Control of Redox Balance by the Stringent Response Regulatory Protein Promotes Antioxidant Defenses of Salmonella

Abstract

We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)⁺ redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H₂O₂), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.

Introduction

Bacterial pathogens have the remarkable ability to survive harsh conditions encountered during the course of infection. For instance, Gram-negative bacteria of the genus Salmonella must endure the extreme acidity of the stomach, osmotic stress in the intestinal lumen, as well as oxygen-dependent and -independent cytotoxicity in the intracellular environment of macrophages. Of all of these stresses, reactive oxygen species are arguably the most extensively studied host defenses. Salmonella infection triggers the assembly of an NADPH phagocyte oxidase respiratory complex (1) for the production of superoxide, a radical that rapidly dismutates to give rise to hydrogen peroxide (H₂O₂). Membrane-permeable H₂O₂ causes extensive damage to DNA bases and [4Fe-4S] prosthetics groups of dehydratases via Fenton-like chemistries that are dependent on the ferrous iron-catalyzed univalent or divalent reduction of H₂O₂ (2, 3). H₂O₂ is also thought to mediate cytotoxicity through the oxidation of thiol groups in redox active cysteines (4). The importance of reactive oxygen species in host defense against Salmonella, as well as an assortment of other fungal and bacterial pathogens, is underscored by the increased incidence of recurrent infections in chronic granulomatous disease patients who lack a functional NADPH phagocyte oxidase (5). A murine model deficient in the gp91phox membrane-bound component of the NADPH oxidase accurately recapitulates the clinical importance of this enzymatic complex in the innate response of humans during Salmonella infections (6).

Bacteria, including Salmonella, have a diverse arsenal of antioxidant defenses. Superoxide dismutases, catalases, alkylhydroperoxidases, glutathione, and thioredoxins all directly or indirectly detoxify reactive oxygen species (7, 8). Because of the membrane-remodeling activity of effectors secreted into the host cell by the type III secretion system encoded within Salmonella pathogenicity island 2, Salmonella can avoid NADPH phagocyte oxidase-containing vesicles (9–11). Recent investigations on the electron transport chain highlight the importance of reduced nucleotides for the resistance of Salmonella against oxidative stress (12). Maintaining cellular reductive power may provide the fuel needed for detoxification of oxyradicals because many of the antioxidant enzymatic systems consume reduced pyridine nucleotides. It is still uncertain whether and to what extent central metabolic pathways contribute reducing power for classical antioxidant defenses. In the present work, we have elucidated a novel role for DksA³ in the control of discrete steps of central metabolism that fuel antioxidant defenses.

Previous studies in Escherichia coli have revealed DksA to have pleiotropic regulatory roles in gene expression, cell division, amino acid biosynthesis, quorum sensing, stress resistance, and virulence (13–19). Some of the global regulatory effects of DksA are dependent on alarmone guanine tetraphosphate (ppGpp) and the stringent response. The stringent response was originally identified as a global adaptation of bacteria to nutritional stress. Starvation for amino acids, glucose, phosphate, or fatty acids leads to the accumulation of ppGpp (20, 21). This alarmone helps the cell adapt to starving conditions by down-regulating transcription of translational machinery, thereby balancing protein production with nutrient availability. Some of the regulatory actions of ppGpp on the activity of RNA polymerase are achieved in conjunction with DksA, a protein with structural homology to Gre transcription factors (22). However, independent and opposing roles for DksA have also been observed, supporting unique functions for DksA (23, 24). DksA has been shown to play a role in the virulence of several pathogens including Salmonella; however, it is still unclear how adjustments in gene expression by DksA contribute to Salmonella pathogenesis (25). We report herein that DksA exerts transcriptional control of discrete steps in central metabolic pathways of the pentose phosphate pathway and tricarboxylic acid cycle during the stationary phase of Salmonella. Our investigations indicate that DksA helps maintain a redox balance critical for Salmonella virulence in response to reactive oxygen species generated intracellularly by the NADPH phagocyte oxidase.

EXPERIMENTAL PROCEDURES

Bacterial Strains

Salmonella enterica sv. typhimurium strain ATCC 14028s was used as wild-type and as a background in the construction of mutants following the method previously described by Datsenko and Wanner (26) (supplemental Table 1). PCR amplicons containing kanamycin or chloramphenicol resistance cassettes, or a 3×FLAG tag, flanked by the flippase (Flp) recognition target (FRT) were generated from pKD13, pKD3, and pSUB11 templates using LA Taq high fidelity polymerase (TaKaRa, Madison, WI) and 60-base-long primers containing homology to the target gene (supplemental Table 2). Purified PCR products were electroporated into S. typhimurium strain TT22236 harboring the plasmid pTP223 that expresses an isopropyl 1-thio-β-d-galactopyranoside-inducible λ Red recombinase system. Mutations were moved into S. typhimurium strain 14028s via P22-mediated transduction, and pseudolysogens were eliminated by streaking on Evans blue uranine agar. The antibiotic cassette was removed by recombining the flanking FRT sites with the Flp recombinase expressed from the temperature-sensitive pCP20 plasmid (27). In-frame deletions were verified by PCR analysis. Strain AV09294 lacking dksA was complemented with the low copy vector pWSK29 encoding a wild-type dksA allele expressed from its native promoter. Transcriptional fusions were constructed by pCP20-mediated integration of pCE36 encoding a promoterless lacZY into FRT scars of selected genes (28).

