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Infect. Immun. doi:10.1128/IAI.00985-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Detection of Bacillus anthracis spore germination in vivo by bioluminescence imaging

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799

^* To whom correspondence should be addressed. Email: aobrien{at}usuhs.mil.

	Abstract

We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspB-lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen (PA) promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored up to 14 days with the Xenogen In Vivo Imaging System (IVIS). While luminescence only became evident in live animals at 18 hours, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 minutes after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By six hours after infection, PMNs with intracellular spores were evident in the alveolar spaces. After 24 hours, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained non-luminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.