Francisella tularensis invasion of lung epithelial cells

Abstract

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site of F. tularensis replication in infected animals. However, recently it was reported that F. tularensis also invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process of F. tularensis invasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixed F. tularensis live vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreased F. tularensis invasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase activity. Collectively, these results suggest that F. tularensis epithelial cell invasion is mediated by a preformed ligand on the bacterial surface and driven entirely by host cell processes. Once internalized, F. tularensis-containing endosomes associated with early endosome antigen 1 (EEA1) followed by lysosome-associated membrane protein 1 (LAMP-1), with peak coassociation frequencies occurring at 30 and 120 min postinoculation, respectively. By 2 h postinoculation, 70.0% (+/- 5.5%) of intracellular bacteria were accessible to antibody delivered to the cytoplasm, indicating vacuolar breakdown and escape into the cytoplasm.

FIG. 1.

F. tularensis LVS association with and internalization by TC-1 lung epithelial cells. (A) Percentage of TC-1 cells with associated live or PFA-fixed LVS at designated times postinoculation. *, Data are significantly different from untreated control data (P < 0.01 by paired two-tailed t test). (B) Percentages of TC-1 cell-associated live or PFA-fixed LVS that are intracellular at the designated times postinoculation.

FIG. 2.

The effect of actin and microtubule polymerization on F. tularensis LVS invasion of lung epithelial cells. TC-1 cells were treated with designated concentrations of cytochalasin D (A) or colchicine (B). Results are expressed as the percentage of LVS, Salmonella, or Campylobacter organisms that survived gentamicin treatment relative to that for the sample without inhibitor inoculated with the same organism (defined as 100% invasion). *, Data are significantly different from untreated control data (P < 0.01 by paired two-tailed t test).

FIG. 3.

The effect of PI 3-kinase and tyrosine kinase activity on LVS invasion of lung epithelial cells. Wortmannin (A) and genistein (B) were added to TC-1 cells at the indicated concentrations. Results are expressed as the percentage of LVS or control organisms that survived gentamicin treatment relative to that for the sample without inhibitor inoculated with the same organism (defined as 100% invasion). *, Data are significantly different from untreated control data (P < 0.01 by paired two-tailed t test).

FIG. 4.

Representative fluorescence microscopy images demonstrating LVSgfp localization within EEA1- or LAMP-1-containing endosomes in TC-1 cells. Images depict TC-1 cells stained for EEA1 30 min after inoculation with LVSgfp (A) or 2 h postinoculation stained for LAMP-1 (B). Single-color images: LVSgfp images depict bacteria alone, EEA1 and LAMP-1 images depict staining for EEA1 or LAMP-1 only, and extracellular images depict staining of extracellular bacteria (no extracellular bacteria were present in panel B). The merged color images depict LVSgfp (green), vacuoles labeled with anti-EEA1 antibody (N19) (red) or anti-LAMP-1 (1D4B) (red), or extracellular LVS labeled with anti-F. tularensis LPS antibody conjugated to Pacific Blue (blue). Extracellular LVSgfp (arrow with round end), intracellular but not EEA1-associated LVSgfp (small arrow), and LVS associated with EEA1-containing vacuoles (large arrowhead). (C) Trafficking of LVSgfp in TC-1 cells. One hundred intracellular bacteria were counted for each condition and scored for association with EEA1- and LAMP-1-containing vacuoles. Three replicates were examined for each time point and condition.

FIG. 5.

Transmission electron micrographs of TC-1 infected with F. tularensis LVS. (A) TC-1 cells 1 h postinoculation showing LVS in a membrane-bound vacuole that in some cases appeared to be degrading (arrow). (B) LVS cells 24 h postinoculation were free in the cytoplasm.

FIG. 6.

LVSgfp was analyzed by flow cytometry for escape from vacuoles into the cytoplasm of TC-1 cells. Extracellular bacteria were labeled with anti-F. tularensis LPS antibody conjugated to Pacific Blue (region R8). Cytoplasmic bacteria were identified by labeling with anti-F. tularensis LPS conjugated to Alexa Fluor 647 after digitonin permeabilization of the cytoplasmic membrane (region R10). Vacuolar bacteria were inaccessible to antibody and therefore GFP positive only (region R9). Representative flow cytometry data of bacteria recovered 10 min (A) or 60 min (B) postinoculation. The value shown in region R10 represents the percentage of intracellular bacteria that are cytoplasmic. (C) Percentages of intracellular bacteria present in the cytoplasm at designated times postinoculation.