Conditional Expression of zwf

Construction of a zwf conditional mutant was achieved by placing the zwf open reading frame under the control of the tetracycline promoter/operator. The tetracycline promoter/operator containing plasmid pASK75 was modified for the vector to be inserted into the Salmonella chromosome. Briefly, the origin of replication in pASK75 was replaced with the R6K origin amplified from plasmid pKD13. The amplicon containing the R6K origin and pASK75 were ligated after SpeI and PfIMI digestion, generating a pir-dependent suicide vector. The FRT site from pCE36 was directionally cloned into the MscI unique site of the modified pASK75. The ampicillin resistance cassette of the modified pASK75 plasmid was disrupted by directionally cloning a gentamicin cassette from pPS856 into ScaI and PvuI unique sites to generate pTX (see Fig. 6A). A zwf conditional mutant was constructed by placing the gene with its native ribosomal binding site into EcoRI and BamHI restriction sites placed downstream of the tet promoter. The final construct was confirmed by sequence analysis. The suicide vector was recombined by Flp expressed from pCP20 into a unique FRT site engineered downstream of putP in the chromosome of Salmonella strain AV09422. The P_tet-controlled zwf was transduced into wild-type and dksA mutant Salmonella strain AV09294. Expression of the allele was initiated with 0.2 μg/ml anhydrotetracycline (Sigma-Aldrich).

FIGURE 6.

DksA increases fitness of Salmonella exposed to the NADPH phagocyte oxidase. A and B, C57BL/6 (A) and congenic NADPH phagocyte oxidase-deficient (phox) (B) mice were inoculated intraperitoneally with ∼400 cfu of either wild-type (WT) or dksA-deficient Salmonella. The percent of mice surviving the infection was evaluated over time. The data represent 10 mice/group from two separate experiments. C and D, the anti-Salmonella activity of periodate-elicited peritoneal macrophages isolated from C57BL/6 mice (C) or their congenic phox-deficient mice (D) was evaluated over time. The number of surviving intracellular bacteria was estimated at the indicated times after culture on LB agar plates as described under “Experimental Procedures. ” The data represent the mean percent survival ± S.E. (error bars) of 6–12 independent observations from at least two independent experiments. *, p < 0.05; **, p < 0.01 compared with WT controls.

Real Time PCR

Bacterial cultures grown in Luria Bertani (LB) broth for 20 h were diluted to 10⁸ cfu/ml in PBS and treated with 1 mm H₂O₂ for 30 min at 37 °C with shaking. Even though the concentration of H₂O₂ used in these experiments is 10 times higher than that used for the killing assays described below, the high bacterial densities used for the isolation of RNA prevented the loss of bacterial viability. Total bacterial RNA was isolated in a bead-beater (Biospec Products, Inc.) as described previously (29) using TRIzol reagent (Invitrogen) containing silicon beads. RNA was extracted with chloroform, precipitated with isopropyl alcohol, washed with ethanol, and dried in a speed vacuum. Pellets were resuspended in RNase-free water and treated with RNase-free DNase (Ambion, Austin, TX). Complimentary cDNA was synthesized at 42 °C using Moloney murine leukemia virus reverse transcriptase (Promega), RNasin (Promega), dNTPs, and random hexamers. cDNA was used as a template for real time and reverse transcription PCR. Transcripts of rpsM and rplN were normalized for the housekeeping gene rpoD.

β-Galactosidase Assays

Strains carrying chromosomal lacZY fusions to genes encoding enzymes of pentose phosphate pathway or tricarboxylic acid cycle known to generate reducing power were constructed as described previously (28). To test whether these metabolic pathways are under the control of DksA, the lacZY fusions were transduced into the dksA mutant strain AV09294. LacZ enzymatic activity was quantified spectrophotometrically in bacterial cultures grown overnight in LB broth as β-galactosidase activity by following the conversion of o-nitrophenyl-β-d-galactopyranoside to o-nitrophenyl. β-Galactosidase activity is expressed as Miller units calculated according to the equation: 1000 × ((A_{420 nm} − 1.75 × A_{550 nm})/(T(min) × V(ml) × A_{600 nm})). Under the experimental conditions used in our assays, the background β-galactosidase activity was ∼1 Miller unit.

Western Blotting

An overnight culture of dksA::3×FLAG grown in LB broth was diluted to 10⁸ cfu/ml and treated for 1 h with 1 mm H₂O₂ in PBS. Samples were taken every 15 min, pelleted, resuspended in lysis buffer, and disrupted by sonication. Samples normalized to 100 ng were resolved using 12% (v/v) SDS-PAGE, transferred electrophoretically to a nitrocellulose membrane, and immunoblotted with anti-FLAG M2 monoclonal antibody (Sigma-Aldrich).

In Vitro Susceptibility to Reactive Oxygen Species

Wild-type and dksA mutant bacteria grown overnight in LB broth were diluted in PBS to a final concentration of 10⁵ cfu/well. Where indicated, the expression of zwf was induced by adding 0.2 μg/ml anhydrotetracycline (Sigma-Aldrich) to bacteria diluted in PBS. Bacteria were challenged with 100 or 250 μm H₂O₂ at 37 °C for 2 h. The cultures were serially diluted in PBS, and the bacteria capable of forming a colony were estimated after overnight culture on LB agar plates. Data are represented as percent survival compared with untreated controls.

H₂O₂ Consumption

Strains grown overnight in LB broth were diluted in prewarmed LB medium to an A_{600 nm} of 1. H₂O₂ was added to a final concentration of 250 μm. H₂O₂ consumption was measured polarographically using a H₂O₂-specific probe (World Precision Instruments, Sarasota, FL). Data are represented as μm H₂O₂ over time.

