FeoB-Mediated Uptake of Iron by Francisella tularensis

Abstract

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.

Iron and Virulence in Francisella tularensis

“…Similar results were obtained using LVS infected hepatocytes (Thomas-Charles et al, 2013), suggesting that the siderophore uptake system contributes to survival within different tissue types. fsl mutants were identified in a negative selection screen of a U112 transposon mutant library in mice (Weiss et al, 2007) and a signature-tagged mutant screen identified fslA and feoB to be important for pulmonary infection by LVS (Su et al, 2007).…”

“…FslC is predicted to be a member of the pyridoxal phosphate-dependent decarboxylases. Mutant analysis demonstrated that both fslA and fslC are required for Francisella rhizoferrin production (Deng et al, 2006; Sullivan et al, 2006; Lindgren et al, 2009; Thomas-Charles et al, 2013). Rhizoferrin biosynthesis in F. tularensis , involving just two dedicated enzymes, may be the simplest siderophore biosynthetic pathway identified thus far.…”

“…Δ feoB mutants of LVS and Schu S4 are deficient for growth on iron-limiting media (Thomas-Charles et al, 2013; Pérez and Ramakrishnan, 2014; Pérez et al, 2016). 55 Fe uptake assays demonstrated that the F. tularensis Δ feoB mutants are completely deficient in ferrous iron uptake (Pérez and Ramakrishnan, 2014; Pérez et al, 2016), implying that the Feo system is the sole ferrous iron transporter across the inner membrane (Figure 2).…”

Successful Protection against Tularemia in C57BL/6 Mice Is Correlated with Expansion of Francisella tularensis-Specific Effector T Cells

“…Similarly, Francisella strains with deletions in purine synthesis genes are defective for intracellular replication and are attenuated in vivo (27,28). In addition to dependence on genes that participate in the purine biosynthesis pathway, Francisella strains with mutations in genes required for efficient iron acquisition, pyrimidine biosynthesis, and glycolysis have exhibited various levels of attenuation (29)(30)(31). Thus, it is possible that the marked difference in the attenuation levels observed in RML LVS and ATCC LVS is due to spontaneous mutations in one or more genes required for efficient metabolism.…”

Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

“…Considering the limited intracellular concentrations of gentamicin and the predominant intracellular localization of F. tularensis, it is likely that the ability of gentamicin to eradicate F. tularensis could be enhanced. In view of the previous demonstration that gallium alone showed efficaciousness in an intranasal challenge model with Francisella novicida (24) in conjunction with the many publications that demonstrate critical roles of iron in the intracellular replication and virulence of F. tularensis (4,5,24,(28)(29)(30)(31)(32), we anticipated that a better understanding of how the requirement for iron affects antibiotic susceptibility of the bacterium would provide important clues to improved treatment regimens, and we hypothesized that gallium could potentiate the effect of gentamicin. We here demonstrate that gallium effectively inhibits the replication of F. tularensis in broth and during intracellular infection as well as in vivo, and these effects were partly synergistic with gentamicin.…”