Francisella tularensis Invasion of Lung Epithelial Cells

Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

^* To whom correspondence should be addressed. Email: kawula{at}med.unc.edu.

	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 (LVS) associated with, and were internalized by, TC-1 cells at a similar frequency and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreased F. tularensis invasion, as did inhibitors of PI3 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 EEA1 followed by LAMP-1 with peak co-association frequencies occurring at 30 and 120 minutes post-inoculation, respectively. By 2 hours post-inoculation 70.0% (±5.5%) of intracellular bacteria were accessible to antibody delivered to the cytoplasm indicating vacuolar breakdown and escape into the cytoplasm.