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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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Sensitivity of Salmonella to acid stress upon exposure to NO donors.(A) Salmonella grown to an OD600 of 0.4 in EG medium, pH 7.0 were acid challenged in fresh EG medium, pH 3.0 (NonAd). Selected groups of mid-log phase cells were adapted (Ad) for 2 h in EG medium, pH 4.4 at 37°C. The % of surviving bacteria was estimated over time. (B) Effects of RNS on the ability of Salmonella to mount an ATR were determined by adding NO donors spermine NONOate (sperNO) and NaNO2 to bacteria during the 2 h adaptation period. The NO donors were removed by pelleting the bacteria before 1 h of challenge in EG medium, pH 3.0. (C) The effects that NO generated from sperNO exert on the viability of Salmonella grown for 2 h in EG medium, pH 4.4 can be seen in panel C. The effect that 10 µM of the NO-scavenger hemoglobin (Hb) had on the sperNO-inhibitable ATR is shown in panel D. The spermine base was used as a negative control. The chemicals were added for 2 h during the adaptation in EG medium, pH 4.4. The data represent the mean±SEM of 4-16 independent observations from 2–4 separate experiments. *, p<0.001 compared to adapted controls.
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pone-0001833-g001: Sensitivity of Salmonella to acid stress upon exposure to NO donors.(A) Salmonella grown to an OD600 of 0.4 in EG medium, pH 7.0 were acid challenged in fresh EG medium, pH 3.0 (NonAd). Selected groups of mid-log phase cells were adapted (Ad) for 2 h in EG medium, pH 4.4 at 37°C. The % of surviving bacteria was estimated over time. (B) Effects of RNS on the ability of Salmonella to mount an ATR were determined by adding NO donors spermine NONOate (sperNO) and NaNO2 to bacteria during the 2 h adaptation period. The NO donors were removed by pelleting the bacteria before 1 h of challenge in EG medium, pH 3.0. (C) The effects that NO generated from sperNO exert on the viability of Salmonella grown for 2 h in EG medium, pH 4.4 can be seen in panel C. The effect that 10 µM of the NO-scavenger hemoglobin (Hb) had on the sperNO-inhibitable ATR is shown in panel D. The spermine base was used as a negative control. The chemicals were added for 2 h during the adaptation in EG medium, pH 4.4. The data represent the mean±SEM of 4-16 independent observations from 2–4 separate experiments. *, p<0.001 compared to adapted controls.

Mentions: As predicted [20], [21], Salmonella adapted for 2 h in EG medium, pH 4.4 exhibited increased survival after 1.5 h of acid challenge in fresh EG medium, pH 3.0 (fig. 1A). In agreement with published investigations [20], [21], [24], [30], [37], between 55 and 90% of the bacteria adapted at pH 4.4 survived acid challenge at pH 3.0. Salivary NO2− and NO3− concentrations ranging from 400 to 1,890 µM generate several RNS with potent antimicrobial activity in the gastric juice [10]. Because the mechanisms for the broad antimicrobial activity exhibited by acidified RNS are poorly understood, we examined the effect that NO donors have on the ability of actively growing Salmonella to mount a productive ATR. Both spermine NONOate and NaNO2 abrogated in a dose dependent manner the acid resistance of adapted Salmonella (fig. 1B). Addition of 250 µM spermine NONOate or 500 µM NO2− during the adaptation period resulted in a 100-fold decrease in Salmonella survival upon challenge in EG medium, pH 3.0 (fig. 1B). Henceforth, 500 µM NaNO2 or 250 µM spermine NONOate were used throughout the remainder of our investigations. These RNS were not directly bactericidal at pH 4.4 because the viability of Salmonella was unaffected even after treatment with 350 µM spermine NONOate (fig. 1C). Similar to pH 4.4, spermine NONOate induced bacteriostasis (not shown). The acid sensitivity resulting from spermine NONOate treatment was dependent on the release of NO, as indicated by the fact that the parent compound spermine lacked inhibitory activity (fig 1D). Furthermore, the NO scavenger ferrous hemoglobin significantly (p<0.001) antagonized the inhibitory effects of spermine NONOate. These results indicate that NO and NO2− are not directly bactericidal under moderately acidic conditions normally encountered in the stomach following the consumption of a meal [19], but instead suggest that nitrogen oxides suppress the adaptive ATR that protects Salmonella from the rigors of the extreme acidity normally found in the stomach.


