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Nitric oxide antagonizes the acid tolerance response that protects Salmonella against innate gastric defenses.

Bourret TJ, Porwollik S, McClelland M, Zhao R, Greco T, Ischiropoulos H, Vázquez-Torres A - PLoS ONE (2008)

Bottom Line: The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids.Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Colorado Health Sciences Center, Aurora, Colorado, United States of America.

ABSTRACT

Background: Reactive nitrogen species (RNS) derived from dietary and salivary inorganic nitrogen oxides foment innate host defenses associated with the acidity of the stomach. The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.

Methodology/principal findings: The genetically tractable acid tolerance response (ATR) that enables enteropathogens to survive harsh acidity was screened for signaling pathways responsive to RNS. The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids. Despite inducing a Fur-mediated adaptive response, acidified RNS largely repressed oral virulence as demonstrated by the fact that Salmonella bacteria exposed to NO donors during mildly acidic conditions were shed in low amounts in feces and exhibited ameliorated oral virulence. NO prevented Salmonella from mounting a de novo ATR, but was unable to suppress an already functional protective response, suggesting that RNS target regulatory cascades but not their effectors. Transcriptional and translational analyses revealed that the PhoPQ signaling cascade is a critical ATR target of NO in rapidly growing Salmonella. Inhibition of PhoPQ signaling appears to contribute to most of the NO-mediated abrogation of the ATR in log phase bacteria, because the augmented acid sensitivity of phoQ-deficient Salmonella was not further enhanced after RNS treatment.

Conclusions/significance: Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses.

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Suppression of PhoPQ-dependent gene transcription is mediated by nitrogen oxides and requires an intact signal transduction cascade.The effect of spermine NONOate on the PhoPQ-dependent induction of the lpxO::lacZ transcriptional fusion is shown in panel A. β∼-galactosidase activity (Miller Units, M.U.) is represented as the mean±SEM of 4–6 independent observations from 2–3 separate experiments. *, p<0.002 compared to adapted controls. The acid-inducible expression of the PhoP-activated loci lpxO, pqaA and pcgE were monitored in the presence or absence of 250 µM spermine, 250 µM spermine NONOate or 500 µM NaNO2 (B–D). (E) The expression of C-terminal 3×FLAG-tagged PhoP and PhoQ proteins was monitored in NonAd, Ad, and Ad+NO Salmonella cultures grown as described in figure 1.
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pone-0001833-g006: Suppression of PhoPQ-dependent gene transcription is mediated by nitrogen oxides and requires an intact signal transduction cascade.The effect of spermine NONOate on the PhoPQ-dependent induction of the lpxO::lacZ transcriptional fusion is shown in panel A. β∼-galactosidase activity (Miller Units, M.U.) is represented as the mean±SEM of 4–6 independent observations from 2–3 separate experiments. *, p<0.002 compared to adapted controls. The acid-inducible expression of the PhoP-activated loci lpxO, pqaA and pcgE were monitored in the presence or absence of 250 µM spermine, 250 µM spermine NONOate or 500 µM NaNO2 (B–D). (E) The expression of C-terminal 3×FLAG-tagged PhoP and PhoQ proteins was monitored in NonAd, Ad, and Ad+NO Salmonella cultures grown as described in figure 1.

Mentions: The effect that NO has on PhoP-mediated transcription were independently studied using lacZ transcriptional fusions. Consistent with the DNA arrays and RT-PCR analysis, NO repressed the expression of lpxO::lacZ, pqaA::lacZ and pcgE::mudJ (fig. 6A–D). In the absence of the sensor kinase PhoQ, lpxO::lacZ was not only unresponsive to a drop in pH but its basal levels of expression were unaffected by spermine NONOate (fig. 6A). The suppressive effects of spermine NONOate on PhoP-dependent gene transcription appear to be directly related to the production of RNS because the polyamine base spermine did not suppress the acid-induced expression of the PhoP-activated loci lpxO, pqaA, and pcgE (fig. 6B–D). Furthermore, these loci were also repressed upon exposure of Salmonella to 500 µM NaNO2 in EG medium, pH 4.4 (fig. 6B–D). Because the expression of the PhoP regulon depends on enzymatic activity and abundance of the PhoQ sensor kinase and the PhoP response regulator, protein levels of the components of this two-component regulatory system were monitored in western blots of Salmonella strains harboring PhoP or PhoQ C-terminal 3×FLAG epitope tags. The amount of PhoP and PhoQ increased by 1.3- and 4.9-fold after adaptation of Salmonella for 2 h in EG medium, pH 4.4 (fig. 6E). Consistent with the transcriptional profiles, acidified spermine NONOate reduced Salmonella PhoP and PhoQ protein levels by 2- and 9.4-fold, respectively. Collectively, these data demonstrate that nitrogen oxides repress the acid-inducible, PhoPQ signaling cascade.


