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Mechanisms utilized by Francisella tularensis to invade hepatocytes

Francisella tularensis is a facultative intracellular bacterium that is the etiological agent of tularemia. This pathogen is classified as a category A select agent, which means it has the potential to be used as a weapon of bioterrorism. Regardless of the route of infection, the liver is an important target of infection in tularemia, and F. tularensis grows to high numbers within hepatocytes, the major cell type of this organ. This study investigates the mechanisms utilized by F. tularensis to invade hepatocytes. Mechanisms of uptake of F. tularensis Live Vaccine Strain (LVS) were investigated in vitro using the murine AML‐12 and human HH4 hepatocytic cell lines. Additionally, an F. tularensis ssp. novicida transposon library was screened for mutants with altered uptake by hepatocytes. Addition of cytochalasin D to the hepatocytes blocked uptake of the LVS nearly completely. In contrast, bacteria killed by heating or formalin fixation were readily ingested by the hepatocytes. Treatment of the bacteria with chloramphenicol also had no effect on invasion. LVS mutants lacking components of pili or the type I secretion system retained their ability to be taken up by the hepatocytes. Screening of the F. tularensis ssp. novicida transposon library led to identification of two unclassified outer‐membrane proteins, the absence of which enhanced uptake by both AML‐12 cells and HH4 human hepatocytes. These results show that polymerization of the actin cytoskeleton is needed for uptake of F. tularensis by hepatocytes. Additionally, there is no requirement for viability of or protein synthesis by the bacteria for this process. The mutants identified by the screen suggest that certain bacterial genes may negatively regulate the uptake of F. tularensis by hepatocytes. Further characterization of these genes is in progress.