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Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis
Published on: September 30, 2017
Francisella tularensis invasion of lung epithelial cells
Robin R Craven1, Joshua D Hall, James R Fuller
1Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7290, USA.
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.
Insights
Francisella tularensis invades lung epithelial cells via host cell processes, not bacterial factors. The pathogen escapes the vacuole into the cytoplasm within two hours, indicating a novel infection pathway.
Area of Science:
- Microbiology
- Cell Biology
- Infectious Diseases
Background:
- Francisella tularensis is a Gram-negative bacterium causing tularemia.
- Macrophages are traditionally considered the primary replication site.
- Recent findings suggest invasion of alveolar epithelial cells.
Purpose of the Study:
- Investigate F. tularensis invasion and intracellular trafficking in nonphagocytic cells.
- Utilize the TC-1 mouse lung epithelial cell line.
- Elucidate the mechanisms of bacterial entry and vacuolar escape.
Main Methods:
- Infection of TC-1 cells with F. tularensis live vaccine strain.
- Assessment of bacterial association and internalization.
- Use of inhibitors for microfilaments, microtubules, PI3K, and tyrosine kinase.
- Immunofluorescence microscopy to track intracellular trafficking (EEA1, LAMP-1).
- Antibody-mediated detection of cytoplasmic bacterial escape.
Main Results:
- F. tularensis efficiently invades TC-1 cells.
- Invasion depends on host cell actin and microtubule dynamics, PI3K, and tyrosine kinase.
- Bacterial entry is mediated by bacterial surface ligands and host cell processes.
- Internalized bacteria traffic through EEA1- and LAMP-1-positive endosomes.
- Significant vacuolar escape into the cytoplasm occurs by 2 hours post-infection.
Conclusions:
- F. tularensis invasion of epithelial cells is host-driven.
- The pathogen utilizes specific intracellular trafficking pathways.
- Rapid vacuolar escape facilitates cytoplasmic replication in nonphagocytic cells.
- This highlights a critical mechanism in tularemia pathogenesis.
