Francisella tularensis, the bacterial cause of tularemia, infects the liver and replicates in hepatocytes in vivo and in vitro. However, the factors that govern adaptation of F. tularensis to the intrahepatocytic niche have not been identified. Using cDNA microarrays, we determined the transcriptional profile of the live vaccine strain (LVS) of F. tularensis grown in the FL83B murine hepatocytic cell line compared to that of F. tularensis cultured in broth. The fslC gene of the fsl operon was the most highly upregulated. Deletion of fslC eliminated the ability of the LVS to produce siderophore, which is involved in uptake of ferric iron, but it did not impair its growth in hepatocytes, A549 epithelial cells, or macrophages. Therefore, we sought an alternative means by which F. tularensis might obtain iron. Deletion of feoB, which encodes a putative ferrous iron transporter, retarded replication of the LVS in iron-restricted media, reduced its growth in hepatocytic and epithelial cells, and impaired its acquisition of iron. Survival of mice infected intradermally with a lethal dose of the LVS was slightly improved by deletion of fslC but was not altered by loss of feoB. However, the ⌬feoB mutant showed diminished ability to colonize the lungs, liver, and spleen of mice that received sublethal inocula. Thus, FeoB represents a previously unidentified mechanism for uptake of iron by F. tularensis. Moreover, failure to produce a mutant strain lacking both feoB and fslC suggests that FeoB and the proteins of the fsl operon are the only major means by which F. tularensis acquires iron.

Francisella tularensis is a Gram‐negative, facultative intracellular bacterium and the agent of tularemia. During infection, F. tularensis targets the liver and grows extensively within hepatocytes. This study investigates the role of iron acquisition pathways in F. tularensis virulence and replication within hepatocytes. Deletion mutants of the candidate fslC and feoB iron acquisition genes and their complemented counterparts were generated in the live vaccine strain (LVS). The ΔfeoB but not the ΔfslC mutant exhibited severely inhibited growth in chemically‐defined medium with restricted iron and FL83B hepatocytes, but replication in macrophages was similar to wild‐type bacteria. Ability of the ΔfeoB mutant to establish infection in mice was studied by measuring organ burden at three days post‐infection. The ΔfeoB mutant showed impaired colonization of the lungs, liver, and spleen compared to wild‐type bacteria, and complementation partially rescued this phenotype. The intracellular environment is reducing, providing an abundance of ferrous iron. These results identify FeoB, which functions to acquire ferrous iron in other bacteria, as a contributor to F. tularensis intracellular replication and virulence. Furthermore, these results designate FeoB as a potential therapeutic target. Funding sources: NIAID T32 A1007539 and P01 AI055621