A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation

Abstract

Exopolysaccharide Psl is a critical biofilm matrix component in Pseudomonas aeruginosa, which forms a fiber-like matrix to enmesh bacterial communities. Iron is important for P. aeruginosa biofilm development, yet it is not clearly understood how iron contributes to biofilm development. Here, we showed that iron promoted biofilm formation via elevating Psl production in P. aeruginosa The high level of iron stimulated the synthesis of Psl by reducing rhamnolipid biosynthesis and inhibiting the expression of AmrZ, a repressor of psl genes. Iron-stimulated Psl biosynthesis and biofilm formation held true in mucoid P. aeruginosa strains. Subsequent experiments indicated that iron bound with Psl in vitro and in biofilms, which suggested that Psl fibers functioned as an iron storage channel in P. aeruginosa biofilms. Moreover, among three matrix exopolysaccharides of P. aeruginosa, Psl is the only exopolysaccharide that can bind with both ferrous and ferric ion, yet with higher affinity for ferrous iron. Our data suggest a survival strategy of P. aeruginosa that uses exopolysaccharide to sequester and store iron to stimulate Psl-dependent biofilm formation.

Importance: Pseudomonas aeruginosa is an environmental microorganism which is also an opportunistic pathogen that can cause severe infections in immunocompromised individuals. It is the predominant airway pathogen causing morbidity and mortality in individuals affected by the genetic disease cystic fibrosis (CF). Increased airway iron and biofilm formation have been proposed to be the potential factors involved in the persistence of P. aeruginosa in CF patients. Here, we showed that a high level of iron enhanced the production of the key biofilm matrix exopolysaccharide Psl to stimulate Psl-dependent biofilm formation. Our results not only make the link between biofilm formation and iron concentration in CF, but also could guide the administration or use of iron chelators to interfere with biofilm formation in P. aeruginosa in CF patients. Furthermore, our data also imply a survival strategy of P. aeruginosa under high-iron environmental conditions.

FIG 1

Psl production of PAO1 and PAO1 ΔpvdS. A representative immune-dot blotting result is shown below each corresponding column. The Psl synthesized by PAO1 ΔpvdS is significantly lower than that of PAO1 (P < 0.01).

FIG 2

Iron promotes biofilm formation via elevating the Psl level. (A) Iron stimulated the Psl-mediated biofilm formation in P. aeruginosa PAO1. PAO1 ΔpelA, Δpsl mutant strains, and PA14 were used for comparison. (B) Psl production of PAO1 and PAO1 pelA mutant at different iron levels. The amount of Psl was quantified by anti-Psl immunoblotting. Con., concentration. (C) The effect of iron on the production of Psl in nonmucoid strain PAO1 and mucoid strains. The amount of Psl was quantified by anti-Psl immunoblotting and has normalized to the corresponding Psl level under the 2 μM iron condition (for PAO1, 1 = 36 μg/ml; for PDO300, 1 = 15 μg/ml; for FRD1, 1 = 12 μg/ml). (D) The CLSM images of PAO1 biofilms grown under iron-depleted (2 μM) and -replete (100 μM) conditions. Biofilm bacteria were stained by SYTO9 (green fluorescence) and their Psl matrix was stained by lectin HHA-TRITC (red fluorescence). The selected horizontal sectioned images (square) and vertical sectioned images are shown. The biomass was indicated on the corresponding image. (E) Comparison of bacterial and Psl biomass. Shown are the ratio of Psl to biomass and the maximum (Maxim) thickness of corresponding biofilms (shown in panel D) at two iron concentrations. (F) Iron stimulated the biofilm formation of mucoid strains. Shown are the biofilm biomass of PAO1-derived mucoid strain PDO300 and mucoid CF isolate FRD1 grown under different iron conditions. **, P < 0.01.

FIG 3

Iron influences on the transcription of psl and amrZ. (A) β-Galactosidase (β-gal) assays for transcriptional pslA-lacZ fusion at different iron levels. Shown are the results detected at early stationary phase (24 h) of bacterial growth. (B) β-Galactosidase assays for translational fusion of amrZ-lacZ at different iron levels on early stationary phase (24 h). (C) Relative amrZ expression of the stationary cultures of PAO1, PDO300, and FRD1 grown at different iron levels. Shown are the results of RT-PCR. The PAO1 level at 2 μM iron was used as a control. **, P < 0.01; *, P < 0.05.

FIG 4

High iron concentration impacts rhamnolipid production and the transcription of rhl genes. (A) Swarming motility of PAO1 at different iron levels. The swarming zone is indicated under each corresponding image. 0, no visible swarming zone. (B) Rhamnolipid production of PAO1 at different iron levels. (C) β-Galactosidase assays for transcriptional fusions of lasR-lacZ, lasI-lacZ, rhlAB-lacZ, rhlR-lacZ, and rhlI-lacZ at different iron levels. **, P < 0.01.

FIG 5

The colocalization analysis of Psl matrix and iron in biofilms. (A) Pellicle biofilms after 1 day of growth under iron-depleted (2 μM) and -replete (100 μM) conditions were observed and imaged by CLSM. The representative images are shown. A highly selective and sensitive iron-specific fluorescent probe (red fluorescence) was used to label and locate the iron distribution in P. aeruginosa biofilms. Lectin FITC-HHA was used to stain Psl (green fluorescence). The deviation map clearly shows that iron was colocalized with Psl in P. aeruginosa biofilms. (B) The scatterplot analysis of fluorescent signals from the corresponding biofilm images shown above to evaluate the colocalization of iron and the Psl matrix. The scatterplot was created by defining the Psl signal as the x axis and the Fe³⁺ signal as the y axis. The information from the images was transformed to a plot chart to reveal the relationship between the two images. The deviation map shown above was created by the data set calculated from the corresponding scatterplot.

FIG 6

Iron can bind to Psl in vitro. (A) Psl binds iron in a dose-dependent manner. Different concentrations of purified Psl were mixed with a 100 μM iron solution. Ferrozine was used as an indicator to determine free iron. (B) Cellulase-treated Psl significantly reduced the iron-binding activity of Psl. Cellulase (4 mg/ml) was incubated with 100 μg/ml purified Psl for 24 h at 25°C prior to mixing with iron solution. The P. aeruginosa genomic DNA was used as a control to test the binding activity. **, P < 0.01.

FIG 7

Comparison of iron-binding ability of exopolysaccharides produced in P. aeruginosa. (A) The binding ability of Psl, Pel, and alginate to ferric (Fe³⁺) and ferrous (Fe²⁺) iron. (B) The interaction of Psl with Fe²⁺ examined by ITC. (C) The ITC examination of the interaction between Psl and Fe³⁺.

FIG 8

Reutilization of iron stored by Psl and a schematic summary. (A) The optical density at 600 nm (OD₆₀₀) of PAO1 after 24 h of growth in M9 medium with different concentrations of iron or Psl prepared from the P. aeruginosa strain WFPA801 grown at 100 μM and 2 μM iron (named Psl¹⁰⁰ and Psl², respectively). (B) A schematic shows the mechanisms of the iron-Psl interplay in P. aeruginosa. Iron ions (both Fe³⁺ and Fe²⁺) are sequestered and stored with exopolysaccharide Psl. Iron can stimulate Psl production by repressing AmrZ expression and reducing rhamnolipid synthesis, which enhances biofilm formation.