Pyridine Nucleotide Quantification

Pyridine nucleotides were extracted and quantified using a modified cycling method described by Husain et al. (12). Briefly, oxidized and reduced nucleotides were extracted in 0.2 m HCl or 0.2 m NaOH, respectively, from bacterial cultures grown for 20–22 h in LB medium or diluted in PBS with or without 0.4% glucose (w/v) or 0.1% casamino acids (w/v). Quantification of oxidized and reduced forms of pyridine nucleotides was achieved utilizing a modified thiazolyl tetrazolium blue cycling assay (12) using NADH/NAD⁺-specific alcohol dehydrogenase or NADPH/NADP⁺ glucose-6-phosphate dehydrogenase (30–34). Nucleotide concentrations calculated by regression analysis of known standards were normalized for bacterial density as measured spectrophotometrically at A_{600 nm}. Intracellular nucleotide concentrations were calculated taking into account a bacterial cell volume of 10⁻¹⁵ liters (35).

Glutathione Quantification

Total (GSH-GSSG) or oxidized glutathione (GSSG) was extracted and measured using a glutathione recycling method described by Baker et al. (36). Samples were prepared by pelleting 250 μl of cultures grown 20–22 h in LB broth. For experiments aimed at measuring the recovery of GSH after oxidative stress, bacterial cultures grown overnight in LB broth were pelleted, resuspended in PBS, and treated with 1 mm H₂O₂. 200 units of catalase were added after 5 min to eliminate the remaining H₂O₂. 250-μl samples collected 5 and 15 min after the addition of catalase were resuspended in an equal volume of 20 mm EDTA. Samples used for the determination of GSSG were resuspended in 20 mm EDTA containing 2 mm N-ethylmaleimide. Bacteria were lysed by sonication using a 40-s pulse. One volume of 10% HClO₄ was added to the lysates to precipitate proteins. Lysates were neutralized with 93.5 μl of KOH, freeze/thawed, and centrifuged to remove KClO₄ and obtain cleared lysates. N-Ethylmaleimide was removed by ether extraction. A reaction mixture was freshly prepared by adding 2.8 ml of 1 mm 5,5′-dithiobis (2-nitrobenzoic acid), 3.75 ml of 1 mm NADPH, 5.85 ml of 100 mm NaH₂PO₄-5 mm EDTA phosphate-EDTA buffer, and 20 units of glutathione reductase (Sigma-Aldrich). 100 μl of reaction mixture was immediately added to 50 μl of sample in a 96-well microtiter plate and read at A_{412 nm} for 5 min. Concentrations were obtained by regression analysis of known standards. Intracellular GSH-GSSG concentrations were calculated taking into account the number of colony-forming units and a bacterial cell volume of 10⁻¹⁵ liters.

Mouse Infections

Six- to 8-week old C57BL/6 or congenic gp91phox-deficient (37) mice bred in our animal facility according to Institutional Animal Care and Use Committee guidelines were used to assess the role of DksA in Salmonella virulence. Briefly, individual animals were inoculated intraperitoneally with ∼400 cfu of Salmonella grown overnight to stationary phase in LB broth. Mouse survival was monitored over time. Mice manifesting signs of distress (i.e. low spontaneous activity and ruffled coat) were humanely euthanized by CO₂ inhalation.

Macrophage Killing Assays

The anti-Salmonella activity of macrophages was evaluated as described previously (29). Briefly, peritoneal exudate cells were harvested from C57BL/6 or gp91phox-deficient mice 4 days after intraperitoneal inoculation of 1 mg/ml sodium periodate. The cells were cultured for 36–48 h in RPMI⁺ 1640 medium (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (BioWhittaker, Walkersville, MD), 15 mm Hepes, 2 mm l-glutamine, 1 mm sodium pyruvate (Sigma-Aldrich), and 100 units/ml penicillin and 100 mg/ml streptomycin (Cellgro). Macrophages washed with media lacking antibiotics were challenged at a multiplicity of infection of 2 with Salmonella that had been opsonized with 10% normal mouse serum for 20 min. After 25 min of infection, the medium was exchanged with prewarmed RPMI⁺ 1640 medium containing 25 μg/ml gentamicin. Macrophages were lysed with 0.25% deoxycholic acid (w/v) at the indicated time points after infection and the surviving intracellular bacteria enumerated on LB agar plates. Killing is expressed as the fraction of bacteria recovered at the indicated time relative to the bacterial burden isolated after 25 min of internalization.

Statistical Analysis

The data were analyzed using Student's paired t test. Determination of statistical significance between multiple comparisons was achieved using analysis of variance (ANOVA) followed by a Bonferroni post test using transformed data. Data were considered statistically significant when p < 0.05.

RESULTS

Hydrogen Peroxide Induces a Stringent Response

Salmonella, as is true for many bacteria, have several defenses with overlapping roles in the detoxification of reactive oxygen and nitrogen species. Published data indicate that the stringent response is induced in Salmonella exposed to reactive nitrogen species (38). In this work, we investigated whether H₂O₂, another reactive species produced in the innate response to Salmonella, may induce the stringent response. The stringent response is characterized by a down-regulation of translational machinery, including ribosomal protein gene expression. Consequently, transcription of the 30 S ribosomal protein gene rpsM was quantified by real time PCR as a readout of stringent regulation. The expression of rpsM was decreased (p < 0.01) 10-fold in wild-type Salmonella exposed to 1 mm H₂O_2, whereas its transcription remained relaxed in a strain lacking the stringent response regulatory protein DksA (Fig. 1A). The transcription of the 50 S ribosomal protein rplN gene also remained relaxed in H₂O₂-treated dksA mutant bacteria (Fig. 1B). These findings indicate that H₂O₂ induces DksA-dependent stringent regulation. H₂O₂ did not, however, alter the amount of DksA protein in the cell (Fig. 1C). Moreover, the differences in ribosomal protein transcription between wild-type bacteria and the dksA mutant do not appear to reflect differences in growth rates as determined in a Bioscreen C growth analyzer (Oy Growth Curves AB Ltd., Helsinki, Finland) (Fig. 1D).

