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C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

Blier AS, Veron W, Bazire A, Gerault E, Taupin L, Vieillard J, Rehel K, Dufour A, Le Derf F, Orange N, Hulen C, Feuilloley MG, Lesouhaitier O - Microbiology (Reading, Engl.) (2011)

Bottom Line: The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment.These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP.Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cold Microbiology - Signals and Micro-environment EA 4312, University of Rouen, 55 Rue Saint Germain, 27000 Evreux, France.

ABSTRACT
Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

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Schematic model representing the mechanism of action of natriuretic peptides on P. aeruginosa PAO1. Activation of the membrane natriuretic peptides sensor (NPS) by CNP induces a rise in intra-bacterial cAMP concentration through adenylate cyclase (AC) activation (Veron et al., 2007). Then, the global regulator Vfr, activated by cAMP, regulates directly or indirectly (after PtxR activation) the expression of several virulence factors such as ETA, HCN and pyocyanin through the modulation of QS signalling molecules. Finally, CNP binding on P. aeruginosa PAO1 increases global bacteria virulence.
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f10: Schematic model representing the mechanism of action of natriuretic peptides on P. aeruginosa PAO1. Activation of the membrane natriuretic peptides sensor (NPS) by CNP induces a rise in intra-bacterial cAMP concentration through adenylate cyclase (AC) activation (Veron et al., 2007). Then, the global regulator Vfr, activated by cAMP, regulates directly or indirectly (after PtxR activation) the expression of several virulence factors such as ETA, HCN and pyocyanin through the modulation of QS signalling molecules. Finally, CNP binding on P. aeruginosa PAO1 increases global bacteria virulence.

Mentions: In order to go further into the mechanism of action of natriuretic peptides in P. aeruginosa we decided to investigate the transcriptional regulator(s) potentially involved in the effect of CNP. In P. aeruginosa, numerous proteins such as Vfr, GacA, MvaT, QscR, PtxR, RsaL, VqsM and VqsR regulate (positively or negatively) the expression of QS genes (Albus et al., 1997; Chugani et al., 2001; Diggle et al., 2002; Dong et al., 2005; Juhas et al., 2004; Reimmann et al., 1997; Schuster & Greenberg, 2006). ETA synthesis itself is controlled both by a cascade of positive regulators, including PvdS and RegA (ToxR) protein (Hamood et al., 2004; Ochsner et al., 1996) or the PtxR regulator (Hamood et al., 1996), and also by direct binding of the PAO1 global virulence regulator Vfr on the toxA promoter region (Davinic et al., 2009). We have demonstrated previously that the Vfr protein is involved in the effect of natriuretic peptides on P. aeruginosa PAO1 virulence (Veron et al., 2007). We observed here that Vfr is not only essential for expression of the lethal effect of PAO1 on C. elegans in both fast and slow killing conditions but also involved in the pro-virulent effect of BNP and CNP. Since the PtxR regulator, which regulates ETA production in PAO1 (Hamood et al., 1996), is under the control of Vfr (Ferrell et al., 2008), and ETA synthesis is promoted by CNP (and BNP) we naturally examined the role of PtxR in CNP action. We observed that CNP induced a 3.5-fold increase of ptxR mRNA synthesis 1 h after its contact with PAO1, suggesting that PtxR is involved in the effect of CNP on PAO1. Moreover, the observation that PtxR negatively regulates the expression of pyocyanin while it positively regulates the production of 3OC12-HSL through lasI (Carty et al., 2006) strongly reinforces our hypothesis that PtxR is a member of the transduction cascade triggered by CNP in PAO1 (Fig. 10). However, PtxR appears to regulate only a part of the effect of CNP, since a ptxR mutation does not lead to a total suppression of bacterial virulence as observed with the vfr mutant, and whereas PtxR is necessary for the expression of the slow-killing activity of PAO1, this protein is not required to kill C. elegans in the fast-killing test (data not shown). We know that Vfr is involved in LPS structural rearrangements induced by CNP (Veron et al., 2007) and an algC mutant is found to be avirulent, an effect which has been specifically ascribed to LPS biosynthesis modifications (Goldberg et al., 1995). In the present study, we observed that not only is an algC mutant less virulent towards C. elegans compared with the wild-type but also CNP pro-virulent activity could be assigned to LPS through algC since CNP enhanced the algC mRNA level in PAO1. It is interesting to note that although the NF-κB signalling pathway is not functional in the immune system of C. elegans (Wang et al., 2006) and although this worm possesses a single Toll-like receptor homologue (Pujol et al., 2001), this worm could be employed as a model to study pathogenesis supported by LPS.


