The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants

Abstract

The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe(3+) transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe(3+) through the periplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations were analyzed using growth rescue and radiolabeled (55)Fe(3+) transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.

FIG. 1.

Multiple-sequence alignments of the conserved permease motifs from multiple permeases of the ABC transporter family. Set 1 includes the free Fe³⁺ transport permeases nFbpB (N. gonorrhoeae [N. gon.]), nFbpB (N. meningitidis [N. men.]), hFbpB (H. influenzae [H. inf.]), SfuB (Serratia marcescens [S. mar.]), YfuB (Yersinia enterocolitica [Y. ent.]), and putative MhFbpB (Mannheimia haemolytica [M. Hae.]) (included in the initial alignment, excluded in subsequent alignments). Set 2 includes the iron-siderophore permeases FepD/G ferric-enterobactin E. coli, FhuB ferric-hydroxymate E. coli, FecC/D ferric-dicitrate E. coli, and FatC/D anguillobactin Vibrio anguillarum (V. ang.). Set 3 includes the thiosulfate/molybdenum/putrescine/glycine-betaine permeases CysT/W thiosulfate E. coli, CysT/W thiosulfate N. meningitidis, ModB molybdenum E. coli, PotB/C putrescine H. influenzae, PotB/C putrescine E. coli, and ProW glycine-betaine E. coli. Set 4 includes the oligosaccharide/glycerophosphate permeases MalF/G maltose Salmonella enterica serovar Typhimurium (S. typ.), MalF/G maltose E. coli, MalF/G Pseudomonas aeruginosa (P. aer.), MalF/G Vibrio cholerae (V. cho.), UgpA/E glycerophosphate E. coli, MalF/G Thermococcus litoralis (T. lit.), MalC/D maltose Streptococcus pneumoniae (S. pneu.), CymF/G Klebsiella oxytoca (K. oxy.), MsmF/G raffinose-melibiose Streptococcus mutans (S. mut), and putative MalF/G1/G2 Streptomyces coelicolor (S. coe.). The numbered sequences of the hFbpB motifs are denoted above the alignments. Asterisks indicate residues in hFbpB that have been targeted for mutagenesis. Homologous residues are enclosed in rectangles, while identical residues are shaded black.

FIG. 2.

Radiolabeled ⁵⁵Fe³⁺ transport assay. (A) Cells grown on NBamp₁₀₀dip₇₅ were washed and incubated at 37°C in iron-free M9 media supplemented with 1 μM ⁵⁵Fe³⁺(NTA)₂. Samples were removed and subjected to filtration, and counts per minute were measured. Radiolabeled iron uptake is plotted versus time. Each strain was tested in triplicate; error bars represent standard errors. (B) Cartoon depiction of the iron transport assay controls shown in panel A. On the left, H-1443/pBR322 is a vector-only control. In the middle, H-1443/pAHIΔC is an FbpA-only control that is missing a functional ABC transport complex (ΔFbpC). On the right, H-1443/pAHIO is a wild-type control expressing a functional FbpABC transporter that can mobilize Fe³⁺(NTA)₂ from the periplasm to the cytosol.

FIG. 3.

⁵⁵Fe³⁺ transport assay results of conserved permease motif mutations (G155 and G418). (A) The conservative mutation G155A results in slightly diminished transport activity compared to that of wild-type pAHIO. The more severe mutations G155V and G155E result in transport activity that is similar to that of the wild type (within standard errors). These results indicate that mutation of the invariant glycine on motif 1 has no discernible effect on activity. (B) The conservative mutation G418A results in an approximately twofold decrease in iron uptake (53.5% of wild-type uptake at 7 min), indicating that mutation of the invariant glycine on motif 2 has a significant effect on activity.

FIG. 4.

⁵⁵Fe³⁺ transport assay results of conserved permease motif mutations (L154 and L417). (A) The motif 1 L154D mutation results in an approximately twofold decrease in transport activity (50.5% of wild-type uptake at 7 min). (B) The motif 2 L417D mutation results in an approximately fourfold decrease in activity (28.3% of that at 7 min); this level is similar to that of the pAHIΔC control.

FIG. 5.

Growth phenotype of the initial gallium selection mutation V497I. (A) The left side shows that strains plated on NBamp₁₀₀dip₇₅ exhibit similar growth phenotypes. Clockwise from top left are H-1443/pAHIO, H-1443/V497I, H-1443/pAHIΔC, and H-1443/I497V. The right side shows the same strains plated on NBamp₁₀₀dip₇₅Ga₁₀₀. The V497I mutant exhibits an uninhibited growth phenotype, while wild-type (WT) pAHIO and the reverse mutant I497V exhibit growth-suppressed phenotypes in the presence of gallium. (B) ⁵⁵Fe³⁺ transport assay results of the V497I and reverse I497V mutations. The V497I mutation results in an ∼2.5-fold decrease in transport activity. The reverse mutation results in activity that is indistinguishable from that of wild-type pAHIO.

FIG. 6.

Topological model of the hFbpB membrane permease. Shaded rectangles represent the 12 putative transmembrane α-helices. Residues that are completely conserved among the free Fe³⁺ permeases are indicated with squares. Residues comprising the conserved permease motifs are shaded gray. Residues identified by mutagenesis are shaded black, and those mutations resulting in affected iron transport activity are labeled. The bottom portion shows alignment between the hFbpB conserved permease motifs and the motif from the single subunit of the vitamin B₁₂ permease BtuC, indicating conservation of the leucine and glycine residues as described in Discussion.