Bacillus anthracis Spore Entry into Epithelial Cells Is an Actin-Dependent Process Requiring c-Src and PI3K

Qiong Xue,Yi Xu,Sarah A Jenkins,Chunfang Gu,Emanuel Smeds,Ranga Vasan,Brooke H Russell,Qing Liu,Andy T Y Lau

doi:10.1371/journal.pone.0011665

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 Δp85α. 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.

Highlights

Inhalational anthrax is a life-threatening infection initiated by pulmonary exposure to Bacillus anthracis spores
B. anthracis translocation through cultured lung epithelial cells was significantly impaired by Src family kinase (SFK) inhibitors, suggesting that this signaling pathway is important for bacterial dissemination
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

Summary

Introduction

Inhalational anthrax is a life-threatening infection initiated by pulmonary exposure to Bacillus anthracis spores. The protection was found to be from cellular rather than humoral immunity, further highlighting the importance of an intracellular stage in the establishment of anthrax infections [5]. Spores encounter three major types of cells, epithelial cells in the alveoli and small airway, resident alveolar macrophages (AMs), and lung dendritic cells (LDCs). 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 escaping from them. Spore entry into lung epithelial cells appears to be an important early event in the development of inhalational anthrax

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Conclusion