Detection of Bacillus anthracis Spore Germination In Vivo by Bioluminescence Imaging

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 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.

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“…Indeed, a number of researchers have described attenuation in lux-and GFPexpressing strains. 47,[75][76][77] Maximizing reporter expression. Bacterial reporter gene expression is exquisitely dependent on transcriptional and translational signals.…”

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“…Infections are also observed to occasionally germinate and initiate in the lumen of the lungs [133,275,276]. This is a surprising finding as the lungs are generally refractory to germination [74,214,[275][276][277].…”

“…This is a surprising finding as the lungs are generally refractory to germination [74,214,[275][276][277]. Compared to the subcutaneous and NALT based infections, lung based infections are detected later and it is unclear how these infections disseminated from the lung lumen.…”

“…Moreover, how these lung-based infections initiate are not well understood. Some have suggested that lung based infections occur at higher doses and initiate from macrophages engulfing too many spores [75,275,276]. The spores would then be able to germinate and kill and escape from the macrophage before it has time to migrate to the mediastinal lymph nodes [275].…”

“…These BLI studies identified two novel sites of establishment and dissemination of infection [81,261,275,276]. In the upper respiratory passage, bacteria were first observed to replicate and disseminate from the nasal associated lymphoid tissue (NALT) [81,260,270,275].…”