Abstract
Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a (55)Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host.
Highlights
FslE and FupA are Francisella-specific paralogous proteins involved in iron acquisition
Generation of Schu ⌬fupA and Schu ⌬fslE ⌬fupA Mutants— Our studies using a Schu ⌬fslE mutant in cross-feeding assays on iron-limiting Chamberlain’s defined medium (CDM) agar suggested that FslE functions as a receptor in siderophore-mediated iron uptake [16]
Using a similar growth assay on agar, Lindgren et al [21] concluded that FupA is involved in siderophore-dependent growth in Schu S4, but phenotypic differences with a siderophore-deficient mutant suggested an additional siderophore-independent role for FupA in iron acquisition
Summary
FslE and FupA are Francisella-specific paralogous proteins involved in iron acquisition. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. We used a 55Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. In a mouse model of tularemia, the ⌬fupA mutant was attenuated, but the ⌬fslE ⌬fupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence These studies highlight the importance of specific iron acquisition functions, that of ferrous iron, for virulence of F. tularensis in the mammalian host. Studies to date suggest that the siderophore-mediated iron uptake impacts virulence to different extents depending on the Francisella strain and/or infection model used. Our results indicate that FupA functions as the principal virulence determinant, both paralogs are required for full virulence in a mouse model of tularemia
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