FIGURE 1.

H₂O₂ induces stringent regulation. A and B, transcription of the stringently controlled ribosomal protein genes rpsM (A) and rplN (B) was evaluated using quantitative real time and reverse transcription PCR, respectively, in wild-type (WT) and dksA mutant bacteria. Transcript levels were normalized to the housekeeping σ factor rpoD. Bacterial cultures grown overnight in LB medium were subcultured 1:10 for 30 min in PBS with or without 1 mm H₂O₂. The data in A represent the mean ± S.E. (error bars) from three independent experiments. **, p < 0.01. C, DksA protein levels were estimated at various times after the bacteria were challenged with 1 mm H₂O₂ in PBS. D, bacterial growth in LB medium is shown.

DksA Increases the Resistance of Salmonella to Oxidative Stress

Because the dksA mutant failed to repress transcription of rpsM and rplN upon exposure to H₂O₂, we tested whether a lack of dksA predisposes this bacterium to reactive oxygen species. H₂O₂, which is generated during the respiratory burst of macrophages in response to Salmonella (1), was chosen in these studies. Cytotoxicity assays were initially conducted using 400 μm H₂O₂. However, this concentration killed all dksA mutant bacteria. Consequently, lower concentrations of H₂O₂ were tested. The dksA mutant exhibited a time-dependent hypersusceptibility to 100 μm H₂O₂ (Fig. 2A). Thirty times fewer dksA-deficient bacteria survived 2-h exposure to H₂O₂ than did wild-type controls. A wild-type dksA allele expressed from the low copy plasmid pWDKS restored the survival of dksA-deficient Salmonella to levels seen in H₂O₂-treated wild-type bacteria. At the low concentrations of bacteria used in these studies (i.e. 10⁵ cfu/well), the decay of H₂O₂ was insignificant over 2 h. The susceptibility of dksA mutant bacteria to H₂O₂-mediated killing is growth-dependent. Logarithmically grown dksA mutant bacteria were remarkably resistant to H₂O₂, whereas they became highly susceptible upon reaching stationary phase (Fig. 2B). The contribution of DksA to the antioxidant defenses of Salmonella appears to be independent of the stringent response alarmone ppGpp because a relA spoT (ppGpp°) mutant was significantly (p < 0.05) less susceptible to H₂O₂ than the isogenic dksA-deficient strain. Moreover, a triple mutant lacking dksA relA spoT was as susceptible to H₂O₂ as the single dksA-deficient Salmonella (p = 0.170). The hypersusceptibility of dksA mutant bacteria to oxidative stress does not appear to be due to a defect in the consumption of H₂O₂ (p = 0.637 compared with wild-type bacteria) (Fig. 2C).

FIGURE 2.

Salmonella lacking dksA are hypersusceptible to H₂O₂. A, survival of wild type (WT), dksA mutant, and dksA mutant complemented with a dksA gene expressed from the low copy vector pWDKS at various times after exposure to 100 μm H₂O₂. The initial counts of WT and dksA mutant at time 0 were ∼10⁵/well. B, killing activity of H₂O₂ against WT Salmonella compared with dksA, relA spoT (ppGpp°), and dksA ppGpp° mutants. The bacteria were grown for 20 h (stationary) or to an A_{600 nm} ∼0.5 (logarithmic). C, H₂O₂-consuming ability of Salmonella grown to stationary phase measured polarographically. The cultures were normalized to bacterial density as determined spectrophotometrically at A_{600 nm}. The data represent the mean percent survival ± S.E. (error bars) of 4–12 independent observations from at least two separate experiments. H₂O₂ consumption represents five independent observations from two separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

DksA Controls the Redox Balance of Stationary Phase Salmonella

Recently, it has been suggested that NADH plays a role in resistance of Salmonella to reactive oxygen species (12). Consequently, the pools of oxidized and reduced pyridine nucleotides were quantified in dksA mutant bacteria. Logarithmically grown wild-type and dksA-deficient Salmonella harbored similar levels of pyridine nucleotides (Table 1). The reduced NADH and NADPH pyridine pools were nonetheless significantly (p < 0.001) lower in stationary phase dksA mutant bacteria than in wild-type controls. Conversely, the oxidized pyridine conjugates were increased in the former (Table 1). The dramatic decrease in reducing power seen in stationary phase dksA-deficient bacteria raises the possibility that DksA controls central metabolic pathways that supply most of the reducing power in the cell. The majority of the NAD(P)H-reducing equivalents are generated in discrete enzymatic reactions of the pentose phosphate pathway and tricarboxylic acid cycle. To test whether steps associated with the production of reducing equivalents may be under control of DksA, lacZY transcriptional fusions were engineered to selected genes. Genes encoding glucose-6-phosphate dehydrogenase (zwf), isocitrate dehydrogenase (icdA), and malic enzyme (maeB) responsible for the generation of NADPH, and the sucAB-encoded subunits of α-oxaloacetate dehydrogenase associated with generation of NADH all showed DksA-dependent regulation (Fig. 3A, bold red font). Several other genes of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle such as gnd, aceEF, and mdh, which are also associated with production of reductive power, do not appear to be regulated by DksA.

