C. difficile Spores and Macrophage Interactions Figure 6. Survival of C. difficile spores and vegetative cells during infection of Raw 264.7 macrophages. Monolayers of Raw 264.7 cells were infected at an MOI of 10 with C. difficile strain 630: A) spores; B) vegetative cells, and unbound spores and vegetative cells rinsed off and further incubated under aerobic conditions for various periods of time and spore or vegetative cell viability was determined as described in Methods section. Results are the average of 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 time 0 h. doi:10.1371/journal.pone.0043635.g006 increase after 48 h of infection. 24 h of infection of Raw 264.7 cells with C. difficile spores at an MOI of 10 produced ,50% of Raw 264.7 cell death (Fig. 9A). Also a significant loss of Raw 264.7 cell’s membrane integrity was observed (Fig. 9A) and this loss continued to increase until 48 h of incubation (Fig. 9A). In- terestingly, when Raw 264.7 cells were infected with C. difficile spores at an MOI of 1 or 10, similar levels of reduction in Raw 264.7 cell viability were observed as measured by esterase activity. For example, after 24 h of infection, viability of Raw 264.7 cells decreased only by ,30% and after 48 h by ,50% (Fig. 9B). When results were confirmed by trypan blue exclusion assay, we observed that in concordance with above results, the majority of uninfected Raw 264.7 cells remained viable even after 48 h of incubation, however, significant cell death was observed on monolayers of Raw 264.7 cells infected with C. difficile spores at an MOI of 10 (Fig. 9C). Similar results were observed at an MOI of 1 (data not shown). Raw 264.7 cells alone incubated for up to 48 h had high levels of calcein fluorescence, and minimal levels of fluorescence due to ethidium homodimer-1 (data not shown) meaning that Raw 264.7 cells remained viable in absence of FBS. Collectively, these results indicate that C. difficile spores not only survive inside macrophages, but are also cytotoxic to Raw 264.7 cells over extended periods of infection. Discussion Successful clearance of bacterial infections requires that the host’s immune system be able to recognize, phagocytose and kill pathogenic cells in host tissues. Although C. difficile is an enteric pathogen, during the course of CDI the enterotoxin TcdA and the cytotoxin TcdB not only induce epithelium damage but also the expression of numerous pro-immflamatory cytokines and chemo- kines, producing a massive recruitment of macrophages and neutrophils and the formation of pseudo-membranes [2]. Even though CDI is an enteric infection, and rarely systemic, macrophages do play a major role in host defense. Several studies have shown that the TLR4 and the Nod1 phagocytic receptors are essential for protection of CDI in mouse [11,12], indicating that C. difficile cells and spores encounter phagocytic cells in the colonic environment. In this context, our current study provides numerous findings on the early stages of interactions between C. difficile spores and Raw 264.7 cells. While the nature of phagocytic receptors [39] mediating C. difficile spore recognition by Raw 264.7 cells was not addressed in this work, we did observe that in absence of serum C. difficile spores were efficiently recognized by Raw 264.7 cells. It was most striking that more than half of C. difficile spores that adhered to Raw 264.7 cells were internalized. Our scanning electron micrographs suggest the uptake of C. difficile spores by Raw 264.7 cells might follow various phagocytic strategies which include membrane ruffling and coiling phagocytosis [32]. Although further studies will be required to fully characterize these pathways, they provide evidence that C. difficile spores are actively internalized by remodeling of the macrophages surface membrane through an actin mediated process [33,39]. The significant reduction in internalization of C. difficile spores by Raw 264.7 cells in the presence of the actin polymerization inhibitor, cytochalasin D, supports that internalization is an actin-polymerization dependent process. It is worth noting that even in presence of cytochalasin D, ,6–7% of adhered C. difficile spores were internalized, indicating that a small fraction of C. difficile spores enter Raw 264.7 cells through a phagocytosis-independent pathway. To the best of our knowledge, there are no reports of internalization of bacterial spores into phagocytic cells through a phagocytosis-independent pathway, thus the precise nature of this actin-polymerization independent internalization pathway is unknown and clearly requires further research. Sonication treatment of C. difficile spores, which removes the majority (.60%) of the outermost exosporium-like structure (this study) including two ,40-kDa protein species reported to have a role in adherence [5], did not affect the ability of Raw 264.7 cells to recognize and phagocytose C. difficile spores. This is presumably PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43635