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Infect. Immun. doi:10.1128/IAI.00282-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells

Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, 77030; Department of Pulmonary Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030

^* To whom correspondence should be addressed. Email: yxu{at}ibt.tamhsc.edu.

	Abstract

Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is dissemination of spores from the lung. We previously demonstrated using in vitro cell culture assays that B. anthracis spores were capable of entering into epithelial cells of the lung and crossing a barrier of lung epithelial cells without apparent disruption of the barrier integrity, suggesting a novel portal for spores to disseminate from the lung. However, in vivo evidence for spore uptake by epithelial cells has been lacking. Here, using a mouse model, we present evidence that B. anthracis spores are taken up by lung epithelial cells in vivo soon after spores are delivered into the lung. Immunofluorescence staining of thin sections of lungs from spore-challenged Balb/c mice revealed that spores were associated with the epithelial surfaces in the airway and the alveoli at 2 and 4 hours post inoculation. Confocal analysis further indicated that some of the associated spores were surrounded by F-actin, demonstrating an intracellular localization. These observations were further confirmed and substantiated by a quantitative method that first isolated lung cells from spore-challenged mice and then stained these cells with antibodies specific for epithelial cells and spores. The results showed that substantial amounts of spores were taken up by lung epithelial cells in vivo. These data combined with our previous reports provided powerful evidence that the lung epithelia were directly targeted by B. anthracis spores at early stages of infection.