In Vivo Demonstration and Quantification of Intracellular Bacillus anthracis in Lung Epithelial Cells

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 the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B. anthracis spores are 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 h postinoculation. Confocal analysis further indicated that some of the associated spores were surrounded by F-actin, demonstrating 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 those in our previous reports, provided powerful evidence that the lung epithelia were directly targeted by B. anthracis spores at early stages of infection.Anthrax is caused by the entry of Bacillus anthracis spores into the host via the lung, the gastrointestinal tract, or cuts or abrasions on the skin. Among the three forms of anthrax, the inhalational form has the highest fatality rate, approximately 50% even with antibiotic treatment. The initial stage of the infection does not display any distinctive symptoms. As the organism spreads and multiplies in the body, the disease rapidly progresses, resulting in damage to vital organs and eventually death.Recent inhalational anthrax animal studies suggested that the infection of the lung is most likely secondary, i.e., that after entering into the respiratory system, spores disseminate into the circulation, germinate to become vegetative bacilli, and then infect the lung via the blood (14,15,23). Therefore, a crucial step in the establishment of anthrax via the pulmonary route is for the spores to breach the respiratory mucosal barrier. To date, a clear understanding of how this breach is achieved remains to be established.Three routes have been proposed: alveolar macrophages (17, 18), dendritic cells (DCs) (6,8), and epithelial cells (31, 32). The macrophage and DC routes are similar in the sense that both macrophages and DCs act as a "Trojan horse," taking up spores from the airway and alveolar space and carrying them to lymph nodes, where spores are released into the circulation via an unknown mechanism. However, stud...

Updating Perspectives on the Initiation of Bacillus anthracis Growth and Dissemination through Its Host

“…These studies have brought to light data which suggest that a reassessment of a number of the Trojan horse model's central tenets, as they were originally defined, is needed. It was shown in vitro that epithelial cells and fibroblasts can engulf and transcytose spores and vegetative bacilli through cell monolayers without disrupting barrier integrity (63,64,72). These data raised questions as to whether phagocytes are the sole cell type that can act as Trojan horses and thus whether phagocytes are an absolute necessity for spores to transit from the lumena of the lungs.…”

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…Spores in the lung are phagocytosed by alveolar macrophages or dendritic cells, which initiate the processes leading to pulmonary anthrax (120,122,123). For pulmonary infections, it has also been shown that B. anthracis spores are capable of entering epithelial cells of the lung and crossing this cell layer without apparent disruption of the barrier integrity (124,125). The gastrointestinal form of anthrax ensues when spores are ingested and migrate to the intestinal tract, where they pass across the gut epithelial cell layer without germination.…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…An alternative model for phagocyte independent dissemination is that the spores and vegetative bacilli can be internalized by epithelial cells directly [62,271,[278][279][280][281]. These studies are supported by in vitro and in vivo studies that demonstrate that the spores are able to adhere, invade, and transcytose through the epithelial layer [271,279,280].…”

“…These studies are supported by in vitro and in vivo studies that demonstrate that the spores are able to adhere, invade, and transcytose through the epithelial layer [271,279,280]. Additionally, B. anthracis spores are unique from related Bacillus spores in that they can reside in the lung for long periods of time [278].…”

“…anthracis has previously been indicated as being capable of internalization and possibly dissemination from lung epithelial cells [271,279,280]. To identify gene products that facilitate adherence and internalization to epithelial cells, we had planned to use TRaSH but first had to confirm the adherence and internalization phenotype.…”