provided another example of interfering bacterial dissemination by inhibiting their entry or passage through epithelial cells. The results also raised the potential of using SFK inhibitors, or inhibitors of other host pathways important for the dissemination of microbial pathogens, as therapeutic agents in conjunction with antibiotics to treat certain infectious diseases. Another potential importance of spore entry into lung epithelial cells is gaining access to an intracellular niche that may allow persistent colonization of the microbes. B. anthracis spores are known to persist in the lung for weeks or months [29,30,31], the basis for the 60-day prolonged antibiotic treatment for patients exposed to spores. It was shown previously that spores and vegetative bacilli can survive inside lung epithelial cells for up to 12 hours [1], suggesting that the intracellular environment of lung epithelial cells can potentially be a site for spore persistence. However, it is not known in which intracellular compartments spores and bacilli reside or how long they can remain viable in those compartments. In macrophages, earlier studies showed that after phagocytosis spores were trafficked along the endocytic pathway and eventually were enclosed within LAMP1+ lysosomal compartments [32,33]. How long spores can survive or remain as spores (i.e., not germinate) in the lysosomal compartments of macrophages remains unclear. The fate of germinated spores and vegetative bacilli inside macrophages has been controversial. Some studies showed bacilli replication inside macrophages [32,33] whereas others indicated efficient killing of vegetative bacilli by macrophages [7,8]. Therefore it would be interesting to investigate the intracellular events following spore entry into host cells not only in the context of microbial passage through host cells but also persistence within host cells. Together, the data presented here elucidate a specific signaling pathway required for B. anthracis spore internalization by epithelial cells. The pathway includes c-Src, PI3K, Cdc42 and the actin cytoskeleton. The results also demonstrate that this pathway is important for B. anthracis translocation through a lung epithelial barrier in vitro, dissemination from the lung to distal organs, and virulence in vivo. Further studies to elucidate the epithelial receptor(s) with which spores interact and the intracellular events following spore entry will not only be important to the pathogenesis of this organism but will also reveal host receptors and signaling pathways that may be used by other respiratory pathogens to overcome the mucosal barrier. Materials and Methods Cell culture, bacterial strain and spore preparation A549 (a human epithelial cell line derived from lung carcinoma, ATCC) and HeLa (a human epithelial cell line derived from cervical cancer cells, ATCC) cells were cultured in F12 and Dulbecco’s Modified Eagle Media (DMEM), respectively, supple- mented with 10% fetal bovine serum (FBS). CloneticsH primary human small airway epithelial cells (hSAECs) (Cambrex) were maintained at 37uC in a humidified chamber with 5% CO2 in SABM media supplemented with reagents from the SAGM SingleQuot kit (Cambrex), following instructions from the supplier. Murine lung epithelial cells (MLE15) were cultured in DMEM with 10% FBS [34]. Murine embryonic fibroblasts (MEFs) from src-/-yes-/-fyn-/- triple knockout mice were obtained from ATCC and cultured according to instructions from ATCC. Primary murine peritoneal macrophages from C57BL/6 mice were kindly provided by Dr. Dekai Zhang, Texas A&M Health Science Center – IBT, Houston, TX, and were used within 24 hr of isolation. B. anthracis Sterne strain 7702 was provided by Dr. T. M. Koehler, UT Health Science Center, Houston, TX. Spores Entry of B. anthracis Spores were prepared from strain 7702 cultured in PA media following a procedure described previously [1]. Inhibitors and plasmid constructs Cytochalasin D, wortmannin, LY294002 and SU6656 were purchased from Sigma. PP2 and PP3 were obtained from Calbiochem. All inhibitors were dissolved in DMSO to prepare stock solutions. Plasmids expressing dominant negative p85a (HA- Dp85a) [17], wild-type p85a (HA-p85a) and the vector control (SRa) were provided by Dr. Lei Wei, Indiana University-Purdue University Indianapolis, Indianapolis, IN. Dominant negative constructs for RhoA (HA-T19NRhoA), Rac1 (HA-T17NRac1) and Cdc42 (HA- T17NCdc42) were purchased from UMR cDNA Resource Center (www.cdna.org) [14]. Akt-PH-GFP construct (in pEGFP-N1 vector) was provided by Dr. Tamas Balla, National Institute of Child Health and Human Development, NIH, Bethesda, MD [19]. Cell transfection A549 and HeLa cells were transfected with DNA plasmids using Lipofectamine LTX (Invitrogen) following the instructions from the supplier. Briefly, the cells were seeded in 24-well plates at a density of 40,000 cells/well 24 hours prior to transfection. DNA (0.25–0.5 mg) and 0.5 ml Plus Reagent (Invitrogen) were mixed and incubated in 100 ml Opti-MEM I medium (Invitrogen) for 10 min, followed by addition of 1–2 ml of LTX and incubated for 25 min. The mixture was then added to the cells. Lipofectamine RNAiMAX (Invitrogen) was used to transfect siRNAs into A549 cells following the instructions from the supplier. Briefly, 1 ml RNAiMAX and 100 ml opti-MEM I were mixed with specific siRNAs against c-Src, c-Yes and Fyn (c-Src and Fyn siRNA were from Santa Cruz Biotechnology whereas c-Yes siRNA and the Allstars negative control siRNA were from Qiagen) and incubated for 12 min. The mixture was then added to each well of a 24-well plate to give a final siRNA concentration of 50 nM. Western Blot Cells were harvested 24 hours post DNA plasmid transfection and 48 hours post siRNA transfection, washed and lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na- deoxycholate, 150 mM NaCl, 1 mM EDTA) containing Com- plete Mini protease inhibitors (Roche). Cell lysates were then mixed with an equal volume of 26 Laemmli sample buffer (BioRad) containing 5% b-mercaptoethanol and boiled at 100uC for 5 min. Samples were subjected to 4%/12% SDS-polyacryl- amide gel electrophoresis, and then transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% non- fat milk in a solution of 100 mM Tris-Cl pH 8.0, 0.9% NaCl, and 0.1% Tween20 (TBST), and then incubated with the appropriate primary and secondary antibodies. For detection, the membrane was incubated with a chemiluminescent substrate HyGLO HRP (Denville) for 1 min at room temperature, exposed to X-ray films (Kodak) and developed in a film processor (Konica SRX-101A). The different primary antibodies were anti-HA (Millipore) (1:1000 dilution in TBST containing 5% milk), anti-Src (Millipore, 1:1000), anti-Yes (BD Biosciences, 1:1000), anti-Fyn (BD Biosci- ences, 1:1000) and anti-actin (Millipore) (1:6000) antibodies. The secondary antibodies were HRP-conjugated goat anti-mouse antibodies (Millipore, 1:3000) and goat anti-rabbit antibodies (BioRad, 1:6000). Gentamicin protection assay This was performed as described previously [1] with slight modifications. Briefly, cells grown in 24-well tissue culture plates PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11665