HeLa cells, respectively. Transfection efficiency was approxi- mately 40% for A549 cells and 80% for HeLa cells, estimated by transfecting cells with a GFP-expressing plasmid. Expression of HA-Dp85a and HA-p85a in transfected cells was confirmed by western blot analysis of cell lysates 24 hours post-transfection using anti-HA antibodies (Fig. 2, D and Fig. S1, C). The mutant migrated slightly faster than wild-type p85a since it lacked the p110 binding site and had a smaller molecular weight. Analysis of spore internalization showed that expression of Dp85a signifi- cantly decreased the spore internalization frequency (by ,45%) in both A549 (Fig. 2, E, **, p,0.01) and HeLa cells (Fig. S1, D, ***, p,0.001) when compared to cells transfected with the vector control. The decrease (,45%) in spore internalization caused by Dp85a expression was less dramatic than that by the two PI3K inhibitors. This was likely due to the moderate transfection efficiency, competition between Dp85a and the endogenous p85a, and the presence of other regulatory subunits of PI3Ks, which could potentially compensate for p85a. Over expression of exogenous wide-type p85a did not increase spore internalization, possibly because the amount of endogenous adaptors was sufficient to mediate spore internalization (Fig. 2, E and Fig. S1, D). Spore adherence to cells was not affected by Dp85a expression (Fig. 2, F and Fig. S1, E). Neither HA-Dp85a nor HA- p85a expression affected cell viability as determined by trypan blue exclusion. Altogether, the above results indicate that internalization of B. anthracis spores by epithelial cells requires PI3K activity, primarily the activity of a class IA PI3K. Entry of B. anthracis Spores Upon activation, class I PI3Ks phosphorylate PtdIns(4,5)P2 to PtdIns(3,4,5)P3, which binds to the PH domain of downstream effectors such as the serine/threonine protein kinase Akt/PKB [18]. In order to examine PI3K recruitment and activation, a construct containing the Akt-PH domain fused to a GFP gene (Akt-PH-GFP) was used as a molecular probe for the PI3K product PtdIns(3,4,5)P3 [19]. A549 cells transfected with the Akt-PH-GFP construct were serum-starved and then incubated with Texas Red-labeled B. anthracis spores. Approximately 28.0% of attached spores recruited Akt-PH- GFP within minutes of incubation (SEM = 1.6%, n = 6 independent assays) (Fig. 2, G – J). Also, significantly less Akt-PH-GFP recruitment was observed (***, p,0.001 compared to no inhibitor) in cells treated with wortmannin or LY294002 (Fig. 2, J), suggesting that the recruitment was due to PI3K activation. The incomplete inhibition of Akt-PH recruitment by the inhibitors has been previously reported [20,21]. Together, these results further confirm that a class IA PI3K is recruited and activated during spore internalization. c-Src, a member of the Src family protein tyrosine kinases, is required for the internalization of B. anthracis spores by epithelial cells To determine if Src family protein tyrosine kinase (SFK) activity was required for spore internalization by epithelial cells, PP2, a specific SFK inhibitor and its negative control compound, PP3, were used to treat A549 cells. Spore internalization was inhibited by PP2 (,70%) but not by PP3 (Fig. 3, A). Spore adherence to A549 cells was not affected by either PP2 or PP3 (Fig. 3, B). Figure 3. Spore internalization by epithelial cells requires the protein tyrosine kinase Src. A and B, spore internalization by A549 cells is inhibited by PP2 but not PP3. Cells were pre-treated with PP2 (10 mM), PP3 (10 mM) or solvent control (Ctrl) for 1 hr and then incubated with labeled 7702 spores in the presence of the inhibitors. Intracellular and extracellular spores were quantified by fluorescence microscopic examination. Relative internalization (A) and relative adherence (B) were calculated as the number of intracellular and extracellular adhered spores vs. total fields counted, respectively, and were normalized to the control. The results are the mean 6 SEM from at least 2 independent assays. C – G, knockdown of c-Src by siRNA significantly reduces spore internalization. A549 cells were transfected with 50 nM siRNA for Src (Src KD), Yes (Yes KD), Fyn (Fyn KD) or control siRNA (Ctrl). Spore internalization (C) and adherence (D) were determined by gentamicin protection assays 48 hr post transfection. The results were from three independent experiments. ***, p,0.001; *, p = 0.0165, Student’s t test, normalized to the control. The knockdown of protein levels in transfected cells was determined by western blot using specific antibodies against Src (E), Yes (F) and Fyn (G). doi:10.1371/journal.pone.0011665.g003 PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11665