Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K Qiong Xue1, Sarah A. Jenkins2, Chunfang Gu1, Emanuel Smeds1, Qing Liu3, Ranga Vasan1, Brooke H. Russell1, Yi Xu 1 * 1 Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, United States of America, 2 Graduate School of Biomedical Sciences, University of Texas Health Science Center, Houston, Texas, United States of America, 3 Department of Thoracic and Cardiovascular Surgery, M.D. Anderson Cancer Center, Houston, Texas, United States of America Abstract Dissemination of Bacillus anthracis from the respiratory mucosa is a critical step in the establishment of inhalational anthrax. Recent in vitro and in vivo studies indicated that this organism was able to penetrate the lung epithelium by directly entering into epithelial cells of the lung; however the molecular details of B. anthracis breaching the epithelium were lacking. Here, using a combination of pharmacological inhibitors, dominant negative mutants, and colocalization experiments, we demonstrated that internalization of spores by epithelial cells was actin-dependent and was mediated by the Rho-family GTPase Cdc42 but not RhoA or Rac1. Phosphatidylinositol 3-kinase (PI3K) activity was also required as indicated by the inhibitory effects of PI3K inhibitors, wortmannin and LY294002, and a PI3K dominant negative (DN) mutant Dp85a. In addition, spore entry into epithelial cells (but not into macrophages) required the activity of Src as indicated by the inhibitory effect of Src family kinase (SFK) inhibitors, PP2 and SU6656, and specific siRNA knockdown of Src. Enrichment of PI3K and F-actin around spore attachment sites was observed and was significantly reduced by treatment with SFK and PI3K inhibitors, respectively. Moreover, B. anthracis translocation through cultured lung epithelial cells was significantly impaired by SFK inhibitors, suggesting that this signaling pathway is important for bacterial dissemination. The effect of the inhibitor on dissemination in vivo was then evaluated. SU6656 treatment of mice significantly reduced B. anthracis dissemination from the lung to distal organs and prolonged the median survival time of mice compared to the untreated control group. Together these results described a signaling pathway specifically required for spore entry into epithelial cells and provided evidence suggesting that this pathway is important for dissemination and virulence in vivo. Citation: Xue Q, Jenkins SA, Gu C, Smeds E, Liu Q, et al. (2010) Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K. PLoS ONE 5(7): e11665. doi:10.1371/journal.pone.0011665 Editor: Andy T. Y. Lau, University of Minnesota, United States of America Received May 12, 2010; Accepted June 26, 2010; Published July 20, 2010 Copyright: 2010 Xue et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants AI061555 and AI082306 (http://www.niaid.nih.gov) to Y. Xu, a career development award from Region VI center for Biodefense and Emerging Infections (U54 AI057156 to D. Walker)(http://www.niaid.nih.gov), and start-up funds from Texas A&M Health Science Center (TAMHSC) (http://www.tamhsc.edu) to Y. Xu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yxu@ibt.tamhsc.edu Introduction Inhalational anthrax is a life-threatening infection initiated by pulmonary exposure to Bacillus anthracis spores. The pathogen then disseminates away from the lung to establish a systemic infection. The systemic spread is thought to come from hematogenous sources; however, how B. anthracis disseminates from the lung, the initial entry site, to the blood remains poorly understood. Although B. anthracis is primarily an extracellular pathogen, studies from multiple groups have indicated that an intracellular stage is necessary for the pathogen to breach the lung epithelial barrier [1,2,3,4]. Mice can be protected by immunization with inactivated spores. The protection was found to be from cellular rather than humoral immunity, further highlighting the impor- tance of an intracellular stage in the establishment of anthrax infections [5]. In the lung, spores encounter three major types of cells, epithelial cells in the alveoli and small airway, resident alveolar macrophages (AMs), and lung dendritic cells (LDCs). AMs and LDCs have been indicated to play roles in the dissemination process by first engulfing spores and then carrying them to regional lymph nodes [2,3]. Spores germinate inside the phagocytes, replicate and eventually escape from them via an undefined mechanism. Another strategy often used by pathogens to breach mucosal barriers is by entering into non-phagocytic host cells and then escaping from them. Recent studies suggested that B. anthracis spores may use this strategy as well [1,4]. Spores of B. anthracis can be internalized by polarized A549 cells (human alveolar type II-like epithelial cells) and primary human small airway epithelial cells (hSAECs) [1,6]. In addition, substantial amounts of spores were found inside epithelial cells of the lung in mice within hours of inoculation [4], indicating that spore entry into lung epithelial cells is relevant in vivo. Furthermore, B. anthracis can cross a barrier of lung epithelial cells in the absence of phagocytes and without compromising the barrier integrity [1]. Spores and vegetative bacilli are also able to survive inside lung epithelial cells [1], in contrast to the finding in macrophages [7,8,9]. Thus spore entry into lung epithelial cells appears to be an important early event in the development of inhalational anthrax. Spore-lung epithelium interactions have also been shown to influence host immune responses. Using a human lung slice model, PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11665