Pyoverdine, the Major Siderophore in Pseudomonas aeruginosa, Evades NGAL Recognition

Abstract

Pseudomonas aeruginosa is the most common pathogen that persists in the cystic fibrosis lungs. Bacteria such as P. aeruginosa secrete siderophores (iron-chelating molecules) and the host limits bacterial growth by producing neutrophil-gelatinase-associated lipocalin (NGAL) that specifically scavenges bacterial siderophores, therefore preventing bacteria from establishing infection. P. aeruginosa produces a major siderophore known as pyoverdine, found to be important for bacterial virulence and biofilm development. We report that pyoverdine did not bind to NGAL, as measured by tryptophan fluorescence quenching, while enterobactin bound to NGAL effectively causing a strong response. The experimental data indicate that pyoverdine evades NGAL recognition. We then employed a molecular modeling approach to simulate the binding of pyoverdine to human NGAL using NGAL's published crystal structures. The docking of pyoverdine to NGAL predicted nine different docking positions; however, neither apo- nor ferric forms of pyoverdine docked into the ligand-binding site in the calyx of NGAL where siderophores are known to bind. The molecular modeling results offer structural support that pyoverdine does not bind to NGAL, confirming the results obtained in the tryptophan quenching assay. The data suggest that pyoverdine is a stealth siderophore that evades NGAL recognition allowing P. aeruginosa to establish chronic infections in CF lungs.

Figure 1

Molecular structures of ferric bacterial siderophores used in this study. (a) Chemical structure of a group I pyoverdine from bacterial species Pseudomonas fluorescens (strain ATCC 13525) illustrating its derivatized peptide structure. The pyoverdine amino acid sequence is Ser*-Lys-Gly-FHO-cyclic [Lys-FHO-Ser*], where Ser* is D-serine and FHO is N-formyl-N-hydroxy-ornithine. Oxygens in red represent atoms that coordinate iron in the 3D structures. (b) Ferric pyoverdine structure taken from the 3D structure of a pyoverdine-Fe transporter protein (FpvA) in complex with pyoverdine (ATCC  13525) and Fe (PDB  2W78). Only the pyoverdine and iron (green) atoms are shown. Both hydroxyl groups on the chromophore and FHO side chain oxygens form interactions with iron. (c) Chemical structure of enterobactin. (d) Ferric enterobactin structure isolated from the 3D structure of a mutant of NGAL in complex with ferric enterobactin (PDB  3CMP). Illustrations for (b) and (d) were made using Python Molecular Viewer 1.5.4. Each ligand is shown as a stick figure with coloring by atom type. Iron atoms are shown in green space-filling representation.

Figure 2

Pyoverdine does not bind to NGAL. The binding of pyoverdine and enterobactin to NGAL was measured by a tryptophan fluorescence-quenching method [13, 15]. Siderophore dilutions were preincubated with recombinant human NGAL (2 μM) for 30 min prior to reading with a plate reader. (a) Optimization of intrinsic tryptophan fluorescence quenching in rhNGAL by Ferric-DHBA complexes in a dose-dependent manner. (b) Percent of reduction in tryptophan fluorescence upon enterobactin binding to NGAL compared to apo-pyoverdine and ferric pyoverdine. Error bars (some smaller than the symbol) represent ±SD from the average of triplicate readouts at each point.

Figure 3

Apo-pyoverdine does bind to NGAL. Docking of apo-pyoverdine (without iron) into the NGAL trimer crystal structure (PDB 1L6M) using AutoDock Vina. NGAL protein in red, blue, and green ribbon diagrams with nine pyoverdine docking modes shown in space-filling representation. Here, each of the binding regions is displayed on only one representative pyoverdine molecule (turquoise, white, or magenta). Tryptophan residues W31 and W79 are highlighted in yellow. The model was rotated 180° for display in the view at right. The binding affinity for each docking mode is listed Table 1.

Figure 4

Ferric pyoverdine does not dock inside the NGAL calyx. (a) Docking of ferric pyoverdine (bound to iron) into the NGAL trimer crystal structure (PDB 1L6M) using AutoDock Vina. NGAL protein in red, blue, and green ribbon diagrams with nine positions of docked pyoverdine shown. For this illustration only one pyoverdine molecule is displayed in each of the predicted binding positions on the rigid NGAL. All simulated docking modes were on the surface and between the units of NGAL but not near or in the ligand-binding site. The binding affinity for each docking mode is listed in Table 1. (b) Ferric pyoverdine docking into the NGAL monomer crystal structure (PDB 1QQS) using AutoDock Vina. Modes 1–8 docked near the opening of the NGAL calyx without fully fitting in the hydrophobic pocket, while mode 9 docked on the opposite surface. The binding affinity for each docking mode is listed Table 1.

Figure 5

Enterobactin binds to NGAL. The docking of ferric enterobactin into the NGAL crystal structure (PDB 1L6M) using AutoDock Vina was performed as a positive control to validate the pyoverdine docking simulation. NGAL crystal structure with each monomer presented in ribbon diagram colored red, blue, and green, plus ferric enterobactin. AutoDock Vina found nine positions in which ferric enterobactin docked, seven of which were in one of the calyx of one of the monomers, that is, the ligand-binding site. The two remaining positions, modes 7 and 8, are between the blue and green monomers (enterobactin is not shown in modes 7 and 8, for clarity). For this illustration only one ferric enterobactin molecule is displayed in each of the predicted binding positions on the rigid NGAL. Tryptophan residues W31 and W79 are highlighted in yellow. The docking of ferric enterobactin to NGAL view was rotated 180° for display in the view at right. The binding affinity for each docking mode is listed in Table 1.

Figure 6

Ferric pyoverdine superimposed on ferric Enterobactin docked into NGAL. The docking of ferric pyoverdine to NGAL is superimposed on ferric enterobactin bound to NGAL. (1) NGAL molecular surface representation with Fe-ENT bound in the hydrophobic calyx where Fe-PVD docking site is buried under the surface. In the surface representation, blue indicates a hydrophobic region, while red indicated a hydrophilic region. Fe-ENT and Fe-PVD are shown as stick figures. (2) NGAL molecular surface representation with 50% transparency. (3) NGAL backbone (fuchsia) with the Fe-PVD surface shown in 50% transparency. This figure was generated using the UCSF Chimera package [25, 26].