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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: 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.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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Related in: MedlinePlus

Acid-inducible PhoPQ-dependent gene transcription is repressed by RNS.Panel A shows the expression of PhoP-activated genes in rapidly growing Salmonella cultured for 1 h in EG medium, pH 4.4 in the presence (Ad+NO) or absence (Ad) of 250 µM spermine NONOate. Microarray data are represented as the mean fold change±SD from 3 independent experiments. (B) Transcription of PhoP-activated genes (pag), PhoP-repressed genes (prg), the control housekeeping rpoD gene and the NO-inducible hmpA gene were assessed by RT-PCR of RNA isolated from NonAd, Ad, and Ad+NO bacterial cultures grown as described in figure 1.
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pone-0001833-g005: Acid-inducible PhoPQ-dependent gene transcription is repressed by RNS.Panel A shows the expression of PhoP-activated genes in rapidly growing Salmonella cultured for 1 h in EG medium, pH 4.4 in the presence (Ad+NO) or absence (Ad) of 250 µM spermine NONOate. Microarray data are represented as the mean fold change±SD from 3 independent experiments. (B) Transcription of PhoP-activated genes (pag), PhoP-repressed genes (prg), the control housekeeping rpoD gene and the NO-inducible hmpA gene were assessed by RT-PCR of RNA isolated from NonAd, Ad, and Ad+NO bacterial cultures grown as described in figure 1.

Mentions: The essential role that the PhoPQ two component regulatory system plays in controlling the ATR is demonstrated by the fact that 1) PhoP is an acid shock protein and 2) strains harboring phoP or phoQ mutations are exquisitely acid sensitive [24], [45]. Because PhoPQ signaling has been shown to dominate the log phase ATR studied herein [24], [46], we examined in more detail the effect that acidified spermine NONOate has on PhoP-dependent gene transcription. Most members of the PhoP regulon, such as phoQ, phoN, virK, pagP, pqaA, cysJIH and cysCND [47], [48], were repressed by NO in Salmonella grown in EG medium, pH 4.4 (fig. 5A). The few PhoP-activated genes that were not repressed by NO may be a consequence of a differential regulation of these loci in EG medium, pH 4.4. RT-PCR analysis independently showed an NO-dependent downregulation in the transcription of PhoP-activated genes phoP, phoQ, mig-14 and phoN, while RNS treatment did not affect (e.g., rpoD) or even increased (e.g., hmpA) the expression of other loci (fig. 5B). According to the idea that NO represses PhoPQ signaling, transcription of the PhoP-repressed genes (prg) fliA (fig. 5B), fliC and hilA was upregulated in response to spermine NONOate treatment (table S2). The low levels at which prg are normally expressed [49] may have contributed to the fact that our array analysis did not show a broader derepression of the PhoP regulon.


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)

Acid-inducible PhoPQ-dependent gene transcription is repressed by RNS.Panel A shows the expression of PhoP-activated genes in rapidly growing Salmonella cultured for 1 h in EG medium, pH 4.4 in the presence (Ad+NO) or absence (Ad) of 250 µM spermine NONOate. Microarray data are represented as the mean fold change±SD from 3 independent experiments. (B) Transcription of PhoP-activated genes (pag), PhoP-repressed genes (prg), the control housekeeping rpoD gene and the NO-inducible hmpA gene were assessed by RT-PCR of RNA isolated from NonAd, Ad, and Ad+NO bacterial cultures grown as described in figure 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001833-g005: Acid-inducible PhoPQ-dependent gene transcription is repressed by RNS.Panel A shows the expression of PhoP-activated genes in rapidly growing Salmonella cultured for 1 h in EG medium, pH 4.4 in the presence (Ad+NO) or absence (Ad) of 250 µM spermine NONOate. Microarray data are represented as the mean fold change±SD from 3 independent experiments. (B) Transcription of PhoP-activated genes (pag), PhoP-repressed genes (prg), the control housekeeping rpoD gene and the NO-inducible hmpA gene were assessed by RT-PCR of RNA isolated from NonAd, Ad, and Ad+NO bacterial cultures grown as described in figure 1.
Mentions: The essential role that the PhoPQ two component regulatory system plays in controlling the ATR is demonstrated by the fact that 1) PhoP is an acid shock protein and 2) strains harboring phoP or phoQ mutations are exquisitely acid sensitive [24], [45]. Because PhoPQ signaling has been shown to dominate the log phase ATR studied herein [24], [46], we examined in more detail the effect that acidified spermine NONOate has on PhoP-dependent gene transcription. Most members of the PhoP regulon, such as phoQ, phoN, virK, pagP, pqaA, cysJIH and cysCND [47], [48], were repressed by NO in Salmonella grown in EG medium, pH 4.4 (fig. 5A). The few PhoP-activated genes that were not repressed by NO may be a consequence of a differential regulation of these loci in EG medium, pH 4.4. RT-PCR analysis independently showed an NO-dependent downregulation in the transcription of PhoP-activated genes phoP, phoQ, mig-14 and phoN, while RNS treatment did not affect (e.g., rpoD) or even increased (e.g., hmpA) the expression of other loci (fig. 5B). According to the idea that NO represses PhoPQ signaling, transcription of the PhoP-repressed genes (prg) fliA (fig. 5B), fliC and hilA was upregulated in response to spermine NONOate treatment (table S2). The low levels at which prg are normally expressed [49] may have contributed to the fact that our array analysis did not show a broader derepression of the PhoP regulon.

Bottom Line: 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.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