Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition

Abstract

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels of iron in excess of what is required for planktonic growth have been shown to promote biofilm formation, and therapies that interfere with ferric iron [Fe(III)] uptake combined with antibiotics may help treat P. aeruginosa infections. However, use of these therapies presumes that iron is in the Fe(III) state in the context of infection. Here we report the ability of phenazine-1-carboxylic acid (PCA), a common phenazine made by all phenazine-producing pseudomonads, to help P. aeruginosa alleviate Fe(III) limitation by reducing Fe(III) to ferrous iron [Fe(II)]. In the presence of PCA, a P. aeruginosa mutant lacking the ability to produce the siderophores pyoverdine and pyochelin can still develop into a biofilm. As has been previously reported (P. K. Singh, M. R. Parsek, E. P. Greenberg, and M. J. Welsh, Nature 417:552-555, 2002), biofilm formation by the wild type is blocked by subinhibitory concentrations of the Fe(III)-binding innate-immunity protein conalbumin, but here we show that this blockage can be rescued by PCA. FeoB, an Fe(II) uptake protein, is required for PCA to enable this rescue. Unlike PCA, the phenazine pyocyanin (PYO) can facilitate biofilm formation via an iron-independent pathway. While siderophore-mediated Fe(III) uptake is undoubtedly important at early stages of infection, these results suggest that at later stages of infection, PCA present in infected tissues may shift the redox equilibrium between Fe(III) and Fe(II), thereby making iron more bioavailable.

Fig. 1.

The phenazine PCA can work together with the siderophore pyoverdine in promoting P. aeruginosa biofilm formation. (A) Confocal microscopic images of biofilms at day 4 (A), total biomass over time as inferred by Auto-COMSTAT analysis (B), and the corresponding release of phenazine(s) and/or siderophore(s) into the biofilm effluents for the YFP-labeled PA14 wild type (WT), the phenazine-null strain (Δphz), the siderophore-null strain (ΔpvdA ΔpchE), and the phenazine-siderophore-null strain (Δphz ΔpvdA ΔpchE) under a flow of 1% TSB medium at 22°C (C) are shown. Confocal images consist of top-down views (x-y plane, top images) and side views (x-z plane, bottom images, enlarged and truncated to emphasize differences in the z dimension). Results are representative of six experiments. Data reported in panels B and C represent means ± standard errors of the means (SEMs). Related quantitative data can be found in Table 3. Scale bars, 100 μm for images with a top-down view and 50 μm for side-view images.

Fig. 2.

PCA and PYO can circumvent the siderophore pathway for promoting P. aeruginosa biofilm development via Fe(II) uptake-dependent and -independent mechanisms, respectively. The effectively insoluble Fe(III) mineral ferrihydrite [Fe(OH)₃(s)] was the Fe(III) source. Confocal microscopic images of the YFP-labeled P. aeruginosa PA14 siderophore-null strain (ΔpvdA ΔpchE) incubated in biofilm flow cells at 22°C for 6 days with no additions or with the addition of 1.0 μM Fe(OH)₃(s), 10 μM phenazine (PCA or PYO), or 1.0 μM Fe(OH)₃(s) together with 10 μM phenazine (PCA or PYO) to 1% TSB medium. Images are top-down views (x-y plane). Results are representative of four experiments. Related quantitative data can be found in Table 4. Scale bar, 100 μm.

Fig. 3.

The P. aeruginosa PA14 feoB::MAR2xT7 mutant (feoB::tn) cannot grow when given Fe(II) as its sole iron source, yet it can grow in the presence of Fe(III); the wild type (WT) can grow regardless of whether iron is in the ferric or ferrous form. Cells were incubated, with anaerobic shaking, in Amberlite-treated 1% TSB medium containing100 mM KNO₃, 50 mM glutamate, 1% glycerol, and 100 μM iron source [either (NH₄)₂Fe(II)(SO₄)₂ or Fe(III)Cl₃] at 37°C for 22 h. Data reported are the means of triplicate experiments ± standard deviations (SDs).

Fig. 4.

(A) The presence of subinhibitory levels of conalbumin alone or together with phenazine (PCA or PYO) does not inhibit planktonic growth of P. aeruginosa PA14 strains. Experiments were performed in batch cultures in 1% TSB-based biofilm medium at 37°C. Data reported are the means of triplicate experiments ± SDs. (B) PCA but not PYO can rescue the conalbumin-induced P. aeruginosa biofilm defect by reducing protein-sequestered Fe(III) with concomitant release of Fe(II). Shown are confocal microscopic images of YFP-labeled P. aeruginosa PA14 wild type (WT) and DIC microscopic images of the P. aeruginosa PA14 feoB::MAR2xT7 mutant (feoB::tn) disrupted in Fe²⁺ transport into the cytoplasm and incubated in biofilm flow cells at 22°C for 6 days, with no addition or with the addition of 40 μg/ml of iron-free conalbumin alone or together with 10 μM phenazine (PCA or PYO) to 1% TSB medium. Confocal images consist of top-down views (x-y plane, top images) and side views (x-z plane, bottom images, enlarged and truncated to emphasize differences in the z dimension). DIC images are top-down views (x-y plane). Results are representative of four experiments. Related quantitative data can be found in Table 5. Scale bars, 100 μm for images with top-down views and 50 μm for side-view images.

Fig. 5.

A highly simplified schematic view of P. aeruginosa iron transport via phenazine-facilitated Fe(II) uptake (right), siderophore-mediated Fe(III) uptake (middle), and heme uptake (left). In Fe(III) uptake, a siderophore (e.g., pyoverdine, indicated by the star) binds extracellular Fe(III) and crosses the outer membrane (OM) via a TonB-dependent transporter (53, 64). In the periplasm, Fe(III) is released from the siderophore, which can then be recycled; Fe(III) is reduced by an unknown mechanism to Fe(II) in the periplasm (P) and transported across the cytoplasmic membrane (CM), presumably by an ABC transport system (53, 64). By contrast, phenazines can reduce extracellular Fe(III) to Fe(II). After entering the periplasm, presumably via an OM porin, Fe(II) is transported across the CM via FeoB (11, 40). Phenazines themselves are recycled (27, 73) and enter and leave the cell through various transporters (not drawn, for simplicity). Intracellularly, phenazine reduction is coupled to NADH oxidation to NAD⁺ (55), although whether this reduction is enzyme mediated is unknown. Reduced phenazine is indicated by the open oval and oxidized phenazine by the filled oval. Additionally, P. aeruginosa can acquire iron from heme and heme-containing proteins (e.g., hemoglobin), which are transported through a specific outer membrane receptor channel in a TonB-dependent manner (31, 48, 72). In P. aeruginosa, two distinct heme uptake systems have been found, although the mechanistic details of heme capture and delivery and the fate of heme after entering the cytoplasm are not fully understood (31, 48).