TABLE 1.

Effect of the dksA stringent response locus on growth phase levels of pyridine nucleotide pools

Data are from 8–20 observations recorded in at least three independent experiments. ***, p < 0.001 compared with wild-type (WT) bacteria.

Nucleotide pool	Logarithmic		Stationary
	WT	dksA	WT	dksA
NADH	257 ± 68a	357 ± 92	131 ± 24	87 ± 15***
NAD+	1632 ± 232	1532 ± 355	731 ± 64	1202 ± 161***
NADH/NAD+	0.158	0.233	0.179	0.073
NADPH	1535 ± 149	1418 ± 240	394 ± 108	280 ± 39***
NADP+	1391 ± 98	1247 ± 173	915 ± 141	1238 ± 230***
NADPH/NADP+	1.103	1.137	0.431	0.227

^a μm/cell.

FIGURE 3.

DksA-mediated control of central metabolism boosts cellular reductive power and resistance to H₂O₂. A, expression of key dehydrogenases associated with generation of reduced pyridine nucleotides in the pentose phosphate pathway (yellow), glycolysis (blue), or the tricarboxylic acid cycle (green) was evaluated by following lacZY transcriptional activity of the indicated genes. Transcriptional activity was measured in wild-type (WT) Salmonella or dksA mutant bacteria grown for 20 h in LB broth. The data represent the ratio of β-galactosidase activity supported by the mutant bacteria over WT. Genes expressed 2-fold or lower in the absence of dksA are shown in bold. The data are the mean of four to nine independent observations from at least two separate experiments. B and C, NAD(P)⁺ and NAD(P)H pyridine nucleotides were measured in strains bearing mutations in genes shown to be differentially regulated by DksA. The data are presented as the mean ratio ± S.D. (error bars) of reduced to oxidized nucleotide levels from at least 2 separate experiments. D, survival of the indicated mutants 2 h after H₂O₂ challenge is shown. E, survival of WT and dksA-deficient Salmonella 2 h after challenge with 100 μm H₂O₂ is shown. Where indicated, PBS was supplemented with 0.4% glucose or 0.1% casamino acids. The data are the mean ± S.E. (error bars) from 4–12 independent observations from at least two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with WT bacteria.

Mutations were constructed in genes whose expression was decreased 2-fold or more in a dksA background. Strains deficient in sucAB or zwf showed decreases in reductive power compared with wild-type bacteria (Fig. 3, B and C). The sucAB and zwf mutants contained significantly (p < 0.05) lower NADPH intracytoplasmic concentrations than wild-type bacteria. These two strains were also hypersusceptible to 100 μm H₂O₂ (Fig. 3D). On the other hand, strain AV08257 lacking icdA harbored slight increases in NADH/NAD⁺ and NADPH/NADP⁺ ratios and was hyperresistant to H₂O₂ (Fig. 3, B–D), whereas the maeB-deficient strain was hypersusceptible to H₂O₂. Reduced cycling of metabolites from the tricarboxylic acid cycle into glycolysis may explain the hypersusceptibility of the maeB mutant to H₂O₂ because pyruvate is known to be an efficient scavenger of reactive oxygen species (39). Similar to overnight cultures grown in LB medium, the dksA mutant strain cultured in PBS for 1 h harbored lower NAD(P)H/NAD(P)⁺ ratios than wild-type controls (Table 2). The addition of glucose or casamino acids to PBS not only raised the pools of reduced nucleotides but also increased the resistance of dksA mutant Salmonella to H₂O₂ (Table 2 and Fig. 3E). Together, these findings are interpreted as evidence in favor of a model in which carbon fluxes through central metabolic pathways increase the pool of reduced nucleotides that can be used for antioxidant defenses. Nonetheless, excessive carbon fluxing through these metabolic pathways appears to predispose against oxidative stress as shown by the fact that glucose and casamino acids enhanced the susceptibility of wild-type Salmonella to H₂O₂ (Fig. 3E).

TABLE 2.

Effect of glucose or amino acids on pyridine nucleotide levels in dksA mutant Salmonella

Data are from 12–16 independent observations recorded in at least four separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p values in the control group are comparisons between wild-type (WT) and dksA mutant. The p values in the glucose and casamino acids groups are comparisons with the corresponding PBS control.

Nucleotide pool	Control		Glucose		Casamino acids
	WT	dksA	WT	dksA	WT	dksA
NADH	90 ± 21a	49 ± 8*	228 ± 8***	243 ± 35***	234 ± 46**	250 ± 92**
NAD+	690 ± 195	1089 ± 218**	942 ± 121	971 ± 122	710 ± 79	1111 ± 155
NADH/NAD+	0.130	0.045	0.242	0.250	0.330	0.225
NADPH	410 ± 89	310 ± 80**	446 ± 114	815 ± 228**	620 ± 43**	707 ± 103**
NADP+	1026 ± 87	1390 ± 254*	485 ± 87	570 ± 148**	456 ± 97***	567 ± 90***
NADPH/NADP+	0.399	0.223	0.922	1.430	1.361	1.248

^a μm/cell.

