Pseudomonas aeruginosa manipulates redox and iron homeostasis of its microbiota partner Aspergillus fumigatus via phenazines

Abstract

The opportunistic fungal pathogen Aspergillus fumigatus is increasingly found as a coinfecting agent along with Pseudomonas aeruginosa in cystic fibrosis patients. Amongst the numerous molecules secreted by P. aeruginosa during its growth, phenazines constitute a major class. P. aeruginosa usually secreted four phenazines, pyocyanin (PYO), phenazine-1-carboxamide (PCN), 1-hydroxyphenazine (1-HP) and phenazine-1-carboxylic acid (PCA). These phenazines inhibited the growth of A. fumigatus but the underlying mechanisms and the impact of these four phenazines on A. fumigatus biology were not known. In the present study, we analyzed the functions of the four phenazines and their mode of action on A. fumigatus. All four phenazines showed A. fumigatus growth inhibitory effects by inducing production of reactive oxygen species (ROS), specifically O2(·-), and reactive nitrogen species (RNS), ONOO(-). A. fumigatus Sod2p was the major factor involved in resistance against the ROS and RNS induced by phenazines. Sub-inhibitory concentrations of PYO, PCA and PCN promote A. fumigatus growth by an independent iron-uptake acquisition. Of the four phenazines 1-HP had a redox-independent function; being able to chelate metal ions 1-HP induced A. fumigatus iron starvation. Our data show the fine-interactions existing between A. fumigatus and P. aeruginosa, which can lead to stimulatory or antagonistic effects.

Figure 1. MIC of phenazines against A. fumigatus.

Growth of A. fumigatus CEA17ΔakuB^KU80 (WT) was determined in the presence of increasing concentrations of the four phenazines (PYO, PCN, 1-HP and PCA). Mycelial growth was estimated by reading absorbance at 560 nm using the crystal violet method after 20 h in 2YT at 37°C, and compared to the control growing in absence of phenazine and normalized at 100% absorbance at 560 nm.

Figure 2. Ultrastructural morphology of A. fumigatus hyphae in the presence of phenazines.

(A) A. fumigatus conidia incubated for 20 h growth at 30°C in the presence of 1% DMSO. (B–F) A. fumigatus conidia incubated for 18 h growth at 37°C in the presence of 1 mM PYO (B, C), 125 μM PCN (D), 62.5 μM 1-HP (E) or 2 mM PCA (F). Arrows show the mitochondria in A. fumigatus cells.

Figure 3. Phenazines penetrate and have redox activity.

(A) Swollen conidia incubated with phenazines (PYO, PCN, 1-HP, PCA) in 2YT at MIC concentrations for 1 h and observed under emission-specific wavelengths. (B) Mycelium was incubated in 2YT 1 h with PYO (MIC concentration). Scale bar represents 5 μm.

Figure 4. Phenazines induce ROS production in A. fumigatus swollen conidia.

H₂DCFDA, a ROS fluorescent probe, was added to swollen conidia prior to the addition of PYO, PCN, 1-HP and PCA at MIC concentrations for 1 h and observed under emission-specific wavelengths. For control swollen conidia were incubated with H₂DCFDA in absence of phenazines. Scale bar represents 5 μm.

Figure 5. Phenazines induce RNS production in A. fumigatus swollen conidia.

DHR123, a RNS fluorescent probe, was added to swollen conidia prior to the addition of PYO, PCN, 1-HP and PCA at MIC concentrations for 1 h and observed under emission-specific wavelengths. For control swollen conidia were incubated with DHR123 in absence of phenazines. Scale bar represents 5 μm.

Figure 6. 1-HP induces an iron starvation response in A. fumigatus.

A. fumigatus was grown for 12 h in liquid 2YT medium, followed by the addition of 0.0625 mM 1-HP and incubated for an additional 2 h. Mycelia were harvested, total RNA isolated and subjected to Northern blot analysis with the indicated probes. Ribosomal RNA (rRNA) is shown as a loading and quality control. Control represents phenazine-untreated mycelia. Full-length blots/gels are presented in the Supplementary Figure 4 (4A – RNA-gels and 4B – Northern blots; all the gels were run under the same experimental condition).

Figure 7. 1-HP chelates iron.

(A) Interaction of 1-HP with iron (FeCl₃) in absence or presence of EDTA, showing an immediate change yellow colored 1-HP in the presence of iron and absence of EDTA, and flocculation of the [1-HP-iron] complex after 24 h incubation. (B) λ_560 nm titration curve of the [1-HP-iron] complex in presence of increasing concentrations of FeCl₃. (C) LC-MS analysis of the [1-HP-iron] flocculation product. (D) Deduced structures of the complexes formed by 1-HP and FeCl₃ based on LC-MS analysis, compared to the exact mass and structure of 1-HP.

Figure 8. 1-HP-iron complex is a quasi-irreversible redox system.

(A) Cyclic voltammetry at 100 mV/s on a gold disc electrode with 1 mM of PYO (red), 1 mM of FeCl₃ (green) and a mixture of PYO-FeCl₃ in the ratio (1:0.1) in violet, (1:0.5) in blue and (1:1.5) in orange. (B) Cyclic voltammetry at 100 mV/s on gold disc electrode with 1 mM of 1-HP (red), 1 mM of FeCl₃ (green) and a mixture of 1HP-FeCl₃ (1:1) (blue). The arrow indicates the direction from initial potential.

Figure 9. Growth of A. fumigatus ΔsidA is stimulated by low concentration of phenazines.

() ΔsidA and () ΔsidAΔftrA mutants incubated in presence of increased concentration of phenazines. (– –) ΔsidA incubated in absence of phenazines. The growth was quantified at absorbance 560 nm following the crystal violet procedure.

Figure 10. Model for phenazines mode of action against A. fumigatus.

The four major phenazines of P. aeruginosa bind to hyphae and penetrate the cell. Few PCA penetrates due its negative charge (Lime green pathway,). Phenazines act at the level of mitochondria and induce the production of superoxide anion (O₂^·−) and peroxynitrite (ONOO⁻) (Red pathway,). MnSod2p enzyme converts O₂^·− to hydrogen peroxide (H₂O₂) and a catalase-independent mechanism allows its detoxification into H₂O. The anion ONOO⁻ formed from O₂^·− and nitric oxide (NO^·) is also detoxified by MnSod2p (Red pathway,). PYO, PCN and PCA reduce Fe(III) to Fe(II) which penetrates the A. fumigatus cell through the iron ferroxidase FetCp/permease FtrAp complex (Green pathway,). 1-HP also reduces Fe(III) to Fe(II) and two 1-HP molecules can chelate the newly formed Fe(II) (Orange pathway,). This chelating activity induces iron starvation which causes HapXp activation. The biosynthetic pathway of triacetylfusarinine C (TAFC) is then activated and allows Fe³⁺ acquisition to stimulate the growth of the fungus (Orange pathway,).