C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

Blier AS, Veron W, Bazire A, Gerault E, Taupin L, Vieillard J, Rehel K, Dufour A, Le Derf F, Orange N, Hulen C, Feuilloley MG, Lesouhaitier O - Microbiology (Reading, Engl.) (2011)

Schematic model representing the mechanism of action of natriuretic peptides on P. aeruginosa PAO1. Activation of the membrane natriuretic peptides sensor (NPS) by CNP induces a rise in intra-bacterial cAMP concentration through adenylate cyclase (AC) activation (Veron et al., 2007). Then, the global regulator Vfr, activated by cAMP, regulates directly or indirectly (after PtxR activation) the expression of several virulence factors such as ETA, HCN and pyocyanin through the modulation of QS signalling molecules. Finally, CNP binding on P. aeruginosa PAO1 increases global bacteria virulence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755537&req=5

f10: Schematic model representing the mechanism of action of natriuretic peptides on P. aeruginosa PAO1. Activation of the membrane natriuretic peptides sensor (NPS) by CNP induces a rise in intra-bacterial cAMP concentration through adenylate cyclase (AC) activation (Veron et al., 2007). Then, the global regulator Vfr, activated by cAMP, regulates directly or indirectly (after PtxR activation) the expression of several virulence factors such as ETA, HCN and pyocyanin through the modulation of QS signalling molecules. Finally, CNP binding on P. aeruginosa PAO1 increases global bacteria virulence.
Mentions: In order to go further into the mechanism of action of natriuretic peptides in P. aeruginosa we decided to investigate the transcriptional regulator(s) potentially involved in the effect of CNP. In P. aeruginosa, numerous proteins such as Vfr, GacA, MvaT, QscR, PtxR, RsaL, VqsM and VqsR regulate (positively or negatively) the expression of QS genes (Albus et al., 1997; Chugani et al., 2001; Diggle et al., 2002; Dong et al., 2005; Juhas et al., 2004; Reimmann et al., 1997; Schuster & Greenberg, 2006). ETA synthesis itself is controlled both by a cascade of positive regulators, including PvdS and RegA (ToxR) protein (Hamood et al., 2004; Ochsner et al., 1996) or the PtxR regulator (Hamood et al., 1996), and also by direct binding of the PAO1 global virulence regulator Vfr on the toxA promoter region (Davinic et al., 2009). We have demonstrated previously that the Vfr protein is involved in the effect of natriuretic peptides on P. aeruginosa PAO1 virulence (Veron et al., 2007). We observed here that Vfr is not only essential for expression of the lethal effect of PAO1 on C. elegans in both fast and slow killing conditions but also involved in the pro-virulent effect of BNP and CNP. Since the PtxR regulator, which regulates ETA production in PAO1 (Hamood et al., 1996), is under the control of Vfr (Ferrell et al., 2008), and ETA synthesis is promoted by CNP (and BNP) we naturally examined the role of PtxR in CNP action. We observed that CNP induced a 3.5-fold increase of ptxR mRNA synthesis 1 h after its contact with PAO1, suggesting that PtxR is involved in the effect of CNP on PAO1. Moreover, the observation that PtxR negatively regulates the expression of pyocyanin while it positively regulates the production of 3OC12-HSL through lasI (Carty et al., 2006) strongly reinforces our hypothesis that PtxR is a member of the transduction cascade triggered by CNP in PAO1 (Fig. 10). However, PtxR appears to regulate only a part of the effect of CNP, since a ptxR mutation does not lead to a total suppression of bacterial virulence as observed with the vfr mutant, and whereas PtxR is necessary for the expression of the slow-killing activity of PAO1, this protein is not required to kill C. elegans in the fast-killing test (data not shown). We know that Vfr is involved in LPS structural rearrangements induced by CNP (Veron et al., 2007) and an algC mutant is found to be avirulent, an effect which has been specifically ascribed to LPS biosynthesis modifications (Goldberg et al., 1995). In the present study, we observed that not only is an algC mutant less virulent towards C. elegans compared with the wild-type but also CNP pro-virulent activity could be assigned to LPS through algC since CNP enhanced the algC mRNA level in PAO1. It is interesting to note that although the NF-κB signalling pathway is not functional in the immune system of C. elegans (Wang et al., 2006) and although this worm possesses a single Toll-like receptor homologue (Pujol et al., 2001), this worm could be employed as a model to study pathogenesis supported by LPS.

Bottom Line: The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment.These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP.Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cold Microbiology - Signals and Micro-environment EA 4312, University of Rouen, 55 Rue Saint Germain, 27000 Evreux, France.

ABSTRACT
Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

Show MeSH
Related in: MedlinePlus