Entry of B. anthracis Spores Figure 1. B. anthracis spore internalization by epithelial cells requires actin polymerization mediated by Cdc42. A – C, the effect of cytochalasin D on spore internalization by A549 (A), HeLa (B) and hSAECs (C). Cells were pretreated with different concentrations (0.01 – 10 mM for A549 cells, 1 and 10 mM for HeLa and hSAECs, respectively) of cytochalasin D (Cyto D) or the solvent control (Ctrl) for 1 hr and then performed fluorescent microscopic examination as described in Materials and Methods. Relative internalization (%) is the ratio of intracellular spores vs. total adhered spores, normalized to the solvent control. The results are the mean 6 SEM from at least two independent experiments. D – F, representative images of colocalization of F-actin with spores. A549 cells were incubated with Texas-Red labeled spores and then stained with phalloidin-Alexa Fluor 488. G, colocalization of F-actin with spores is inhibited by Cyto D. Cyto D (10 mM) was included in the colocalization examination as described in Materials and Methods. The percentage of spores with enriched F-actin staining vs. total attached spores is denoted as %Colocalization. The results shown are combined from five independent experiments. ***, p,0.0001, t test. Scale bars represent 5 mm. H – J, Cdc42 is the primary Rho-family GTPase required for spore uptake. A549 cells were transfected with pcDNA3.1(+) vector control (Ctrl), HA-T19NRhoA (T19NRhoA), HA-T17NRac1 (T17NRac1) or HA-T17NCdc42 (T17NCdc42). The expression of the transfected constructs was verified using western blot analysis 24 hr post transfection (H). Dominant negative GTPases were detected by anti-HA antibodies and secondary antibodies as described in Materials and Methods. The actin level was used as a loading control. Spore uptake by (I) and adherence to (J) transfected cells were determined by gentamicin protection assays 24 hr post transfection as described in Materials and Methods. Relative internalization (%) is the ratio of intracellular spores vs. total spores added, normalized to the solvent control. Relative association (%) is the ratio of associated spores (extracellular adhered + intracellular) vs. total spores added, normalized to the solvent control. The results shown are the mean 6 SEM, combined from three independent experiments. **, p = 0.0018, t test. doi:10.1371/journal.pone.0011665.g001 reduction on spore internalization strongly suggests that PI3K activity is required for spore internalization. Neither wortmannin nor LY294002 affected the viability of cells or spores. As PI3Ks are also involved in other cellular processes such as intracellular vesicle trafficking, we tested if the reduction of intracellular bacteria in inhibitor treated cells was due to alterations in some intracellular processes post spore entry. Spores were allowed to be taken up by A549 cells in the absence of the inhibitor and then treated with wortmannin. No significant difference in the number of intracellular bacteria was observed between wortman- PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11665