Cell aggregation promotes pyoverdine-dependent iron uptake and virulence in Pseudomonas aeruginosa

Abstract

In Pseudomonas aeruginosa the Gac signaling system and the second messenger cyclic diguanylate (c-di-GMP) participate in the control of the switch between planktonic and biofilm lifestyles, by regulating the production of the two exopolysaccharides Pel and Psl. The Gac and c-di-GMP regulatory networks also coordinately promote the production of the pyoverdine siderophore, and the extracellular polysaccharides Pel and Psl have recently been found to mediate c-di-GMP-dependent regulation of pyoverdine genes. Here we demonstrate that Pel and Psl are also essential for Gac-mediated activation of pyoverdine production. A pel psl double mutant produces very low levels of pyoverdine and shows a marked reduction in the expression of the pyoverdine-dependent virulence factors exotoxin A and PrpL protease. While the exopolysaccharide-proficient parent strain forms multicellular planktonic aggregates in liquid cultures, the Pel and Psl-deficient mutant mainly grows as dispersed cells. Notably, artificially induced cell aggregation is able to restore pyoverdine-dependent gene expression in the pel psl mutant, in a way that appears to be independent of iron diffusion or siderophore signaling, as well as of recently described contact-dependent mechanosensitive systems. This study demonstrates that cell aggregation represents an important cue triggering the expression of pyoverdine-related genes in P. aeruginosa, suggesting a novel link between virulence gene expression, cell-cell interaction and the multicellular community lifestyle.

Keywords: Pseudomonas aeruginosa; cell aggregates; extracellular polysaccharide; gene regulation; iron uptake; mechanosensor; siderophore; virulence.

FIGURE 1

The exopolysaccharides Pel and Psl are essential for Gac-mediated control on pyoverdine production and positively regulate pyoverdine-dependent virulence factors. (A) Pyoverdine production by Pseudomonas aeruginosa PAO1, pel, psl, and pel psl mutants, and their derivatives deleted in the rsmA or in the rsmY and rsmZ genes. (B) Intracellular levels of c-di-GMP (relative to wild type PAO1) in P. aeruginosa PAO1, the pel psl mutant, and their derivatives deleted in the rsmA or the rsmY and rsmZ genes. (C) Relative mRNA levels of pvdD, toxA, and prpL as determined by qRT-PCR, and (D) PrpL enzymatic activity in culture supernatants of P. aeruginosa PAO1 and the pel psl mutant. (E) Western-blot showing ToxA levels in culture supernatants of P. aeruginosa PAO1, the pel psl mutant, and the toxA mutant (used as negative control). Bacteria were grown in TSBD at 37°C under static conditions for 14 h. Values in (A–D) are the mean (±SD) of at least three independent assays. Asterisks indicate statistically significant differences with respect to the corresponding parental strain (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). The image in panel (E) is representative of four independent experiments giving similar results.

FIGURE 2

Fur and pyoverdine signaling are not involved in exopolysaccharide-mediated pyoverdine regulation. Pyoverdine production in (A) P. aeruginosa PAO1, the pel psl mutant, and their derivatives in which the native fur gene was replaced by an arabinose-inducible allele (P_BADfur), and (B) P. aeruginosa PAO1 fpvR and pel psl fpvR mutants. Bacteria were cultured in TSBD without arabinose at 37°C under static conditions for 14 h, and values are the mean (±SD) of at least three independent assays. Asterisks indicate statistically significant differences with respect to the corresponding parental strain (^∗∗∗p < 0.001).

FIGURE 3

Role of Pel and Psl exopolysaccharides in planktonic aggregation of P. aeruginosa cells. (A) Confocal microscopy images of P. aeruginosa PAO1 and pel psl cells harboring the GFP-expressing vector pMMG cultured in TSBD at 37°C under static conditions for 14 h. The images are representative of several micrographs from five independent experiments. Bar: 50 μm. (B) Representative laser diffraction analysis (LDA) particle-size scans of P. aeruginosa PAO1 and pel psl liquid cultures in TSBD after 14 h of growth at 37°C.

FIGURE 4

Cell aggregation is involved in Pel and Psl-mediated control of pyoverdine-dependent virulence factors. (A) Fluorescent phenotype upon UV light exposure of P. aeruginosa PAO1, the pel psl mutant and the pvdA mutant (used as pyoverdine-deficient negative control) grown in liquid TSBD medium (Control) or on TSBD solidified with 1.5% agar, 1% phytagel, or 10% polyacrylamide. (B) Pyoverdine production by P. aeruginosa PAO1 and the pel psl mutant grown in TSBD supplemented with increasing concentrations of agar (0–0.2%) and/or β-agarase I (3.3 units/ml). (C) Representative LDA particle-size scans of P. aeruginosa PAO1 and pel psl cultures in TSBD supplemented with 0.2% agar after 14 h of growth at 37°C. (D) Activity of the PpvdD::lacZ and PproC’-‘lacZ reporter fusions, (E) PrpL enzymatic activity and (F) ToxA levels in culture supernatants from P. aeruginosa PAO1 and pel psl cultures in TSBD supplemented or not with 0.2% agar. Bacteria were cultured in TSBD at 37°C under static conditions for 14 h. (G) Activity of the PpvdD::lacZ transcriptional fusion in P. aeruginosa PAO1 and pel psl cells cultured on TSBD solidified with 10% polyacrylamide. Values in (B,D,E,G) are the mean (±SD) of at least three independent assays. Asterisks indicate statistically significant differences with respect to the wild type strain (PAO1) grown under the same culture conditions (^∗∗p < 0.01, ^∗∗∗p < 0.001). Images in (A,F) are representative of two independent experiments giving similar results.

FIGURE 5

Aggregation does not stimulate pyoverdine production thorough non-specific physical contacts or the mechanosensors PilY1 and type IV pili. (A) Confocal microscopy images of P. aeruginosa PAO1 and pel psl cells harboring the GFP-expressing vector pMMG cultured in TSBD in the presence of 5 × 10⁸ polystyrene beads/ml. Images are representative of several micrographs showing similar results. Bar: 30 μm. The inset is a 2× magnification of the highlighted area. (B) Pyoverdine production by P. aeruginosa PAO1 and pel psl in the presence or in the absence of 3-μm size polystyrene beads (5 × 10⁷ or 5 × 10⁸ beads/ml), normalized to the number of colony forming units/ml and expressed as percentage with respect to the untreated wild type. Bacteria were grown in TSBD at 37°C for 14 h under vigorous shaking (220 rpm). (C) Pyoverdine production by P. aeruginosa PAO1, pel psl and their corresponding pilY1 or pilA deletion mutants after 14 h of growth in TSBD at 37°C under static conditions. Values are the mean (±SD) of at least three independent assays. Asterisks indicate statistically significant differences with respect to the corresponding parental strain grown under the same culture conditions (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).