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. 2008 Mar 19;3(3):e1833.
doi: 10.1371/journal.pone.0001833.

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

Travis J Bourret  1 , Steffen Porwollik, Michael McClelland, Rui Zhao, Todd Greco, Harry Ischiropoulos, Andrés Vázquez-Torres
Affiliations

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

Travis J Bourret et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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.
Figure 2
Figure 2. RNS inhibit the Salmonella ATR.
The susceptibility of adapted (Ad) and nonadapted (NonAd) Salmonella cultures to acid challenge in EG medium, pH 3.0 was determined as described in figure 1. (A) Selected groups of Ad and NonAd bacterial cultures were treated with 250 µM spermine NONOate (NO) for 2 h before acid challenge. (B) The acid sensitivity of Ad and Ad+NO Salmonella was compared to controls grown for 1 h in EG medium, pH 4.4 before exposure to 250 µM spermine NONOate for 1 h (Ad 1 h, Ad+NO 1 h). The data represent the mean±SEM of 4–8 independent observations from 2–3 separate experiments.
Figure 3
Figure 3. RNS suppress the increased oral virulence of ATR-adapted Salmonella.
C57BL/6 mice were inoculated orally with ∼5×105 CFU of NonAd, Ad and Ad+NO Salmonella grown as described in figure 1. (A) Fecal shedding of nalidixic acid resistant Salmonella was monitored in individual mice for 3 days after oral inoculation. Panel B shows the % of mice that survived after oral challenge with Salmonella. Data represent 10 mice per group from 2 separate experiments.
Figure 4
Figure 4. Responses of Salmonella to acidified RNS.
The acid-inducible expression of the SPI2-encoded spiC::lacZ transcriptional fusion was compared in nonadapted (NonAd), adapted (Ad) and adapted+250 µM spermine NONOate (Ad+NO) Salmonella cultures (A). The survival of NonAd, Ad and Ad+NO Salmonella cultures exposed for 1 h to 400 µM H2O2 in PBS is shown in panel B. Oxygen consumption was monitored in Ad and Ad+NO Salmonella cultures (C). The transcription of the ribosomal protein-encoding gene rplN relative to the housekeeping gene rpoD was monitored by semi-quantitative (D) and quantitative RT-PCR (E). Relative expression is represented as the ratio of rplN/rpoD transcripts. Data represent the mean±SEM of 3–6 independent observations. *, p<0.05 by one-way ANOVA.
Figure 5
Figure 5. 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.
Figure 6
Figure 6. 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.
Figure 7
Figure 7. Acidified RNS suppress the PhoPQ-dependent ATR.
Killing of wild-type Salmonella and its isogenic ΔphoQ::FRT control in EG medium, pH 3.0 is shown in panel A. The ΔphoQ::FRT mutation was complemented with a wild-type allele expressed from pWSQ. Selected groups of bacteria were treated with 250 µM spermine NONOate (Ad+NO) during the adaptation in EG medium, pH 4.4. Panel B compares the acid sensitivity of Ad and Ad+NO groups of Salmonella strains isogenic for ΔrpoS::km, ompR::Tn10 and Δfur::FRT. The data represent the mean±SEM of 6–9 independent observations from 2–3 separate experiments. *, p<0.05 compared to adapted controls. **, no viable cells were detected after the adapted+NO group was challenged for 1 h in EG medium, pH 3.0.
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