Redox Balance Associated with a Functional DksA Supports the Proper Function of Antioxidant Defenses

Intracellular redox potentials can be calculated with the Nernst equation (E = E°- RT/nF × ln(reduced/oxidized)), where E° is the potential of the redox couple, R is the gas constant (8.3145 J·K⁻¹·mol⁻¹), n is the number of electrons transferred in the reaction, and F is Faraday's constant (9.6485 × 10⁴ C·mol⁻¹) (40). Using the intracytoplasmic NADPH/NADP⁺ concentrations found in our studies, we calculated that wild-type and dksA mutant bacteria have an intracellular redox environment of −305 and −287 mV, respectively (Fig. 4A). Although noticeably lower than wild-type bacteria, the values in the dksA mutant should still be thermodynamically favorable for fueling glutathione (GSH) or thioredoxin redox couples with E values of −240 and −270 mV, respectively (41, 42). Given this potential paradox, we investigated whether the decreased reductive power seen in dksA mutant Salmonella may be of consequence to the redox state of GSH. Compared with wild-type controls grown to stationary phase in LB broth, the dksA mutant had significantly (p < 0.001) lower concentrations of reduced GSH, although it contained higher (p < 0.05) pools of oxidized GSH (GSSG) (Fig. 4B). Overall, the dksA mutant had a 3.5-fold lower GSH/GSSG ratio than wild-type bacteria (Fig. 4C). Next, we studied the effect of H₂O₂ on glutathione levels in wild-type and dksA-deficient Salmonella. These studies were performed with 1 mm H₂O₂, which at the high bacterial densities used to biochemically analyze GSH/GSSG ratios, did not affect bacterial viability. The GSH/GSSG ratio decreased in both wild-type and dksA-deficient Salmonella upon challenge with H₂O₂ in PBS (Fig. 4D), demonstrating that GSH is oxidized in response to H₂O₂. To test the turnover of GSSG, 200 units/ml catalase were added to the bacteria 5 min after 1 mm H₂O₂ treatment. The redox GSH/GSSG balance quickly recovered to resting levels in wild-type Salmonella (Fig. 4D). This recovery was accompanied by a 2-fold increase in the total glutathione pool (GSH+GSSG) (Fig. 4E). In contrast, the GSH/GSSG ratio only recovered partially in dksA-deficient Salmonella, which exhibited slower kinetics compared with wild-type bacteria. Moreover, its already smaller GSH+GSSG pool dropped by half in the dksA mutant 10 min after challenge with 1 mm H₂O₂, rebounding to resting levels 10 min later (Fig. 4E). The pool of GSH+GSSG did not decrease appreciably in wild-type bacteria 10 min after exposure to H₂O₂, whereas it increased (p < 0.01) by close to 2-fold 10 min after the addition of catalase.

FIGURE 4.

dksA-deficient Salmonella exhibit a slow turnover of oxidized glutathione. A, redox potentials in wild-type (WT) and dksA mutant Salmonella were calculated according to the Nernst equation factoring NADPH/NADP⁺ intracellular concentrations of stationary phase bacterial cultures. Redox potentials of NADPH, glutathione (GSH), and thioredoxin (Trx) are shown for reference. B, concentrations of reduced GSH and oxidized GSH (GSSG) were quantified in bacterial cultures grown for 18–20 h in LB broth. C, GSH/GSSG ratio is represented. The data are the mean ± S.D. (error bars) of six independent observations from three separate experiments. D and E, recovery of GSH was studied after the bacteria had been exposed to 1 mm H₂O₂ in PBS. 200 units/ml catalase (arrows) were added after 5 min of exposure to H₂O₂. The data are shown as mean GSH/GSSG ratios (D) or total (GSH+GSSG) glutathione (E) from six independent observations collected in three separate experiments. Differences in GSH+GSSG reported in B and E likely reflect growth of the bacteria in LB medium or PBS. F, expression of genes involved in glutathione synthesis was measured as β-galactosidase activity of the indicated transcriptional fusions after the bacteria were grown for 20 h in LB broth. The data are the mean ± S.E. (error bars) of four independent observations from two separate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

The lower GSH+GSSG pool seen in dksA mutant bacteria suggests that de novo GSH biosynthesis may also be regulated by DksA. To test this hypothesis, lacZY fusions were constructed to the genes gshA and gshB encoding glutamate-cysteine ligase and glutathione synthetase, respectively. Fig. 4F shows that the transcription of both steps in the biosynthesis of GSH, and especially the second step catalyzed by GshB, are under control of DksA. Collectively, these data indicate that DksA does not only help maintain a proper reduced nucleotide pool needed in the recycling of GSSG, but also coordinates the expression of glutathione biosynthetic genes.

Conditional Expression of the Glucose-6-phosphate Dehydrogenase Increases the Resistance of the dksA Mutant to H₂O₂

The majority of NADPH-reducing equivalents are generated in the pentose phosphate pathway by Zwf glucose-6-phosphate and Gnd 6-phosphogluconate dehydrogenases. The step catalyzed by Zwf rate limits the flux of carbon through the pentose phosphate pathway. Consequently, it is not surprising that a mutation in the zwf locus lowers cellular reductive power needed for resistance to H₂O₂. We therefore selected zwf as a candidate for the conditional complementation of the dksA mutation. The zwf gene containing its native ribosomal binding site was put under the control of the heterologous tetracycline promoter/operator, generating plasmid pTZWF (Fig. 5A). The pTZWF and pTX suicide plasmids containing a unique FRT site were integrated into the Salmonella chromosome in an intergenic region downstream of the putP gene (43). The constructs were transduced into the dksA mutant strain AV09294, generating AV10080 and AV10158, respectively. The induction of pTZWF with 0.2 μg/ml anhydrotetracycline resulted in the production of large amounts of glucose-6-phosphate dehydrogenase as indicated by Coomassie Blue staining of cytoplasmic soluble proteins (data not shown). Conditional expression of zwf increased the survival of the dksA mutant expressing put::pTZWF by 10-fold when treated with 250 μm H₂O₂ (Fig. 5B). The increased resistance of the dksA mutant expressing zwf under the tet promoter coincided with lower (p = 0.0027) NADP⁺ (dksA put::pTX versus dksA put::pTZWF; 1388 ± 7 μm versus 1255 ± 26 μm) and higher (p = 0.0306) NADPH (dksA put::pTX versus dksA put::pTZWF; 153 ± 4 μm versus 186 ± 12 μm) levels at 30 and 60 min after induction. Consequently, the NADPH/NADP⁺ ratios increased upon induction with anhydrotetracycline (Fig. 5C).

