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Infection and Immunity, March 2008, p. 1036-1047, Vol. 76, No. 3
0019-9567/08/$08.00+0     doi:10.1128/IAI.00985-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Detection of Bacillus anthracis Spore Germination In Vivo by Bioluminescence Imaging{triangledown} ,{dagger}

Patrick Sanz, Louise D. Teel, Farhang Alem, Humberto M. Carvalho,{ddagger} Stephen C. Darnell, and Alison D. O'Brien*

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

Received 18 July 2007/ Returned for modification 21 August 2007/ Accepted 20 December 2007

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 sspBp::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 promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min 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 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, 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 nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.

* Corresponding author. Mailing address: 4301 Jones Bridge Road, Bethesda, MD 20814. Phone: (301) 295-3400. Fax: (301) 295-3773. E-mail: aobrien{at}usuhs.mil

{triangledown} Published ahead of print on 14 January 2008.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: R. P. Morrison

{ddagger} Present address: NIH/NIAID, 6700-B Rockledge Dr., Room 4218, Bethesda, MD 20892.

Infection and Immunity, March 2008, p. 1036-1047, Vol. 76, No. 3
0019-9567/08/$08.00+0     doi:10.1128/IAI.00985-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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