difficile strain Pitt177 (Fig. 2D), there was a slight, but significant (p,0.05) decrease in the number of Pitt177 spores per macrophage-spore complex (Fig. 2E). However, a significant (p,0.001) decrease in internalization of spores by Raw 264.7 cell was observed (Fig. 2F), indicating that the internalization of C. difficile spores by Raw 264.7 cells is dependent on actin polymerization. Complement Decreases Internalization of C. difficile Spores by Raw 264.7 Cells Complement proteins are constitutively present in the serum and can opsonize bacteria nonspecifically contributing to pathogen clearance [29]. Therefore, we evaluated if comple- ment might play a role in recognition and phagocytosis of C. difficile spores by Raw 264.7 cells. Surprisingly, although the adherence of C. difficile spores to Raw 264.7 cells was not significantly affected by the presence of fetal bovine serum (FBS) than in absence of FBS (Fig. 3A), it was significantly (p,0.05) less in presence of heat-inactivated FBS (absence of comple- ment) than in absence of FBS (Fig. 3A). The number of average spores per Raw 264.7 cell in presence of FBS significantly (p 0.01) decreased for spores of C. difficile strain 630 but not for spores of C. difficile strain Pitt177 (Fig. 3B). Heat-inactivated FBS produced a slight decrease in the number of average spores per Raw 264.7 cells for both C. difficile strains 630 and Pitt177 than that in DMEM alone (Fig. 3B). It was also striking to note that the presence of FBS or heat inactivated FBS also decreased the percentage of spore internalization by Raw 264.7 cells (Fig. 3C). Addition of complement did not restore the adherence and internalization of spores of both C. difficile strains 630 and Pitt177 by Raw 264.7 cells (Fig. 3A,B,C), indicating that Raw 264.7 cells are less able to recognize and internalize C. difficile spores through complement-mediated phagocytosis. Sonication Effects on Adherence and Internalization of C. difficile Spores by Raw 264.7 Cells Several species, such as B. anthracis and B. cereus, have a balloon- like structure that overlays the coats, called the exosporium [30]. Indeed, C. difficile seems to have an exosporium-like structure [5,31]. Therefore, to evaluate if this structure might have motifs recognized by Raw 264.7 cells, C. difficile spores were sonicated, treatment that removes at least some of the exosporium material [5], and tested for adherence. There was no significant decrease in adherence of sonicated spores of C. difficile strains 630 and Pitt177 to Raw 264.7 cells (Fig. 4A). The number of spores that adhered to Raw 264.7 cells was not affected by sonication (Fig. 4B). Furthermore, there was no significant decrease in internalization of spores of C. difficile strains 630 and Pitt177 by Raw 264.7 cells (Fig. 4C). These results suggest that the motifs recognized by Raw 264.7 cells to bind and internalize C. difficile spores are not localized in the spores’ outermost layer that is affected by sonication; or alternatively, and most likely, the remaining exosporium structure contained sufficient conserved motifs recog- nized by Raw 264.7 cells. Raw 264.7 Cells Efficiently Recognize and Phagocytose C. difficile Spores Scanning electron micrographs (SEM) confirmed our above observations that Raw 264.7 cells can bind more than one C. difficile spores (Fig. 5A). During the first 30 min of infection, Raw 264.7 cells actively phagocytosed spores, and this seems to be associated in part by an actin-dependent mechanism that produces PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43635