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)

Sensitivity of Salmonella to acid stress upon exposure to NO donors.(A) Salmonella grown to an OD600 of 0.4 in EG medium, pH 7.0 were acid challenged in fresh EG medium, pH 3.0 (NonAd). Selected groups of mid-log phase cells were adapted (Ad) for 2 h in EG medium, pH 4.4 at 37°C. The % of surviving bacteria was estimated over time. (B) Effects of RNS on the ability of Salmonella to mount an ATR were determined by adding NO donors spermine NONOate (sperNO) and NaNO2 to bacteria during the 2 h adaptation period. The NO donors were removed by pelleting the bacteria before 1 h of challenge in EG medium, pH 3.0. (C) The effects that NO generated from sperNO exert on the viability of Salmonella grown for 2 h in EG medium, pH 4.4 can be seen in panel C. The effect that 10 µM of the NO-scavenger hemoglobin (Hb) had on the sperNO-inhibitable ATR is shown in panel D. The spermine base was used as a negative control. The chemicals were added for 2 h during the adaptation in EG medium, pH 4.4. The data represent the mean±SEM of 4-16 independent observations from 2–4 separate experiments. *, p<0.001 compared to adapted controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266805&req=5

pone-0001833-g001: Sensitivity of Salmonella to acid stress upon exposure to NO donors.(A) Salmonella grown to an OD600 of 0.4 in EG medium, pH 7.0 were acid challenged in fresh EG medium, pH 3.0 (NonAd). Selected groups of mid-log phase cells were adapted (Ad) for 2 h in EG medium, pH 4.4 at 37°C. The % of surviving bacteria was estimated over time. (B) Effects of RNS on the ability of Salmonella to mount an ATR were determined by adding NO donors spermine NONOate (sperNO) and NaNO2 to bacteria during the 2 h adaptation period. The NO donors were removed by pelleting the bacteria before 1 h of challenge in EG medium, pH 3.0. (C) The effects that NO generated from sperNO exert on the viability of Salmonella grown for 2 h in EG medium, pH 4.4 can be seen in panel C. The effect that 10 µM of the NO-scavenger hemoglobin (Hb) had on the sperNO-inhibitable ATR is shown in panel D. The spermine base was used as a negative control. The chemicals were added for 2 h during the adaptation in EG medium, pH 4.4. The data represent the mean±SEM of 4-16 independent observations from 2–4 separate experiments. *, p<0.001 compared to adapted controls.
Mentions: As predicted [20], [21], Salmonella adapted for 2 h in EG medium, pH 4.4 exhibited increased survival after 1.5 h of acid challenge in fresh EG medium, pH 3.0 (fig. 1A). In agreement with published investigations [20], [21], [24], [30], [37], between 55 and 90% of the bacteria adapted at pH 4.4 survived acid challenge at pH 3.0. Salivary NO2− and NO3− concentrations ranging from 400 to 1,890 µM generate several RNS with potent antimicrobial activity in the gastric juice [10]. Because the mechanisms for the broad antimicrobial activity exhibited by acidified RNS are poorly understood, we examined the effect that NO donors have on the ability of actively growing Salmonella to mount a productive ATR. Both spermine NONOate and NaNO2 abrogated in a dose dependent manner the acid resistance of adapted Salmonella (fig. 1B). Addition of 250 µM spermine NONOate or 500 µM NO2− during the adaptation period resulted in a 100-fold decrease in Salmonella survival upon challenge in EG medium, pH 3.0 (fig. 1B). Henceforth, 500 µM NaNO2 or 250 µM spermine NONOate were used throughout the remainder of our investigations. These RNS were not directly bactericidal at pH 4.4 because the viability of Salmonella was unaffected even after treatment with 350 µM spermine NONOate (fig. 1C). Similar to pH 4.4, spermine NONOate induced bacteriostasis (not shown). The acid sensitivity resulting from spermine NONOate treatment was dependent on the release of NO, as indicated by the fact that the parent compound spermine lacked inhibitory activity (fig 1D). Furthermore, the NO scavenger ferrous hemoglobin significantly (p<0.001) antagonized the inhibitory effects of spermine NONOate. These results indicate that NO and NO2− are not directly bactericidal under moderately acidic conditions normally encountered in the stomach following the consumption of a meal [19], but instead suggest that nitrogen oxides suppress the adaptive ATR that protects Salmonella from the rigors of the extreme acidity normally found in the stomach.

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