Gallium(III)-Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition

Abstract

Pseudomonas aeruginosa is an opportunistic bacterium that causes life-threatening infections in immunocompromised patients. In infection, it uses heme as a primary iron source and senses the availability of exogenous heme through the heme assimilation system (Has), an extra cytoplasmic function σ-factor system. A secreted hemophore HasAp scavenges heme and, upon interaction with the outer-membrane receptor HasR, activates a signaling cascade, which in turn creates a positive feedback loop critical for sensing and adaptation within the host. The ability to sense and respond to heme as an iron source contributes to virulence. Consequently, the inhibition of this system will lead to a disruption in iron homeostasis, decreasing virulence. We have identified a salophen scaffold that successfully inhibits the activation of the Has signaling system while simultaneously targeting iron uptake via xenosiderophore receptors. We propose this dual mechanism wherein free Ga³⁺-salophen reduces growth through uptake and iron mimicry. A dual mechanism targeting extracellular heme signaling and uptake together with Ga³⁺-induced toxicity following active Ga³⁺salophen uptake provides a significant therapeutic advantage while reducing the propensity to develop resistance.

Keywords: Pseudomonas aeruginosa; antimicrobials; heme sensing; heme uptake; metallotherapeutics.

Figure 1.

(A) Transcriptional Activation of the hasR Promoter with holo– and FeSal–HasAp. FeSal shows decreased transcriptional activation relative to heme. Both cultures were supplemented with preformed HasAp complexes and data represent the average of three independent experiments*, p < 0.05; **, p < 0.005 by t test between holo and FeSal–HasAp. (B) Growth of PAO1 Wild-Type and the ΔhasRΔphuR Strain with FeSal and FeSal–HasAp. Cultures were grown in a 96-well plate with 1 μM supplements, as described in the experimental section.

Figure 2.

(A) Transcriptional Activation of the hasR Promoter with holo– and GaSal–HasAp. GaSal shows decreased transcriptional activation relative to heme. Both cultures were supplemented with preformed HasAp complexes and represent the average of three independent experiments: *, p < 0.05; **, p < 0.005, by a t test between holo and GaSal–HasAp. (B) GaSal antibacterial activity in the PAO1WT and ΔhasRΔphuR strains. Data represent the average of three independent cultures with blanks (media only) subtracted. Cultures were grown in M9 minimal media with 1 μM supplements. (C) HasAp titration alleviates GaSal toxicity in PAO1 ΔhasAp. Cultures were prepared in M9 minimal media with FeCl₃ (400 nM) and GaSal (7 μM), with HasAp supplemented at increasing concentrations. Growth was calculated relative to gallium-free cultures in stationary-phase growth, and data represent the average of three independent experiments: *, p < 0.05; **, p < 0.005, by a t test between 0 μM HasAp and specified concentrations.

Figure 3.

(A) ¹H NMR spectrum of GaSal in M9 minimal Media. Proton peaks were assigned and labeled, as depicted for comparison in the STD spectrum. (B) STD NMR spectrum using GaSal (1 mM) and HasAp (10 μM) in PAO1 ΔhasAp supernatant.

Figure 4.

(A) Transcriptional activation of the hasR promoter with holo– and GaPPIX–HasAp. GaPPIX shows increased (2 h) or similar transcriptional activation to heme. Cultures were supplemented with preformed HasAp complexes, and data represent the average of three independent experiments: **, p < 0.005, by a t test between holo– and GaPPIX–HasAp. (B) Growth of PAO1 Supplemented with either 1 μM Heme or GaPPIX. Supplementation with GaPPIX inhibits growth in M9 minimal media. Cultures were grown in a 96-well plate, as described in Methods.

Figure 5.

Conformational changes of HasAp upon ligand binding. (A) Alignment of apo–, holo–, and FeSal–HasAp, highlighting the closure of the H32 loop from the apo– (orange, PDB 3MOK) to the holo– (blue, PDB 3ELL) and FeSal–HasAp (red, PDB 3W8M) forms. (B) Alignment of holo– and FeSal–HasAp, highlighting the closed-loop conformation with minimal structural differences. Relative deuteration of HasAp bound to heme (C) and GaSal (D). Individual peptides are plotted from the N- to C-terminus based on the first residue number. For each peptide, differences in the percent deuteration at each time point, color coded according to the legend, with the sum of all differences integrated over time are represented in gray bars. Also, 98% confidence intervals are represented as dashed (for individual time points) and solid (for total sums) lines. Peptides exceeding both confidence intervals were considered to display a statistically significant difference in deuterium uptake between the apo and ligand bound form and were mapped onto the crystal structure of holo–HasAp (panel C, inset; PDB 3ELL). Positive values indicate protection from deuteration upon ligand binding, and negative values indicate increased deuteration of a region upon ligand binding. Significant regions are highlighted and color coded on the HasAp structure (panel C, inset).

Figure 6.

Deuteration differences between ligand-bound forms. (A) Percent deuteration of GaSal–HasAp minus holo–HasAp. Positive peaks represent greater deuteration in the salophen-bound form, and negative peaks indicate increased deuteration in the heme-bound form. Inset: representation of protein regions that are different between all three HasAp states, using FeSal–HasAp as a representation of GaSal–HasAp. (B) Deuteration of a peptide comprising residues 26–54. (C) Heme binding site with heme (blue) and FeSal (red) overlaid. Potential contacts between the H32 loop and heme are drawn in red.

Figure 7.

Thermal denaturation of HasAp bound to heme or GaSal. (A) Comparison of three ligand states at 25 °C. (B) Thermal denaturation profiles at 222 nm fit to a sigmoidal distribution. CD spectra were recorded as described in Methods.