Entry of B. anthracis Spores Figure 6. Src activity is important for bacterial dissemination in vivo. A/J mice were treated with SU6656 (7.5 mg/g body weight) or equivalent volumes of solvent 24 hr prior to spore inoculation as described in Materials and Methods. A – C, bacterial burden in the spleen (A), blood (B) and lungs (C) of mice inoculated by intranasal instillation. Each mouse was inoculated with 1 - 36106 spores. Organs were harvested at 72 hr post inoculation, homogenized and plated. The results are combined from 2 – 3 independent experiments with a total of 7–15 mice per treatment group. D, mice were inoculated with 16105 spores/mouse by i.p. injection. Bacterial burden in the spleen was determined at 72 hr post inoculation. The results are combined from 2 independent experiments with a total of 12 mice per treatment group. E, mice were inoculated with 16104 spores/ mouse by injection into the tail vein. Bacterial counts in the blood were determined at 84 hr post inoculation. The results were combined from 2 independent experiments with a total of 13 mice per treatment group. Statistical significance was calculated using t test. *, p,0.05. F, SU6656 treatment improves survival in i.n. inoculated mice. Mice were inoculated with ,86106 spores/mouse intranasally (10 mice per group). Statistical significance was calculated using the Logrank test. Similar trend was observed in three other experiments. G, mice were inoculated with ,16105 spores/mouse by i.p. injection. The results are from two experiments with a total of 20 mice per group. doi:10.1371/journal.pone.0011665.g006 to AMs and LDCs as portals for dissemination through the barrier [1,4]. The recent finding that lung epithelial cells rather than macrophages or neutrophils were responsible for the induction of innate resistance to pulmonary exposure of spores highlighted the importance of B. anthracis-epithelium interactions in the patho- genesis of this organism. The report that mice immunized with inactivated spores were protected by cellular rather than humoral immunity further emphasized the importance of an intracellular stage during the establishment of anthrax infections [5]. Although a fair amount of information is available on spore-macrophage interactions, there are few previous reports on the molecular mechanisms underlying the interactions between spores and lung epithelial cells. In this study we sought to determine the molecular events involved in spore entry into lung epithelial cells. The results indicate that spore uptake by epithelial cells is dependent on the actin cytoskeleton and a signaling pathway involving Src, PI3K and Cdc42. Furthermore, we provide evidence that Src activity is important for B. anthracis translocation through a barrier of lung epithelial cells in culture as well as dissemination from the lung to distal organs in mice. The involvement of the actin cytoskeleton in spore internalization is supported by three lines of evidence. Uptake of spores by A549, HeLa and primary hSAECs was virtually abolished in the presence of cytochalasin D. Local F-actin enrichment was observed at spore attachment sites, suggesting a ‘‘zipper’’-like entry mechanism [25]. Furthermore, spore internalization was specifically inhibited by a DN mutant of Cdc42, but not by DN mutants of Rac1 or RhoA, suggesting that Cdc42 is the major Rho-family GTPase regulating the actin polymerization events during spore entry. The require- ment for class IA PI3K activities is supported by the following evidence. Spore internalization was inhibited by two structur- ally and mechanistically distinct PI3K inhibitors, wortmannin and LY294002, and by a DN mutant of the p85 regulatory subunit of class IA PI3K. In addition, Akt-PH-GFP was rapidly recruited to the spore attachment sites, and the recruitment was reduced in the presence of wortmannin or LY294002. The requirement for Src activity is supported by the inhibitory effects of two distinct SFK inhibitors PP2 and SU6656, and specific siRNA knockdown of Src. The results also showed that inhibition of either PI3K or Src decreased F-actin enrichment around spore attachment sites. Inhibition of both PI3K and Src did not result in additional decreases of F-actin enrichment as compared to individual inhibitor treatment only, suggesting that PI3K and Src are in the same signaling pathway for spore internalization. In addition, inhibition of Src activity reduced PI3K recruitment and activation, suggesting that Src likely acts upstream of PI3K in the signaling pathway. Based on these results we propose a working model for the signaling pathway responsible for spore entry into epithelial cells (Fig. 7). PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11665