collected and dilution plated to determine bacterial counts in the blood. Mice were monitored twice daily for the survival studies. Statistical analysis The Logrank test was used to evaluate the statistical significance in mouse survival studies. Student’s t-test was used to calculate statistical significance in all the other data. The GraphPad Prism 4 software was used for these analyses. Supporting Information Figure S1 PI3K activity is required for B. anthracis spore internalization by HeLa cells. The experiments were performed as described in Figure 2 legend. A, spore internalization by HeLa cells was inhibited by WM (50 nM) and LY (50 mM). B, spore adherence to HeLa cells was not affected by WM or LY significantly. C, western blot analysis of the expression of mutant p85a and p85a in transfected HeLa cells. D, spore internalization by HeLa cells was inhibited by the expression of mutant p85a. E, spore adherence to HeLa cells was not affected by mutant p85a. Found at: doi:10.1371/journal.pone.0011665.s001 (0.07 MB PDF) Figure S2 Src activity is specifically required for spore entry into epithelial cells. A, PP2 (10 mM) was added to cells prior to and during the 1 hour incubation with spores. Spore uptake was assessed following the procedures described in the legends for Figures 1 and 2. Relative uptake was calculated as the percentage References 1. Russell BH, Vasan R, Keene DR, Koehler TM, Xu Y (2008) Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells. Cell Microbiol 10: 945–957. 2. Cleret A, Quesnel-Hellmann A, Vallon-Eberhard A, Verrier B, Jung S, et al. (2007) Lung dendritic cells rapidly mediate anthrax spore entry through the pulmonary route. J Immunol 178: 7994–8001. 3. Guidi-Rontani C (2002) The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol 10: 405–409. 4. Russell BH, Liu Q, Jenkins SA, Tuvim MJ, Dickey BF, et al. (2008) In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells. Infect Immun 76: 3975–3983. 5. Glomski IJ, Corre JP, Mock M, Goossens PL (2007) Cutting Edge: IFN-gamma- producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis. J Immunol 178: 2646–2650. 6. Russell BH, Vasan R, Keene DR, Xu Y (2007) Bacillus anthracis internalization by human fibroblasts and epithelial cells. Cell Microbiol 9: 1262–1274. 7. Hu H, Sa Q, Koehler TM, Aronson AI, Zhou D (2006) Inactivation of Bacillus anthracis spores in murine primary macrophages. Cell Microbiol 8: 1634–1642. 8. Kang TJ, Fenton MJ, Weiner MA, Hibbs S, Basu S, et al. (2005) Murine macrophages kill the vegetative form of Bacillus anthracis. Infect Immun 73: 7495–7501. 9. Welkos S, Friedlander A, Weeks S, Little S, Mendelson I (2002) In-vitro characterization of the phagocytosis and fate of anthrax spores in macrophage and the effects of anti-PA antibody. J Med Microbiol 51: 821–831. 10. Chakrabarty K, Wu W, Booth JL, Duggan ES, Nagle NN, et al. (2007) Human lung innate immune response to Bacillus anthracis spore infection. Infect Immun 75: 3729–3738. 11. Evans SE, Scott BL, Clement CG, Larson DT, Kontoyiannis D, et al. (2009) Stimulated innate resistance of lung epithelium protects mice broadly against bacteria and fungi. Am J Respir Cell Mol Biol 42: 40–50. 12. Jewett TJ, Fischer ER, Mead DJ, Hackstadt T (2006) Chlamydial TARP is a bacterial nucleator of actin. Proc Natl Acad Sci U S A 103: 15599–15604. 13. Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21: 247–269. 14. Feig LA (1999) Tools of the trade: use of dominant-inhibitory mutants of Ras- family GTPases. Nat Cell Biol 1: E25–27. 15. Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, et al. (2000) Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 6: 909–919. 16. Vanhaesebroeck B, Waterfield MD (1999) Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253: 239–254. 