FIGURE 5.

Conditional expression of the glucose-6-phosphate dehydrogenase zwf gene boosts reductive power and increases resistance to H₂O₂. A, diagram of the suicide expression vector pTX containing the tetracycline promoter/operator is shown. The position of R6K origin, gentamicin resistance cassette (GM^r), tetracycline repressor (tetR), FRT, tetracycline promoter elements (tet^p/o), and multiple cloning site are shown. B, pTX empty vector and pTZWF integrated into a neutral site downstream of putP were transduced into wild-type and dksA mutant bacteria. The bacteria were treated with 0.2 μg/ml anhydrotetracycline at the time of challenge with H₂O₂ in PBS. The data represent the mean percent survival ± S.E. (error bars) of three independent observations. C, NADP⁺ and NADPH pyridine nucleotides were measured at the indicated times after the addition of anhydrotetracycline to bacteria cultured in PBS. The data are the mean NADPH/NADP⁺ ratio of four independent observations collected in 3 separate days. *, p < 0.05.

DksA Contributes to Salmonella Virulence in a Murine Model of Acute Salmonellosis

The induction of stringent regulation by H₂O₂ and the high susceptibility of dksA mutant bacteria to H₂O₂ raise the possibility that DksA may play a role in resistance against reactive species generated by NADPH oxidase. To evaluate the role of DksA during acute salmonellosis, C57BL/6 and congenic gp91phox-deficient mice were challenged intraperitoneally with wild-type or its isogenic dksA mutant Salmonella strain AV09294. Bacteria lacking dksA were highly attenuated in C57BL/6 mice (Fig. 6A). About 8,000 cfu of the dksA-deficient strain could be recovered from spleens of infected mice 14 days after inoculation. The isolated bacteria did not grow in E salts minimal medium supplemented with glucose, indicating that suppressor mutations had not developed during the course of the infection. In addition, PCR analysis indicated that the bacteria recovered from viscera of infected mice carried a mutated dksA allele. Although wild-type bacteria killed gp91phox-deficient mice 2 days earlier than the isogenic dksA-deficient Salmonella, the latter regained virulence in gp91phox-deficient mice (Fig. 6B). Together, these data indicate that DksA helps Salmonella withstand the innate host response associated with a functional NADPH oxidase.

DksA Mediates Salmonella Fitness inside Professional Phagocytes

The early innate response of macrophages against Salmonella is dominated by reactive oxygen species generated through the enzymatic activity of the NADPH phagocyte oxidase. Given the dramatic attenuation of a dksA mutant in mice bearing a functional NADPH oxidase, the intracellular survival of wild-type and dksA mutant Salmonella was compared in primary macrophages. The number of Salmonella lacking dksA that were isolated from macrophages 2 h after infection was 3-fold lower than that of wild-type bacteria. Differences in bacterial burden of wild-type and dksA mutant Salmonella augmented at later times in the course of the infection, when wild-type bacteria increased whereas the number of dksA-deficient bacteria remained unchanged (Fig. 6C). The early disadvantage of dksA-deficient Salmonella in the intracellular environment of macrophages suggests that DksA helps antagonize the antimicrobial activity emanating from NADPH oxidase. To test this hypothesis, the intracellular survival of wild-type and dksA mutant bacteria was studied in gp91phox-deficient macrophages. Similar numbers of wild-type and dksA mutant bacteria were recovered from gp91phox-deficient macrophages (Fig. 6D), supporting a role for DksA in defense against products of the respiratory burst.

DISCUSSION

Repression of genes encoding ribosomal proteins, rRNA, and tRNA is a hallmark of the stringent response of bacteria undergoing nutritional deprivation. Findings reported herein indicate that oxidative stress also represses transcription of the rpsM- and rplN-encoded 30 S and 50 S ribosomal proteins. In analogy to NO (38), the apparent induction of stringent regulation by H₂O₂ may be related to nutritional stress. By modifying redox active centers, NO and H₂O₂ affect a variety of cellular processes. In particular, the [4Fe-4S] prosthetic group of the IlvD dihydroxyacid dehydratase has been shown to react with both reactive oxygen and nitrogen species, whereas the redox active thiol group of the MetE cobalamin-independent methionine synthase is a primary target of oxidative stress (2, 44). Oxidation of redox active centers in these two proteins is expected to decrease the biosynthesis of branch-chain amino acids and methionine, thereby providing a mechanism for the H₂O₂-dependent induction of the stringent response.

Investigations presented herein indicate that DksA improves the resistance of Salmonella to the antimicrobial activity of the NADPH phagocyte oxidase. The antioxidant defenses mediated by DksA in Salmonella are functional within macrophages and in an acute model of infection. By exerting transcriptional regulation of central metabolic pathways, DksA controls the production of reducing power that helps detoxify oxyradicals and repair biomolecules damaged by reactive oxygen species. Our investigations indicate that the antioxidant defenses associated with DksA are mostly independent of the traditional stringent response alarmone ppGpp. This is in keeping with the idea that both DksA and ppGpp can have different modes of action and can compensate for amino acid auxotrophies, cell-cell aggregation, motility, filamentation, and stimulation of RpoS accumulation when the other is absent (24). As is the case for the latter examples, further studies are needed to delineate the mechanisms by which DksA regulates redox homeostasis independently of ppGpp.