Nitric oxide antagonizes the acid tolerance response that protects Salmonella against innate gastric defenses.

Bourret TJ, Porwollik S, McClelland M, Zhao R, Greco T, Ischiropoulos H, Vázquez-Torres A - PLoS ONE (2008)

Suppression of PhoPQ-dependent gene transcription is mediated by nitrogen oxides and requires an intact signal transduction cascade.The effect of spermine NONOate on the PhoPQ-dependent induction of the lpxO::lacZ transcriptional fusion is shown in panel A. β∼-galactosidase activity (Miller Units, M.U.) is represented as the mean±SEM of 4–6 independent observations from 2–3 separate experiments. *, p<0.002 compared to adapted controls. The acid-inducible expression of the PhoP-activated loci lpxO, pqaA and pcgE were monitored in the presence or absence of 250 µM spermine, 250 µM spermine NONOate or 500 µM NaNO2 (B–D). (E) The expression of C-terminal 3×FLAG-tagged PhoP and PhoQ proteins was monitored in NonAd, Ad, and Ad+NO Salmonella cultures grown as described in figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266805&req=5

pone-0001833-g006: Suppression of PhoPQ-dependent gene transcription is mediated by nitrogen oxides and requires an intact signal transduction cascade.The effect of spermine NONOate on the PhoPQ-dependent induction of the lpxO::lacZ transcriptional fusion is shown in panel A. β∼-galactosidase activity (Miller Units, M.U.) is represented as the mean±SEM of 4–6 independent observations from 2–3 separate experiments. *, p<0.002 compared to adapted controls. The acid-inducible expression of the PhoP-activated loci lpxO, pqaA and pcgE were monitored in the presence or absence of 250 µM spermine, 250 µM spermine NONOate or 500 µM NaNO2 (B–D). (E) The expression of C-terminal 3×FLAG-tagged PhoP and PhoQ proteins was monitored in NonAd, Ad, and Ad+NO Salmonella cultures grown as described in figure 1.
Mentions: The effect that NO has on PhoP-mediated transcription were independently studied using lacZ transcriptional fusions. Consistent with the DNA arrays and RT-PCR analysis, NO repressed the expression of lpxO::lacZ, pqaA::lacZ and pcgE::mudJ (fig. 6A–D). In the absence of the sensor kinase PhoQ, lpxO::lacZ was not only unresponsive to a drop in pH but its basal levels of expression were unaffected by spermine NONOate (fig. 6A). The suppressive effects of spermine NONOate on PhoP-dependent gene transcription appear to be directly related to the production of RNS because the polyamine base spermine did not suppress the acid-induced expression of the PhoP-activated loci lpxO, pqaA, and pcgE (fig. 6B–D). Furthermore, these loci were also repressed upon exposure of Salmonella to 500 µM NaNO2 in EG medium, pH 4.4 (fig. 6B–D). Because the expression of the PhoP regulon depends on enzymatic activity and abundance of the PhoQ sensor kinase and the PhoP response regulator, protein levels of the components of this two-component regulatory system were monitored in western blots of Salmonella strains harboring PhoP or PhoQ C-terminal 3×FLAG epitope tags. The amount of PhoP and PhoQ increased by 1.3- and 4.9-fold after adaptation of Salmonella for 2 h in EG medium, pH 4.4 (fig. 6E). Consistent with the transcriptional profiles, acidified spermine NONOate reduced Salmonella PhoP and PhoQ protein levels by 2- and 9.4-fold, respectively. Collectively, these data demonstrate that nitrogen oxides repress the acid-inducible, PhoPQ signaling cascade.

Bottom Line: The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids.Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Colorado Health Sciences Center, Aurora, Colorado, United States of America.

ABSTRACT

Background: Reactive nitrogen species (RNS) derived from dietary and salivary inorganic nitrogen oxides foment innate host defenses associated with the acidity of the stomach. The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood.

Methodology/principal findings: The genetically tractable acid tolerance response (ATR) that enables enteropathogens to survive harsh acidity was screened for signaling pathways responsive to RNS. The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids. Despite inducing a Fur-mediated adaptive response, acidified RNS largely repressed oral virulence as demonstrated by the fact that Salmonella bacteria exposed to NO donors during mildly acidic conditions were shed in low amounts in feces and exhibited ameliorated oral virulence. NO prevented Salmonella from mounting a de novo ATR, but was unable to suppress an already functional protective response, suggesting that RNS target regulatory cascades but not their effectors. Transcriptional and translational analyses revealed that the PhoPQ signaling cascade is a critical ATR target of NO in rapidly growing Salmonella. Inhibition of PhoPQ signaling appears to contribute to most of the NO-mediated abrogation of the ATR in log phase bacteria, because the augmented acid sensitivity of phoQ-deficient Salmonella was not further enhanced after RNS treatment.

Conclusions/significance: Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses.

Show MeSH
Related in: MedlinePlus