The fslE homolog, FTL_0439 (fupA/B), mediates siderophore-dependent iron uptake in Francisella tularensis LVS

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 ΔfslE mutant for siderophore utilization. We generated a deletion of fupA/B in LVS and in the LVS ΔfslE background. The ΔfupA/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 ΔfupA/B mutants showed a replication defect in J774.1A cells and decreased virulence following intraperitoneal infection in mice. Complementation of the ΔfupA/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.

FIG. 1.

Growth deficiency of ΔfupA/B mutants under conditions of iron limitation. (A) Growth on iron-replete (MHA) and iron-limiting (CDM-Fe) agar plates. LVS and the mutants were grown in CDM, and serially diluted cultures were spotted on iron-replete and iron-limiting plates in parallel. (B) Growth in liquid medium. Washed cells were inoculated in iron-replete and iron-limiting che-CDM, and growth was followed over a period of 48 h. Cultures were grown in triplicate, and means and standard deviations of data representing the results of one representative experiment are shown in the growth plot. The strains used were LVS, GR13 (LVS ΔfslE), GR16 (LVS ΔfupA/B), and GR17 (LVS ΔfslE ΔfupA/B).

FIG. 2.

Siderophore utilization by mutants and complemented strains. Plates were seeded with strains as noted. (A) Siderophore-dependent growth of LVS and GR13 (LVS ΔfslE), showing growth around the siderophore-secreting LVS spot and lack of growth around siderophore-deficient ΔfslA strain GR7. (B) Deficiency of GR16 (LVS ΔfupA/B) and GR17 (LVS ΔfslE ΔfupA/B) mutants in utilization of siderophore secreted by LVS in the central spot. (C) The siderophore utilization defect is fully or partially restored in complemented strains. Siderophore-dependent growth of the ΔfupA/B strains was fully restored by reintroduction of fupA/B in cis, whereas reintroduction of fslE in GR17 partially complemented the growth defect. The siderophore source in this set of assays was LVS (vector).

FIG. 3.

Partial complementation of GR211 (Schu ΔfslE) by LVS homolog FTL_0439. (A) The FslE protein family in F. tularensis strain Schu S4 and LVS. The paralogs within Schu S4 share sequence similarity of 52 to 68% and identity of 45 to 62%. Orthologs between SchuS4 and LVS share 98 to 99% identity. FTL_0439 and FTL_0147 show variations from their orthologs in Schu S4. FTL_0439 (FupA/B) is a hybrid consisting of the amino-terminal half of FupA fused to the carboxy-terminal half of FupB. The amino-terminal end of FTL_0147 from LVS is deleted in the ortholog FTT0267 in Schu S4. The black boxes represent potential signal peptides. (B) Growth on iron-limiting CDM agar of serially diluted cultures of Schu S4, Schu S4 ΔfslE (GR211), and complemented strains. (C) Siderophore utilization by Schu ΔfslE partially complemented by fupA/B. Iron-limiting plates were seeded with GR211 harboring the vector or with the fslE or fupA/B complement as noted. SchuS4 cells were spotted as a source of siderophore, and growth halos around the spots were recorded.

FIG. 4.

Chromosomal organization of the FTL_0439 (fupA/B) region in F. tularensis LVS and PCR analysis of deletion. Arrows indicate the direction of transcription. The arrowheads represent primers 263-272 and 266-267 used in amplifying the flanking regions for generation of the pBAS1B deletion construct depicted underneath. The deletion includes the two IS elements (gray boxes) in addition to fupA/B (black box). Primer 242 located upstream of the fupA/B gene and primer 281 located downstream of ISftu2 were used for analyzing genomic DNA from LVS and the mutants. Lane M shows a 1-kb DNA ladder (Bioline).

FIG. 5.

Complementation of ΔfupA/B mutants for growth under conditions of iron limitation. (A) Complementation of GR16 (LVS ΔfupA/B). Growth of serially diluted cultures of GR16 transformed with vector or fupA/B plasmid was compared to that of LVS with vector (LVS-V) on iron-rich MHA and iron-limiting CDM-Fe agar. (B) Complementation of GR17 (LVS ΔfslE ΔfupA/B). Growth of serially diluted cultures of GR17 transformed with vector, fupA/B, or fslE plasmid was compared to that of LVS with vector on iron-rich and iron-limiting agar. (C) Complementation of GR16 in iron-limiting liquid media. Growth of GR16 harboring vector or the fupA/B plasmid was compared to that of LVS with vector (LVS-V). (D) Complementation of GR17 in iron-limiting liquid media. Growth of GR17 with control vector or the fupA/B or fslE plasmid was compared to that of LVS with vector (LVS-V).

FIG. 6.

Deregulation of siderophore and FslE levels in ΔfupA/B mutants. (A) Specific levels of CAS activity representing siderophore levels in culture supernatants of LVS and the mutants were determined after growth in iron-replete CDM (black bars) or iron-limiting CDM (hatched bars). The strains harboring complements were similarly assayed. Cultures were grown in triplicate, and the means and standard deviations of data representing the results of representative experiments are shown. *, P < 0.001; **, P < 0.002. (B) Western blotting with FslE antibodies. Cultures were grown in iron-replete (Hi) and iron-limiting (Lo) media. Whole-cell lysates normalized for cell density were subjected to SDS-PAGE, transferred to a PVDF membrane, and probed with polyclonal antiserum raised against an FslE peptide. The locations of the prestained standards run on the gel are indicated. The strains used were LVS, GR13 (LVS ΔfslE), GR16 (LVS ΔfupA/B), and GR17 (LVS ΔfslE ΔfupA/B).

FIG. 7.

Intracellular replication of LVS and the mutants. J774A.1 macrophages in a 24-well plate were infected with bacteria at an MOI of 10 to 30 in quadruplicate. Bacterial entry and replication were determined over a 25-h period; the results are expressed as mean log numbers of colony-forming units per milliliter. The error bars indicate standard deviations. Mutants found to be significantly defective in intracellular growth are indicated by asterisks (P < 0.02). The strains used were LVS, GR13 (LVS ΔfslE), GR16 (LVS ΔfupA/B), and GR17 (LVS ΔfslE ΔfupA/B).