Fig 1.
Growth of F. tularensis Schu S4, F. tularensis waaY::TrgTn and F. tularensis LVS in cultured human alveolar type II cells.
Immortalized primary AT-II tissue culture cells were used to perform gentamicin protection assays. The ability of the three Francisella strains to enter and replicate with these epithelial lung cells was measured after infecting at an MOI 100:1. Fold growth was calculated as the difference between the number of bacteria surviving gentamicin treatment at 4 hours and 24 hours post-infection. (A) Data are a single experiment representative of three separate experiments. (B) Data are an average of four independent experiments.
Fig 2.
Confocal microscopy of F. tularensis Schu S4, F. tularensis waaY::TrgTn and F. tularensis LVS growth in cultured human alveolar type II cells.
Immortalized primary AT-II cells were infected at an MOI of 100:1 and confocal microscopy was performed with cells stained for actin (red). In addition, each Francisella strain contained a plasmid encoding GFP and is represented as green. The thin white arrows point to single Francisella within cells.
Fig 3.
Scoring of growth patterns observed in confocal imaging.
Infected cells were classified into three separate categories based on the amount of bacteria each cell contained at 24 hpi. Cells infected with Schu S4 had significantly more bacteria per cell compared to cells infected with LVS 24 hpi (*P <. 001). Percentages were calculated from greater than 300 cells containing organisms from three independent experiments.
Fig 4.
Identification of F. tularensis in AT-II cells.
TEM images of infected mouse lungs at 24 hours post infection. To differentiate between lamellar bodies and bacteria in AT-II cells, infected cells were observed under high magnification. Lamellar bodies were classified as dark stratified stained organelles whereas; bacteria were identified by smooth dark staining that lacked stratification. Panel A—Cell containing a F. tularensis organism magnified 8,000-fold. White rectangle marks area of interest that is shown in panels B and C. Panel B is the area of the white rectangle in Panel A magnified 25,000-fold. A bacterium and the membrane surrounding the organism are shown. Panel C—The white arrows are pointing to the double membrane (outer and inner membrane) of F. tularensis. (Panel D) An intracellular organism magnified 8,000-fold. The white rectangle marks an area of interest shown in Panel E. Panel E is the area of the white rectangle in Panel D magnified 25,000-fold. Panel F—25,000-fold magnification of a lamellar body demonstrating the stratified staining from the packaged surfactant.
Fig 5.
Electron microscopy images of Francisella tularensis strains within murine alveolar type II epithelial cells.
TEM images of infected mouse lungs at 24 hours post-infection. Mice were infected intranasally with either F. tularensis Schu S4 (A-F), F. tularensis Schu S4 waaY::TrgTn (G-K), and F. tularensis LVS (I,L). Each image shows an AT-II cell containing organisms. The AT-II cell can be identified by the presence of microvilli at the cell-air interface and by the presence of lamellar granules. It is also worth noting that each infected AT-II cell was immediately adjacent to a pulmonary capillary. The area containing the internalized bacteria is within the white rectangle which is shown at higher magnification immediately beneath the corresponding image. The arrows identify the bacteria in each field.
Fig 6.
TEM of F. tularensis in dying AT-II cells in vivo.
TEM images of infected mouse lungs at both 32 and 48 hpi infected with Schu S4. (A-C) AT-II cells containing Francisella going through cell death characterized by lack of electron density of the cytosol and swollen mitochondria. (D-F) A separate AT-II cell undergoing cell death infected with Francisella. The open arrows identify the bacteria in each field and the solid black arrows indicate lamellar bodies in the cells.
Fig 7.
Schu S4 waaY::TrgTn increases cellular debris within the alveoli.
(A-C). TEM imaging of 24 hours demonstrating clean airspace with the alveoli in lung tissue that is infected with either PBS (A), Schu S4 (B), or LVS (C) and airspace that is representative of lung tissue infected with Schu S4 waaY::TrgTn (D-F). B. Graphical representation of scoring of the airspace for cellular debris. There was an increase in cellular debris in lungs infected with Schu S4 waaY:TrgTn compared to PBS control (P < 0.001). Averages were calculated from analysis of greater than 50 scored airspaces from 2 separate lung sections per strain and time point.
Fig 8.
Model of early Francisella infection within the lung.
(1) Francisella enters into the alveoli from an aerosolized infection where it either gets phagocytosed by alveolar macrophages (2a) or interacts with AT-II cells (2b). Upon uptake into alveolar macrophages there are at least two possible outcomes, either alveolar macrophages allow bacterial growth and release into the airspace (2a) or alveolar macrophages detach and are removed from the alveoli by the mucociliary escalator (2c). Growth and release from the alveolar macrophages allows reinfection with surrounding tissue including AT-II cells (3). Internalization with AT-II cells acts as a mechanism to get past the epithelial barrier and allows for interaction with endothelial cells and eventually dissemination to the liver and spleen.