The Haemophilus influenzae hFbpABC Fe ³⁺ 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.Pathogenic bacteria employ a number of acquisition strategies in competition for host iron (Fe 3ϩ ). Siderophore-dependent iron transport is a widely used strategy that involves the secretion of organic siderophore molecules that compete for iron bound to the high-affinity host transferrin (Tf) and lactoferrin (Lf) proteins (18). Fe 3ϩ -siderophore complexes are subsequently recovered by the bacteria through the activity of siderophore-specific surface receptors and transporters. As an alternate strategy, several gram-negative pathogens, including Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae, utilize a siderophore-independent (free) Fe 3ϩ

Genome Analysis of Moraxella catarrhalis Strain RH4, a Human Respiratory Tract Pathogen

“…port system for transport of Fe 3ϩ from the periplasm to the cytosol, encoded by the fbpABC gene cluster, was identified based on homology to the corresponding locus in H. influenzae (2). Further, the afeABCD gene cluster was identified in RH4; this cluster has been proposed to be involved in the acquisition of chelated iron, as described previously for Actinobacillus actinomycetemcomitans and Yersinia pestis, and is regulated by Fur in these species (5,57).…”

Moonlighting of Haemophilus influenzae heme acquisition systems contributes to the host airway-pathogen interplay in a coordinated manner

“…We next asked if heme interbacterial donation relates to its binding. To do so, we employed the free iron-binding system HitABC, in which HitA is a periplasmic protein that binds free iron linked to the HitBC transporter located at the inner membrane [98][99][100][101]. Inactivation of the hitABC Figure 4.…”

Silica-Induced Protein (Sip) in Thermophilic Bacterium Thermus thermophilus Responds to Low Iron Availability

“…As we reported previously (8), the amino acid sequence of Sip is similar to that of a solute-binding protein, the Fe 3ϩ -binding ABC transporter, predicted from the HB8 genome. The Fe 3ϩ -binding ABC transporter functions in the process of Fe 3ϩ uptake, and it has been analyzed in detail in such pathogenic bacteria as Serratia marcescens (20), Actinobacillus actinomycetemcomitans (21), Haemophilus influenzae (22), and Vibrio cholerae (23). These Fe 3ϩ uptake systems consist of a periplasmic substrate-binding protein, permease, and ATPase, and the genes involved are usually regulated as an operon; sip has a similar arrangement but with a palindromic structure situated between the sip and permease genes (Fig.…”