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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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Effect of BNP and CNP on pyocyanin, PQS, HHQ and HNQ production by P. aeruginosa PAO1. (a) P. aeruginosa PAO1 pyocyanin production in response to natriuretic peptides (BNP and CNP). Data are the mean±sem of seven values obtained from three independent experiments. The mean pyocyanin level in the control was 0.0071±0.0016 OD520/OD580. Significant difference is indicated by asterisks (***P<0.001); ns, not significantly different. (b) Expression levels of phzC1 in PAO1-treated bacteria relative to those in the PAO1 control. RNA was extracted 1 h after PAO1 exposure to physiological water or natriuretic peptides (BNP and CNP) and assayed by qRT-PCR. See the legend to Fig. 2 for further information. (c, d) Relative amounts of PQS and HHQ in P. aeruginosa PAO1 supernatant 3 h after exposure of PAO1 to physiological water or CNP. Data are the mean±sd of three independent experiments. The mean PQS and HHQ concentration in control conditions were 741×103 Area/OD580 and 1410×103 Area/OD580, respectively. *P<0.05; ns, not significantly different. (e) Relative amounts of HNQ in P. aeruginosa PAO1 supernatant, 3 h after exposure of PAO1 to physiological water or CNP. Data are the means of three independent experiments. The mean HNQ concentration in the control was 1520×103 Area/OD580. *P<0.05. 1, Control; 2, BNP-treated PAO1; 3, CNP-treated PAO1.
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f3: Effect of BNP and CNP on pyocyanin, PQS, HHQ and HNQ production by P. aeruginosa PAO1. (a) P. aeruginosa PAO1 pyocyanin production in response to natriuretic peptides (BNP and CNP). Data are the mean±sem of seven values obtained from three independent experiments. The mean pyocyanin level in the control was 0.0071±0.0016 OD520/OD580. Significant difference is indicated by asterisks (***P<0.001); ns, not significantly different. (b) Expression levels of phzC1 in PAO1-treated bacteria relative to those in the PAO1 control. RNA was extracted 1 h after PAO1 exposure to physiological water or natriuretic peptides (BNP and CNP) and assayed by qRT-PCR. See the legend to Fig. 2 for further information. (c, d) Relative amounts of PQS and HHQ in P. aeruginosa PAO1 supernatant 3 h after exposure of PAO1 to physiological water or CNP. Data are the mean±sd of three independent experiments. The mean PQS and HHQ concentration in control conditions were 741×103 Area/OD580 and 1410×103 Area/OD580, respectively. *P<0.05; ns, not significantly different. (e) Relative amounts of HNQ in P. aeruginosa PAO1 supernatant, 3 h after exposure of PAO1 to physiological water or CNP. Data are the means of three independent experiments. The mean HNQ concentration in the control was 1520×103 Area/OD580. *P<0.05. 1, Control; 2, BNP-treated PAO1; 3, CNP-treated PAO1.

Mentions: We first observed that the phnB mutant of PAO1 grown in ONB medium killed C. elegans with the same kinetics as the wild-type strain (data not shown). Pyocyanin production was measured in culture supernatant (24 h, 37 °C) after exposure of bacteria to BNP or CNP (10−6 M). We observed a decrease in pyocyanin production of 65.1±4.3 % for CNP-treated bacteria (Fig. 3a). In contrast, when bacteria were exposed to BNP, no significant variation in pyocyanin was observed (Fig. 3a). The phzC1 mRNA level was halved after CNP exposure compared with untreated PAO1, whereas BNP did not affect the amount of phzC1 mRNA (Fig. 3b).


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)

Effect of BNP and CNP on pyocyanin, PQS, HHQ and HNQ production by P. aeruginosa PAO1. (a) P. aeruginosa PAO1 pyocyanin production in response to natriuretic peptides (BNP and CNP). Data are the mean±sem of seven values obtained from three independent experiments. The mean pyocyanin level in the control was 0.0071±0.0016 OD520/OD580. Significant difference is indicated by asterisks (***P<0.001); ns, not significantly different. (b) Expression levels of phzC1 in PAO1-treated bacteria relative to those in the PAO1 control. RNA was extracted 1 h after PAO1 exposure to physiological water or natriuretic peptides (BNP and CNP) and assayed by qRT-PCR. See the legend to Fig. 2 for further information. (c, d) Relative amounts of PQS and HHQ in P. aeruginosa PAO1 supernatant 3 h after exposure of PAO1 to physiological water or CNP. Data are the mean±sd of three independent experiments. The mean PQS and HHQ concentration in control conditions were 741×103 Area/OD580 and 1410×103 Area/OD580, respectively. *P<0.05; ns, not significantly different. (e) Relative amounts of HNQ in P. aeruginosa PAO1 supernatant, 3 h after exposure of PAO1 to physiological water or CNP. Data are the means of three independent experiments. The mean HNQ concentration in the control was 1520×103 Area/OD580. *P<0.05. 1, Control; 2, BNP-treated PAO1; 3, CNP-treated PAO1.
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f3: Effect of BNP and CNP on pyocyanin, PQS, HHQ and HNQ production by P. aeruginosa PAO1. (a) P. aeruginosa PAO1 pyocyanin production in response to natriuretic peptides (BNP and CNP). Data are the mean±sem of seven values obtained from three independent experiments. The mean pyocyanin level in the control was 0.0071±0.0016 OD520/OD580. Significant difference is indicated by asterisks (***P<0.001); ns, not significantly different. (b) Expression levels of phzC1 in PAO1-treated bacteria relative to those in the PAO1 control. RNA was extracted 1 h after PAO1 exposure to physiological water or natriuretic peptides (BNP and CNP) and assayed by qRT-PCR. See the legend to Fig. 2 for further information. (c, d) Relative amounts of PQS and HHQ in P. aeruginosa PAO1 supernatant 3 h after exposure of PAO1 to physiological water or CNP. Data are the mean±sd of three independent experiments. The mean PQS and HHQ concentration in control conditions were 741×103 Area/OD580 and 1410×103 Area/OD580, respectively. *P<0.05; ns, not significantly different. (e) Relative amounts of HNQ in P. aeruginosa PAO1 supernatant, 3 h after exposure of PAO1 to physiological water or CNP. Data are the means of three independent experiments. The mean HNQ concentration in the control was 1520×103 Area/OD580. *P<0.05. 1, Control; 2, BNP-treated PAO1; 3, CNP-treated PAO1.
Mentions: We first observed that the phnB mutant of PAO1 grown in ONB medium killed C. elegans with the same kinetics as the wild-type strain (data not shown). Pyocyanin production was measured in culture supernatant (24 h, 37 °C) after exposure of bacteria to BNP or CNP (10−6 M). We observed a decrease in pyocyanin production of 65.1±4.3 % for CNP-treated bacteria (Fig. 3a). In contrast, when bacteria were exposed to BNP, no significant variation in pyocyanin was observed (Fig. 3a). The phzC1 mRNA level was halved after CNP exposure compared with untreated PAO1, whereas BNP did not affect the amount of phzC1 mRNA (Fig. 3b).

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