ThefslEHomolog,FTL_0439(fupA/B), Mediates Siderophore-Dependent Iron Uptake inFrancisella tularensisLVS

Abstract

The Gram-negative pathogen Francisella tularensis secretes a siderophore to obtain essential iron by a TonB-independent mechanism. The fslABCDE locus, encoding siderophore-related functions, is conserved among different Francisella strains. In the virulent strain Schu S4, fslE is essential for siderophore utilization and for growth under conditions of iron limitation. In contrast, we found that deletion of fslE did not affect siderophore utilization by the attenuated live vaccine strain (LVS). We found that one of the fslE paralogs encoded in the LVS genome, FTL_0439 (fupA/B), was able to partially complement a Schu S4 ⌬fslE mutant for siderophore utilization. We generated a deletion of fupA/B in LVS and in the LVS ⌬fslE background. The ⌬fupA/B mutant showed reduced growth under conditions of iron limitation. It was able to secrete but was unable to utilize siderophore. Mutation of both fupA/B and fslE resulted in a growth defect of greater severity. The ⌬fupA/B mutants showed a replication defect in J774.1A cells and decreased virulence following intraperitoneal infection in mice. Complementation of the ⌬fupA/B mutation in cis restored the ability to utilize siderophore and concomitantly restored virulence. Our results indicate that fupA/B plays a significant role in the siderophore-mediated iron uptake mechanism of LVS whereas fslE appears to play a secondary role. Variation in iron acquisition mechanisms may contribute to virulence differences between the strains.

Paralogous Outer Membrane Proteins Mediate Uptake of Different Forms of Iron and Synergistically Govern Virulence in Francisella tularensis tularensis

“…An insertion element ISFTU1 is located 65 bp downstream of the fupB stop codon. As in our previous work, we avoided use of the transposon sequences in the 3Ј-flanking sequences (28), and instead, amplified the 1.532 kb sequence located downstream of the insertion element using primers 360 and 361. The 5Ј-and 3Ј-flanking sequences were cloned in suicide vector pGIR463 to generate plasmids pGIR476 (for fupA deletion) and pBAS4 (for fupB deletion).…”

“…The integrative plasmids had the promoter of fslA driving expression of the different genes. Plasmid pGIR474 expressing fslE has been described previously (28). For construction of the fupA complementing plasmid pGIR477, the fupA sequences were amplified with primers 256 and 257 using FastStart High Fidelity polymerase (Roche Applied Science).…”

“…Growth in Iron-replete and Iron-limiting Media-The bacteria were grown in che-CDM containing 2.5 g/ml (3.36 M, iron-replete) or 0.125 g/ml (0.168 M, iron-limiting) FePPi for analysis of growth under iron limitation as previously described (28). In specific experiments as noted, FeSO 4 was added at 2 or 0.2 g/ml to generate iron-replete and iron-limiting media, respectively.…”

“…Complementation in cis was achieved by integration of a suicide plasmid targeted to the 3Ј region of feoB as previously detailed (28). The integrative plasmids had the promoter of fslA driving expression of the different genes.…”

“…The paralog with the greatest similarity, FupA was initially characterized as a virulence factor in Schu S4 based on attenuation of a fupA mutant in a mouse model of tularemia (27). fupA was found to be important for intracellular replication of Schu S4 as well as F. novicida (23,27), and the related fupA/B hybrid protein in LVS has also been linked to virulence in mice (28).…”