Bacteria lacking DksA exhibit a normal rate of consumption of H₂O₂ at concentrations of the reactive species frequently seen in inflammatory processes. Thus, DksA does not appear to have a major effect on high rates of H₂O₂ detoxification. In contrast, DksA helps regulate the production of reducing power in stationary phase bacteria. Transcriptional analysis has revealed that DksA enhances the expression of certain steps of the pentose phosphate pathway and tricarboxylic acid cycle that generate NAD(P)H. DksA was found to fine-tune the transcription of zwf, icdA, sucAB, and maeB. Conditional expression of zwf encoding the glucose-6-phosphate dehydrogenase boosted the redox potential and antioxidant defenses of dksA mutant bacteria. Given the fact that DksA plays a global regulatory role and is involved in the transcription of many steps of central metabolism, we found it remarkable that the conditional expression of zwf has such a profound effect on the resistance to H₂O₂ in the dksA mutant. The resistance to H₂O₂ associated with the heterologous expression of zwf was nonetheless delayed 30 min after NADPH accumulated in the bacteria, suggesting that NADPH fuels downstream enzymes. Together, the work presented herein provides evidence in favor of a model in which the generation of reductive power through the DksA-mediated control of central metabolic pathways boosts antioxidant defenses.

Application of the Nernst equation to the calculated intracellular concentrations of the NADPH/NADP⁺ redox couple indicates that the intracellular environment of dksA-deficient bacteria is −287 mV compared with −305 mV of wild type. The diminished redox power of the dksA mutant should still be thermodynamically favorable for fueling glutathione and thioredoxin reductases. Nonetheless, the cytoplasmic environment of dksA-deficient Salmonella is poor in reduced GSH, whereas it harbors high concentrations of GSSG. This observation suggests that the reduced nucleotide pool in dksA mutant bacteria is indeed detrimental to downstream antioxidant defenses, including the glutathione oxidoreductase. Further supporting this idea, the dksA mutant exhibits lower rates in the reduction of GSSG than wild-type bacteria. The pool of ∼200 μm NADPH in dksA mutant Salmonella is still above the estimated K_m of 20 μm that glutathione reductase has for NADPH (45). According to Michaelis-Menten kinetics, the velocity of an enzymatic reaction is dependent on substrate level (V = V_max [S]/K_m + [S], where V is velocity, V_max is maximal velocity, and S is substrate concentration). Considering V_max and K_m constant and taking into account the large pool of GSSG in the dksA mutant, it follows that limited NADPH availability likely drives the slow reduction of GSSG seen in this mutant. Collectively, these findings indicate that low NAD(P)H/NAD(P)⁺ ratios negatively affect the antioxidant defenses of dksA-deficient bacteria.

In addition to low pools of NADPH and NADH, the dksA mutant contains low levels of reduced GSH. This tripeptide is synthesized from cysteine, glutamate, and glycine. Auxotrophies for several amino acids, including glycine, have been described for dksA mutant bacteria (46). Therefore, the reduced transcription of glutathione synthase reported herein and limited glycine availability likely underlie the low GSH pool in the dksA mutant. The defect in GSH biosynthesis might shift the prime targets of oxidative stress from the tripeptide to more essential biomolecules. This idea is supported by the observed drop in measurable GSH+GSSG (possibly through the formation of mixed disulfides) when dksA mutant bacteria were challenged with H₂O₂. A defect in GSH biosynthesis might aggravate susceptibility to oxidative stress of nutritionally deprived bacteria because they appear to synthesize GSH de novo upon treatment with H₂O₂. Because of the global role for DksA in the regulation of gene transcription, it is still possible that DksA controls the expression of other antioxidant defenses as well.

Control of antioxidant defenses by DksA is growth-dependent. Rapidly growing, dksA-deficient Salmonella are even more resistant to H₂O₂ than wild-type bacteria. In contrast, stationary phase dksA mutant bacteria suffer a considerable loss of viability when exposed to H₂O₂. These findings are consistent with the idea that DksA exerts transcriptional control in stationary phase when nutrients become limited. Accordingly, the addition of carbon in the form of glucose or amino acids enhances both the reductive power and the resistance of dksA-deficient Salmonella to H₂O₂. The pool of reduced pyridine nucleotides must nonetheless be tightly controlled to minimize collateral damage. On one hand, NADH and NADPH reductive power fuels the antioxidant activities of alkyhydroperoxidase, and glutathione and thioredoxin reductases. On the other, NADH can act as a pro-oxidant that feeds reducing equivalents to flavoproteins, which can adventitiously generate the Fenton catalyst ferrous iron (47). Accordingly, experiments presented herein show that glucose and amino acids increase the cytotoxicity of H₂O₂ against stationary phase wild-type Salmonella. Therefore, the fine-tuning of central metabolism by DksA during periods of nutritional starvation helps maintain a delicate balance of reduced nucleotides for use in antioxidant defenses while limiting excessive buildup of reducing power that could generate undesired damage.

In conclusion, our investigations have elucidated a novel role for DksA in the antioxidant defenses of Salmonella. The regulation of central metabolic pathways by DksA coordinates the supply of reducing power that fuels antioxidant defenses crucial for resistance of Salmonella to reactive oxygen species produced during the innate host response.