Temporal transcriptional response during infection of type II alveolar epithelial cells with Francisella tularensis live vaccine strain (LVS) supports a general host suppression and bacterial uptake by macropinocytosis

Abstract

Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2-6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell.

FIGURE 1.

Immunostaining of A549 epithelial cells infected with F. tularensis LVS shows an intracellular, cytoplasm-localized infection. A549 cells were infected with F. tularensis LVS at a 1:100 MOI for the indicated times, permeabilized, and fixed. Cells were stained with anti-F. tularensis rabbit IgG primary antibody and then with a secondary goat anti-rabbit IgG (H + L) antibody labeled with R-Phycoerythrin. Nuclei were stained with DAPI, and actin were stained with phalloidin. Images were taken at ×40. A, 2 h; B, 6 h; C, 16 h; D, 24 h. E, close up view of one 16-h infected A549 cell with many bacteria, surrounded by uninfected cells. Note the localization of bacteria in the cytoplasm and exclusion from the nuclei. F, confocal microscopy of uninfected A549 cells. The confocal image is the composite of 25–40 z-stacked images, taken at ×60. Blue, DAPI-stained nucleus; green, FITC-phalloidin-stained actin.

FIGURE 2.

F. tularensis LVS invasion and replication within A549 cells. Infection was performed at 100 MOI for each sample using the spin protocol at the time courses evaluated in this study and up to 24 h. Infected cells were lysed at the indicated time points, and cfu were determined by dilution plating on chocolate agar. The experiment was performed in triplicate. The 6, 16, and 24 h time points were significantly different than control (t = 0) and significantly higher than 15 min (p < 4.3 × 10⁻⁵). The 2 h time point was not significantly different than the 15 min time point (p = 0.155). Error bars, S.D.

FIGURE 3.

Comparison of transcripts. Venn diagram showing the up- and down-regulated transcript quantifications (represented in red and blue, respectively) at each of the time points. Differentiation between infected samples and uninfected controls is significant at the earlier time points (15 h and 2 min) and then falls by 6 h, at which point very little difference from the uninfected control is detectable.

FIGURE 4.

Macropinocytosis of F. tularensis LVS in A549 cells. A, inhibition of macropinosomes by amiloride in A549 cells reduced entry of F. tularensis LVS. Cells were preincubated either with or without 1 mm amiloride 2 h prior to infection. Infection was performed at 100 MOI and co-incubated with or without 1 mm amiloride up to 24 h. Cells preincubated or co-incubated with amiloride had reduced intracellular F. tularensis LVS. B and C, F. tularensis LVS induces macropinocytosis in A549 cells. Confocal microscopic images were taken at ×10 with z-stack analysis of 8–12 slices. B, uninfected A549 cells coincubated with FITC-dextran. C, F. tularensis LVS-infected A549 cells coincubated with FITC-dextran. Induced macropinosomes were labeled with FITC-dextran (green). Bacteria were unlabeled in this experiment. D, F. tularensis LVS co-localizes with macropinocytosis in A549 cells. A549 cells were infected with F. tularensis LVS at a 1:10 MOI. Red, anti-Francisella rabbit IgG primary antibody, secondary goat anti-rabbit IgG (H + L) antibody labeled with R-phycoerythrin. Green, induced macropinosomes were labeled with FITC-dextran. Blue, actin labeled with phalloidin. The three panels show a close-up of ×60 of each color scan and then a merged image of an A549 cell infected with Francisella LVS and labeled with FITC-dextran. Notice co-localization of bacteria and FITC-dextran, suggesting that bacteria are within an induced macropinosome. Co-localization of macropinosome signal and Francisella signal was seen for ∼50% of the cells in the infected monolayer (data not shown). Error bars, S.D.