Chakrabarty et al. observed activation of the mitogen-activated protein kinase signaling pathways and increases in the cytokines levels (e.g., IL-6, TNF-a, IL8, MIP-1a/b, and MCP-1) upon exposure to spores. Lung epithelial cells as well as alveolar macrophages were the main sources for the increased cytokines and chemokines [10]. A recent report by Evans et al. showed that mice treated with bacterial lysates developed an innate immunity to infections by B. anthracis spores. Interestingly, lung epithelial cells not macrophages or neutrophils were responsible for the induced resistance [11]. These results further underscored the importance of spore-epithelium interactions in the pathogenesis of B. anthracis. However, prior to this study little information was available regarding the molecular mechanism of spore-epithelium interactions, what factors mediate spore entry into epithelial cells or the biological consequence of disrupting the entry process. We previously showed that spore germination was not required for internalization by non-phagocytic cells, and that spores of B. subtilis were internalized by host cells at a significantly lower frequency than that of B. anthracis spores [1,6]. These results indicated that specific components on B. anthracis spores were necessary and sufficient to induce spore entry into non-phagocytic cells. Therefore, in this study we sought to investigate the entry mechanism of wild-type spores by elucidating the cellular components and signaling molecules in epithelial cells required for the internalization process. Using a combination of specific pharmacological inhibitors, dominant negative mutants, colocali- zation experiments and specific siRNA knockdown, a signaling pathway responsible for mediating the internalization of spores by epithelial cells was uncovered. The importance of this signaling pathway in bacterial dissemination in vitro and in vivo was also investigated. Results B. anthracis spore internalization by epithelial cells is actin-dependent We first examined if spore internalization by epithelial cells was dependent on the actin cytoskeleton. Cytochalasin D, an inhibitor of actin polymerization, inhibited spore uptake by A549 cells in a dose-dependent manner (Fig. 1, A). Uptake of spores was nearly abolished in the presence of 10 mM cytochalasin D. Similar results were observed in HeLa cells and hSAECs (Fig. 1, B and C). Cell viability was not affected by cytochalasin D at the concentrations used, as assessed by trypan blue exclusion. Nor was spore viability affected, as determined by plating and colony counts. We further investigated if spores colocalized with F-actin during entry by fluorescence microscopy. After 30 minutes of incubation, approximately 17.4% of the total attached spores were seen surrounded by enriched F-actin staining (SEM = 1.9%, n = 6 independent assays) (Fig. 1, D – G), suggesting that there was local activation of actin polymerization or reorganization at these sites. In general, the ratio of internalization spores vs. attached spores under our assay conditions is approximately 1:10–1:5. The percentage of attached spores with enriched F-actin is consistent with this ratio. We determined that spores did not emit green fluorescence by themselves by examining spores attached to coverslips in the absence of cells. To rule out the possibility that the colocalization was due to preferential attachment of spores to pre-existing actin-rich patches, we performed the experiment in the presence of cytochalasin D. Colocalization of spores with F- actin was significantly reduced (by ,61%) in cytochalasin D- treated cells (***, p,0.001 compared to no inhibitor), suggesting that there was active polymerization of F-actin at these spore attachment sites (Fig. 1, G). Cytochalasin D did not completely Entry of B. anthracis Spores abolish F-actin enrichment around spores. This could be due to the possibility that cytochalasin D prevented short actin filaments from polymerization. However, these short actin filaments could still be recruited to the spore attachment sites, although they were not able to drive the internalization process [12]. Together the above results indicated that spore internalization by epithelial cells required actin polymerization. The Rho-family GTPase Cdc42 is required for spore uptake The Rho family of small GTPases regulates the polymerization and reorganization of the actin cytoskeleton. RhoA, Rac1 and Cdc42 are the three major Rho GTPases. RhoA mainly mediates stress fiber formation, Rac1 lamellipodia and filopodia, and Cdc42 filopodia [13]. We investigated which of the three Rho GTPases was responsible for spore internalization by epithelial cells. T19NRhoA, T17NRac1 and T17NCdc42 are mutants of these GTPases that lack the ability to adopt the active GTP-bound form, but maintain the ability to bind guanine nucleotide exchange factors (GEFs). They are widely used as dominant negative (DN) mutants for the respective proteins [14]. HeLa cells were transfected with plasmids expressing either HA-tagged T19NRhoA, T17NRac1, T17NCdc42 or the vector control, respectively. The expression of the three DN mutant proteins in transfected cells was confirmed by western blot analysis of cell lysates 24 hours post-transfection (Fig. 1, H). Spore internalization was significantly reduced in cells transfected with T17NCdc42, but not in cells transfected with T19NRhoA or T17NRac1 (Fig. 1, I). None of the three DN mutants affected spore adherence to cells (Fig. 1, J), as expected. Transfection efficiency was approximately 80%, as determined by transfecting cells with a GFP expressing plasmid. The relatively moderate inhibition by DN Cdc42 mutant compared to that by cytochalasin D treatment could be due to incomplete transfection and/or incomplete inhibition of the endogenous Cdc42 activity. Similar results were observed in A549 cells transfected with the respective plasmids, i.e., approx- imately 35% decreases in spore internalization were only observed in A549 cells transfected with T17NCdc42 but not in cells transfected with the other two DN mutants (data not shown). Transfection did not affect cell viability, assessed by trypan blue exclusion. These results indicate that Cdc42, but not Rac1 or RhoA, regulates actin polymerization during spore uptake. Internalization of B. anthracis spores by epithelial cells requires phosphatidylinositol 3-kinase (PI3K) PI3K is required for the internalization of a number of bacteria by host non-phagocytic cells. We tested the effect of different concentrations (0–50 nM) of wortmannin, a specific PI3K inhibitor, on spore internalization by A549 cells. A dose- dependent inhibition was observed (Fig. 2, A). The IC50 was calculated to be ,6–10 nM (non-linear regression curve fit, GraphPad Prism software), consistent with the reported IC50 of wortmannin for PI3K [15]. The effect was not specific to A549 cells. Spore uptake by HeLa cells was also dramatically reduced (,80%) by wortmannin (100 nM) (Fig. S1, A). LY294002 inhibits PI3K enzymatic activities by a mechanism distinct from that of wortmannin [15]. The effect of LY294002 was also tested. LY294002 had a similar effect on spore internalization as that of wortmannin, ,60% reduction on spore internalization by A549 cells (Fig. 2, B) and ,70% by HeLa cells at the concentration used (Fig. S1, A). As expected, no significant effect on spore adherence to A549 (Fig. 2, C) or HeLa cells (Fig. S1, B) by either inhibitor was observed. The fact that both inhibitors caused significant PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11665