Entry of B. anthracis Spores Figure 2. B. anthracis spore internalization by epithelial cells requires PI3K activity. A – C, spore internalization by A549 cells is inhibited by PI3K inhibitors, wortmannin (WM) and LY294002 (LY). Cells were pre-incubated with 0–50 nM WM, 50 mM LY, or the solvent control (Ctrl) for 1 hr and infected with spores in the presence of the inhibitor. Intracellular and extracellular spores were determined by gentamicin protection assay (A) and fluorescence microscopic examination (B and C). Relative internalization (%) is as described in the legend for Fig. 1. Relative adherence (%) is the ratio of extracellular bound spores vs. total fields counted, normalized to the control. The gentamicin protection result is the mean 6 SEM from a representative experiment. The fluorescence microscopic results are combined from at least two independent experiments. D – F, spore internalization by A549 cells is inhibited by dominant negative p85. A549 cells were transfected with SRa vector control (Ctrl), HA-Dp85a (Dp85a) and HA-p85a (p85a). The expression of HA-Dp85a and HA-p85a in transfected cells was analyzed by western blot using anti-HA antibodies and appropriate secondary antibodies 24 hr post transfection (D). Spore internalization by (E) and association with (F) transfected cells were determined using gentamicin protection assays 24 hr post transfection. The results are the mean 6 SEM, combined from three independent experiments. ***, p,0.001; **, p,0.01, t test. G – I, PI3K is rapidly recruited and activated at spore entry sites. A549 cells were transfected with Akt-PH-GFP, incubated with Texas Red-labeled spores for 8 min and processed for confocal examination. Representative images are shown. Arrows indicate the spore attachment sites where Akt-PH-GFP was recruited. Scale bars represent 5 mm. J, Quantitation of Akt-PH-GFP recruitment at spore attachment sites in the absence (Ctrl) or presence of the PI3K inhibitors. WM (100 nM) or LY (50 mM) was included in the colocalization assay. The percentage of spores surrounded with enhanced green fluorescence vs. total attached spores is denoted as %Recruitment. The results shown are combined from four independent experiments. ***, p,0.001, t test. doi:10.1371/journal.pone.0011665.g002 nin-treated cells and the control cells (data not shown), suggesting that the reduction of intracellular bacteria caused by the PI3K inhibitors was due to impaired spore entry. There are three classes of PI3Ks, among which class I PI3Ks are involved in regulating the actin cytoskeleton. This class is further divided into two sub-groups, IA and IB. Class IA enzymes are ubiquitously expressed and are made up of a 110 kDa catalytic subunit (p110) and an adaptor/regulatory subunit, the most abundant of which in mammalian cells is p85a [16]. Class IB PI3K is primarily expressed in white blood cells. We tested if spore entry into epithelial cells was mediated by a class IA PI3K. A dominant negative construct for class IA PI3Ks, Dp85a, was employed for this purpose. This mutant lacks the binding site for the catalytic p110 subunit and therefore cannot recruit p110 upon activation [17]. Plasmid constructs of HA-tagged Dp85a, wild-type p85a, or SRa (vector) were transfected into A549 and PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11665