17. Hara K, Yonezawa K, Sakaue H, Ando A, Kotani K, et al. (1994) 1- Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A 91: 7415–7419. Entry of B. anthracis Spores of uptake in the presence of PP2 normalized to the no inhibitor control for each type of cells. The results are compiled from at least 3 independent experiments. A549, human alveolar epithelial cell line; HeLa, human cervical epithelial cell line; hSAEC, primary human small airway epithelial cells (Cambrex); MLE, murine lung epithelial cell line MLE15; RAW, murine macro- phage cell line RAW264.7; PPM, primary peritoneal macrophages from C57BL/6 mice. B, phagocytosis of spores by RAW264.7 was inhibited by LY294002 (LY). RAW264.7 were pre-treated with LY (50 mM) for 1 hr. Spore phagocytosis was performed using gentamicin protection assays described in Materials and Methods. The phagocytosis assays were performed in the presence of the inhibitor. Found at: doi:10.1371/journal.pone.0011665.s002 (0.04 MB PDF) Acknowledgments We are grateful to Lei Wei, IUPUI, Indianapolis, IN, Tamas Balla, NICHD, NIH, Bethesda, MD, Mingyao Liu and Dekai Zhang, TAMHSC-IBT for their generous help in providing constructs, primary macrophages and technical advice. Author Contributions Conceived and designed the experiments: QX SAJ BHR YX. Performed the experiments: QX SAJ CG ES QL RV. Analyzed the data: QX SAJ YX. Wrote the paper: QX YX. 18. Hawkins PT, Anderson KE, Davidson K, Stephens LR (2006) Signalling through Class I PI3Ks in mammalian cells. Biochem Soc Trans 34: 647–662. 19. Varnai P, Balla T (2006) Live cell imaging of phosphoinositide dynamics with fluorescent protein domains. Biochim Biophys Acta 1761: 957–967. 20. Varnai P, Bondeva T, Tamas P, Toth B, Buday L, et al. (2005) Selective cellular effects of overexpressed pleckstrin-homology domains that recognize PtdIns(3,4,5)P3 suggest their interaction with protein binding partners. J Cell Sci 118: 4879–4888. 21. Tanaka M, Konishi H, Touhara K, Sakane F, Hirata M, et al. (1999) Identification of myosin II as a binding protein to the PH domain of protein kinase B. Biochem Biophys Res Commun 255: 169–174. 22. Oliva C, Turnbough CL, Jr., Kearney JF (2009) CD14-Mac-1 interactions in Bacillus anthracis spore internalization by macrophages. Proc Natl Acad Sci U S A 106: 13957–13962. 23. Thomas SM, Brugge JS (1997) Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13: 513–609. 24. Severgnini M, Takahashi S, Tu P, Perides G, Homer RJ, et al. (2005) Inhibition of the Src and Jak kinases protects against lipopolysaccharide-induced acute lung injury. Am J Respir Crit Care Med 171: 858–867. 25. Cossart P, Sansonetti PJ (2004) Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304: 242–248. 26. Oliva CR, Swiecki MK, Griguer CE, Lisanby MW, Bullard DC, et al. (2008) The integrin Mac-1 (CR3) mediates internalization and directs Bacillus anthracis spores into professional phagocytes. Proc Natl Acad Sci U S A 105: 1261–1266. 27. Blake RA, Broome MA, Liu X, Wu J, Gishizky M, et al. (2000) SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol Cell Biol 20: 9018–9027. 28. Zhang JR, Mostov KE, Lamm ME, Nanno M, Shimida S, et al. (2000) The polymeric immunoglobulin receptor translocates pneumococci across human nasopharyngeal epithelial cells. Cell 102: 827–837. 29. Glassman HN (1966) Discussion. Bacteriol Rev 30: 657–659. 30. Henderson DW, Peacock S, Belton FC (1956) Observations on the prophylaxis of experimental pulmonary anthrax in the monkey. J Hyg 54: 28–36. 31. Vietri NJ, Purcell BK, Lawler JV, Leffel EK, Rico P, et al. (2006) Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax. Proc Natl Acad Sci U S A 103: 7813–7816. 32. Dixon TC, Fadl AA, Koehler TM, Swanson JA, Hanna PC (2000) Early Bacillus anthracis-macrophage interactions: intracellular survival and escape. Cell Microbiol 2: 453–463. 33. Guidi-Rontani C, Levy M, Ohayon H, Mock M (2001) The fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol Microbiol 42: 931–938. 34. Barrett EG, Johnston C, Oberdorster G, Finkelstein JN (1998) Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-alpha. Am J Physiol 275: L1110–1119. PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11665