Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2

Biochemical Journal (Impact Factor: 4.4). 10/2012; 449(3). DOI: 10.1042/BJ20121426
Source: PubMed


Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. Here we explore the structure and function of FhuD2, a staphylococcal surface lipoprotein mediating iron-uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2-ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully Fe3+-dependent. We determined the x-ray crystallographic structure of ligand-bound FhuD2 at 1.9Å resolution, revealing the bilobate fold of class III solute-binding proteins (SBPs). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2-siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other Class III SBPs might also be exploited as vaccine candidates.

Download full-text


Available from: Ravi Mishra
  • [Show abstract] [Hide abstract]
    ABSTRACT: The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B12 transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by "charge swapping" experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.
    No preview · Article · Dec 2013 · Biology of Metals
  • [Show abstract] [Hide abstract]
    ABSTRACT: An improved synthesis of petrobactin and analogues has been developed that provides reliable access to siderophores in sufficient quantities for biological studies. The determination of the iron affinity of different petrobactin derivatives showed that functionalization at the central amino group of the spermidine sidechain has only a minor influence on the iron binding properties of the siderophore. Accordingly, such derivatives are promising starting points for the development of biological probes to study bacterial siderophore transport, a possible new target for antibiotics.
    No preview · Article · Jan 2014 · European Journal of Organic Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Iron acquisition is a central process for virtually all organisms. In Staphylococcus aureus, FhuD2 is a lipoprotein that is a high-affinity receptor for iron-bound hydroxamate siderophores. In this study, FhuD2 was crystallized bound to ferrioxamine-B (FXB), and also in its ligand-free state; the latter structures are the first for hydroxamate-binding receptors within this protein family. The structure of FhuD2-FXB shows that residues W197 and R199 from the C-terminal domain donate hydrogen bonds to the hydroxamate oxygens, and a ring of aromatic residues cradles the aliphatic arms connecting the hydroxamate moieties of the siderophore. The available ligand-bound structures of FhuD from E. coli and YfiY from B. cereus show that, despite a high degree of structural conservation, three protein families have evolved with critical siderophore binding residues on either the C-terminal domain (S. aureus), the N-terminal domain (E. coli), or on both domains (B. cereus). Unliganded FhuD2 was crystallized in 5 conformations related by rigid-body movements of the N- and C-terminal domains. Small-angle X-ray scattering indicates that the solution conformation of unliganded FhuD2 is more compact than the conformations observed in crystals. The ligand-induced conformational changes for FhuD2 in solution are relatively modest and depend on the identity of the siderophore. The crystallographic and SAXS results are used to discuss roles for the liganded and unliganded forms of FhuD2 in the siderophore transport mechanism.
    No preview · Article · Mar 2014 · Biochemistry
Show more