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

Visaggio D, Pasqua M, Bonchi C, Kaever V, Visca P, Imperi F - Front Microbiol (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome Rome, Italy ; Department of Sciences, Universita degli Studi Roma Tre Rome, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: In order to verify whether the Pel and Psl exopolysaccharides are also involved in the Gac-mediated control of pyoverdine gene expression, we generated single and double deletion mutants in pel and psl genes in P. aeruginosa PAO1 (wild-type) and in isogenic mutants in which the Gac signaling is inactive (rsmY rsmZ mutant) or constitutively active (rsmA mutant; Supplementary Table S1). In agreement with previous observations (Frangipani et al., 2014), pyoverdine production in the iron-poor TSBD medium was higher in the rsmA mutant and strongly reduced in the rsmY rsmZ mutant compared with the wild type (WT; Figure 1A). This pyoverdine production profile was maintained in strains lacking either Pel or Psl, while pyoverdine production was drastically reduced in the pel psl double mutant irrespective of the activation state of the Gac system (Figure 1A). This response appeared to be independent of the previously reported effect of the Gac system on c-di-GMP production (Moscoso et al., 2011; Frangipani et al., 2014), since mass spectrometry analysis revealed that the Gac system is still able to control intracellular c-di-GMP levels in a Pel/Psl deficient background (Figure 1B). Thus, both the Gac system (Figure 1A) and the c-di-GMP second messenger (Chen et al., 2015) require at least one of two exopolysaccharides Pel and Psl to exert their control on pyoverdine biosynthesis, indicating that these exopolysaccharides, or an exopolysaccharide-dependent phenotype, are implicated in pyoverdine gene regulation. Indeed, we observed that the expression of the pyoverdine biosynthetic gene pvdD was greatly reduced in the pel psl mutant relative to the WT, as well as that of the pyoverdine-dependent virulence genes toxA and prpL (Figure 1C). Accordingly, the levels of the corresponding virulence factors in culture supernatants were significantly lower in the pel psl double mutant as compared to the WT strain (Figures 1D,E), as also observed for pyoverdine (Figure 1A). Therefore, lack of the Pel and Psl exopolysaccharides in P. aeruginosa causes a significant reduction not only in the production of the siderophore pyoverdine but also of pyoverdine-dependent virulence factors.


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

Visaggio D, Pasqua M, Bonchi C, Kaever V, Visca P, Imperi F - Front Microbiol (2015)

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.
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Related In: Results  -  Collection

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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.
Mentions: In order to verify whether the Pel and Psl exopolysaccharides are also involved in the Gac-mediated control of pyoverdine gene expression, we generated single and double deletion mutants in pel and psl genes in P. aeruginosa PAO1 (wild-type) and in isogenic mutants in which the Gac signaling is inactive (rsmY rsmZ mutant) or constitutively active (rsmA mutant; Supplementary Table S1). In agreement with previous observations (Frangipani et al., 2014), pyoverdine production in the iron-poor TSBD medium was higher in the rsmA mutant and strongly reduced in the rsmY rsmZ mutant compared with the wild type (WT; Figure 1A). This pyoverdine production profile was maintained in strains lacking either Pel or Psl, while pyoverdine production was drastically reduced in the pel psl double mutant irrespective of the activation state of the Gac system (Figure 1A). This response appeared to be independent of the previously reported effect of the Gac system on c-di-GMP production (Moscoso et al., 2011; Frangipani et al., 2014), since mass spectrometry analysis revealed that the Gac system is still able to control intracellular c-di-GMP levels in a Pel/Psl deficient background (Figure 1B). Thus, both the Gac system (Figure 1A) and the c-di-GMP second messenger (Chen et al., 2015) require at least one of two exopolysaccharides Pel and Psl to exert their control on pyoverdine biosynthesis, indicating that these exopolysaccharides, or an exopolysaccharide-dependent phenotype, are implicated in pyoverdine gene regulation. Indeed, we observed that the expression of the pyoverdine biosynthetic gene pvdD was greatly reduced in the pel psl mutant relative to the WT, as well as that of the pyoverdine-dependent virulence genes toxA and prpL (Figure 1C). Accordingly, the levels of the corresponding virulence factors in culture supernatants were significantly lower in the pel psl double mutant as compared to the WT strain (Figures 1D,E), as also observed for pyoverdine (Figure 1A). Therefore, lack of the Pel and Psl exopolysaccharides in P. aeruginosa causes a significant reduction not only in the production of the siderophore pyoverdine but also of pyoverdine-dependent virulence factors.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome Rome, Italy ; Department of Sciences, Universita degli Studi Roma Tre Rome, Italy.

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.

No MeSH data available.


Related in: MedlinePlus