Francisella tularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation

Abstract

Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells.

FIG. 1.

F. tularensis LVS invades and replicates within ATII cell lines in vitro. (A) Intracellular bacteria recovered from A549, MLE-12, Tc-1, and J774A.1 cells 6 and 24 h postinoculation. The percentages above the bars represent percentages of infected cells. (B and C) Fluorescence imaging of A549 cells inoculated with GFP-expressing LVS 6 h (B) and 24 h (C) following inoculation. Cell borders were visualized by rhodamine-phalloidin (Molecular Probes) staining (red), and nuclei were visualized with 4′,6′-diamidino-2-phenylindole (DAPI; blue). (D and E) Fluorescence imaging of J774A.1 cells inoculated with GFP-expressing LVS (green) 6 h (D) and 24 h (E) postinoculation. Cell borders were visualized using biotinylated lectin from Lens culinaris and streptavidin-conjugated Alexa Fluor 647 (Molecular Probes) (red), and nuclei were stained with DAPI (blue). Intracellular replication experiments were carried out in triplicate; error bars represent standard deviations of the means.

FIG. 2.

F. tularensis localizes to the alveolus following inhalation. Mice were intranasally inoculated with 10⁵ CFU of F. tularensis LVS expressing GFP. One, three, and seven days postinoculation, lungs were harvested and prepared for immunofluorescence analysis. (A to C) Bacterial localization was determined by probing lung sections with a fluorescently labeled antibody to GFP (green). Nuclei were stained with DAPI (blue) to visualize lung cells. Representative images of the alveoli of infected mice 1 (A), 3 (B), and 7 (C) days postinoculation. (D) Bacterial recovery from lungs 1, 3, and 7 days following intranasal inoculation with 10⁵ CFU LVS. Each bar represents mean recovery from three mice; error bars represent standard deviations of the means.

FIG. 3.

Following inhalation, F. tularensis LVS expressing GFP colocalized with proSP-B and proSP-C, proteins produced by ATII epithelial cells. Bacterial localization was determined with a fluorescently labeled antibody to GFP (green). Nuclei were stained with DAPI (blue). Sections were probed with fluorescently labeled antibody to proSP-B (red) (A and B), proSP-C (red) (C and D) to identify ATII cells and β-tubulin (red) (E). Representative images are from lung sections 3 days postinoculation.

FIG. 4.

F. tularensis LVS expressing GFP colocalized with cells expressing the macrophage marker F4/80, the dendritic cell marker CD11c, and the ATII cell markers proSP-B and proSP-C. Nuclei were stained with DAPI (blue). Mouse lung cells were probed with fluorescently labeled antibody to F4/80 (red) (A and B), CD11c (red) (C and D), proSP-B (red) (E), and proSP-C (red) (F). Representative images are from lung cells 3 days postinoculation.