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

Christopher E Bradburne,Anne B Verhoeven,Ganiraju C Manyam,Saira A Chaudhry,Eddie L Chang,Dzung C Thach,Charles L Bailey,Monique L Van Hoek

doi:10.1074/jbc.m112.362178

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.

Highlights

The mechanism of Francisella live vaccine strain (LVS) entry into A549 cells is unknown
F. tularensis LVS Establishes an Intracellular Infection of A549 Cells—In order to confirm the establishment of a stable intracellular infection by F. tularensis LVS over the time line evaluated in this microarray study, we performed immunofluorescence microscopy to detect intracellular bacteria
Active infection was confirmed by MOI counts of F. tularensis LVS at each time point evaluated by microarray, indicating steadily increasing bacterial numbers over 24 h (Fig. 2)

Summary

Background

The mechanism of Francisella LVS entry into A549 cells is unknown. Results: Microarrays were performed at early infection time points, supported by phenotypic observations and inhibition experiments. 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. Type II alveolar epithelial cells are integral components of the lung mucosa, are non-phagocytic, and are able to detect and respond to the presence of pathogens and pathogen-associated molecules They modulate the majority of the epithelial cellular response during infections (compared with Type I), and they may play an important role in Francisella infection. Our results suggest one mechanism of Francisella LVS entry into lung epithelial cells and may reveal regulation by Francisella LVS of important host pathways in human Type II alveolar epithelial cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION