SU6656, an SFK inhibitor that has a narrower set of targets than PP2, was also tested. SU6656 at a concentration of 50 mM significantly inhibited spore entry into A549 cells by approximately 70% (data not shown). None of the compounds affected the viability of cells or spores. Next we tested the effect of PP2 on spore uptake by epithelial cells of different origin and spore phagocytosis by macrophages. PP2 inhibited spore uptake by HeLa, hSAECS, and MLE15 (a murine lung epithelial cell line) cells, suggesting that SFK activity is required for spore internalization by epithelial cells from different origins. In contrast the same concentration of PP2 did not affect spore phagocytosis by RAW264.7 macrophages or murine primary peritoneal macrophages (Fig. S2, A), suggesting that SFK activity is specifically required for spore entry into epithelial cells. It was recently reported that PI3K activity was involved in spore phagocytosis by macrophages [22]. We tested the effect of LY294002 and found that spore phagocytosis in fact was inhibited by LY294002 (Fig. S2, B), consistent with the previous report [22]. Of the nine Src family tyrosine kinases, c-Src, c-Yes and Fyn are ubiquitously expressed, while others are primarily expressed in cells of the hematopoietic lineage [23]. Therefore, these three kinases are likely candidates involved in spore internalization. We first tested murine embryonic fibroblasts (MEFs) from src-/-yes-/- fyn-/- triple knockout mice (obtained from ATCC). Spore internalization by the triple knockout MEFs was ,80% lower compared to that by wild type MEFs (data not shown). To determine which one of the three kinases was required for spore internalization by epithelial cells, A549 cells were transfected with specific siRNAs for c-Src, c-Yes and Fyn. Analysis of spore internalization in cells transfected with the different siRNAs indicated that knockdown of c-Src decreased spore internaliza- tion by approximately 60% (***, p,0.001 compared to the control), whereas knockdown of c-Yes or Fyn had no or only marginal inhibitory effect (Fig. 3, C). Spore adherence was not affected by knockdown of any of the three kinases (Fig. 3, D). Specific knockdown of the targeted protein kinase was confirmed using western blot analysis (Fig. 3, E – G). These results indicate that c-Src is the primary SFK involved in spore uptake by epithelial cells. Src and PI3K function in the same signaling pathway to mediate spore internalization To understand the connection between the different signaling molecules elucidated above, we first investigated whether PI3K and c-Src were required for actin polymerization during spore entry. F-actin enrichment around spore attachment sites was significantly reduced in cells treated with the PI3K inhibitors, wortmannin and LY294002, and the SFK inhibitor, PP2, but not in PP3-treated cells (Fig. 4, A). None of these inhibitors disrupted the actin cytoskeleton at the concentration tested as judged by the phalloidin staining pattern in uninfected cells. These results suggested that both PI3K and c-Src are involved in regulating actin polymerization during spore entry. PI3K and SFKs can act in the same signaling pathway or in independent pathways to transduce signals to downstream effectors leading to the activation of actin cytoskeleton. Therefore, we investigated the connection between PI3K and c-Src in mediating spore uptake by epithelial cells. If PI3K and c-Src function in the same signaling pathway, treating cells with inhibitors for both PI3K and c-Src simultaneously should have the same effect on spore internalization as treating cells with individual inhibitor alone. On the other hand, if they function in independent pathways, inhibition of both kinases would have a synergistic effect compared to inhibition of the individual kinase. Entry of B. anthracis Spores Figure 4. Src and PI3K act in a linear signaling pathway. A, F- actin colocalization with spores is reduced in the presence of WM, LY and PP2. A549 cells were pre-incubated with WM (100 nM), LY (50 mM), PP2 (10 mM), PP3 (10 mM), LY (50 mM) + PP2 (10 mM) or solvent control for 1 hr and then incubated with Texas Red-labeled 7702 spores in the presence of the respective inhibitors for 30 min and then stained for F- actin. The samples were examined using confocal fluorescence microscopy. Relative F-actin colocalization is the ratio of spores that associated with enriched F-actin staining vs. total spores that attached to the cells, normalized to the solvent control. B, Akt-PH-GFP recruitment is reduced in the presence of LY and PP2. A549 cells transfected with Akt-PH-GFP were pre-incubated with LY (50 mM), PP2 (10 mM), PP3 (10 mM), LY (50 mM) + PP2 (10 mM) or solvent control for 1 hr and then incubated with labeled spores in the presence of the respective inhibitors for 8 min. Relative Akt-PH-GFP recruitment is the ratio of spores that recruited Akt-PH-GFP vs. total spores that attached to the cells, normalized to the solvent control. The results are the mean 6 SEM from at least three experiments. ***, p,0.001; **, p,0.01, t test. doi:10.1371/journal.pone.0011665.g004 The results showed that treatment of A549 cells with both LY294002 and PP2 caused a similar level of reduction in F-actin enrichment around spores compared to the reductions seen with individual inhibitor only (,40% reduction; **, p,0.01) (Fig. 4, A), suggesting that PI3K and SFKs functioned in the same signaling pathway for spore internalization. To further investigate the order of PI3K and c-Src activation in the signaling cascade, we examined the recruitment of Akt-PH- GFP upon spore attachment in cells treated with PP2. In PP2 treated cells, the recruitment of Akt-PH-GFP was reduced (***, p,0.001 compared to the no inhibitor control) to a similar level as those in cells treated with wortmannin or LY294002 (Fig. 4, B). Treatment of cells with both PP2 and LY294002 did not cause any further reduction in Akt-PH-GFP recruitment (,40%, ***, p,0.001) compared to cells treated with the individual inhibitor (Fig. 4, B). PP3 had no effect on Akt-PH-GFP recruitment. These results suggest that PI3K likely act downstream of c-Src in the signaling pathway involved in spore internalization by epithelial cells. PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11665