RationaleThe lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. B. anthracis spores must escape from the alveolus, pass to the regional lymph nodes, germinate and enter the circulatory system as vegetative bacteria to cause systemic disease. Of the resident lung cells, three have been reported to take up B. anthracis spores: the antigen presenting cells (APC) alveolar macrophages and dendritic cells, and alveolar epithelial cells (AEC). Also, B. anthracis produces the exotoxins lethal factor and protective antigen (PA) which combine to form lethal toxin (LT), a metalloproteinase important in pathogenicity. The roles of carrier cells and the effects of B. anthracis toxins in escape of spores from the alveolus are unclear, especially in humans.MethodsWe employed a human lung organ culture model and a human A549 alveolar epithelial cell culture model, along with fluorescent confocal imaging to quantitate spore partitioning between APC and AEC, and the effects of B. anthracis LT and PA on this process. Cell types were distinguished by positive staining for HLA-DR (APC) and cytokeratin (AEC).ResultsWe found that spores progressed through the lung slice over time, and that spore movement was not dependent on cell internalization. Both free and cell-associated spores moved through slices between 2 and 48 hrs of incubation. However, partitioning of spores between AEC, APC, and the extracellular space did not significantly change over this time. After 2 hrs, 4.7% of spores were in APC; 13.8% in AEC; and 81.5% were not cell-associated. By 48 hrs, 2.9% were in APC; 12.7% were in AEC; and 84.4% were not cell-associated. Spores also internalized in a non-uniform manner, with more variable spore internalization into AEC than into APC. At all incubation times, the majority of cell-associated spores were in AEC, not in APC. PA and LT did not affect transit of the spores through the lung tissue or the distribution of spores into AEC and APC. In A549 cells, spore internalization increased significantly after 24 hrs incubation. However, there was no statistically consistent effects of PA or LT on spore internalization in A549 cells.ConclusionsOverall, our results support a “Jailbreak”-like model of spore escape from the alveolus that involves transient passage of spores, although this occurs through intact AEC. However, subsequent transport of spores by APC from the lung to the lymph nodes may occur.
Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin.
