Magnesium homeostasis protects Salmonella against nitrooxidative stress

The PhoPQ two-component regulatory system coordinates the response of Salmonella enterica serovar Typhimurium to diverse environmental challenges encountered during infection of hosts, including changes in Mg2+ concentrations, pH, and antimicrobial peptides. Moreover, PhoPQ-dependent regulation of gene expression promotes intracellular survival of Salmonella in macrophages, and contributes to the resistance of this pathogen to reactive nitrogen species (RNS) generated from the nitric oxide produced by the inducible nitric oxide (NO) synthase of macrophages. We report here that Salmonella strains with mutations of phoPQ are hypersensitive to killing by RNS generated in vitro. The increased susceptibility of ∆phoQ Salmonella to RNS requires molecular O2 and coincides with the nitrotyrosine formation, the oxidation of [4Fe-4S] clusters of dehydratases, and DNA damage. Mutations of respiratory NADH dehydrogenases prevent nitrotyrosine formation and abrogate the cytotoxicity of RNS against ∆phoQ Salmonella, presumably by limiting the formation of peroxynitrite (ONOO−) arising from the diffusion-limited reaction of exogenous NO and endogenous superoxide (O2 •−) produced in the electron transport chain. The mechanism underlying PhoPQ-mediated resistance to RNS is linked to the coordination of Mg2+ homeostasis through the PhoPQ-regulated MgtA transporter. Collectively, our investigations are consistent with a model in which PhoPQ-dependent Mg2+ homeostasis protects Salmonella against nitrooxidative stress.

system enhances the resistance of Salmonella against the nitrooxidative stress generated in the interaction of exogenous NO with endogenously produced O 2 •− through its regulation of intracellular Mg 2+ concentrations.

Results
PhoPQ-deficient Salmonella are hypersusceptible to NO. The PhoPQ signaling cascade coordinates important aspects of the antioxidant and antinitrosative defenses of Salmonella 12,13 . The PhoPQ two component regulatory system is involved in Salmonella defense against Fenton-mediated oxidative stress 7 , however, it is unclear how PhoPQ signaling promotes resistance to RNS. To learn more about the role of PhoPQ in resistance of Salmonella to RNS, we investigated the survival of a ∆phoQ mutant exposed to the NO generator spermine NONOate (sperNO). Most wild-type Salmonella survived 6 h after challenge with 250 µM sperNO, while ∼99% of ∆phoQ Salmonella were killed upon sperNO treatment (Fig. 1A). The NO-mediated killing of ∆phoQ Salmonella was already noted after 4 h of challenge. The susceptibility of ∆phoQ Salmonella to sperNO appears to rely on the generation of NO, because the polyamine spermine control lacked antimicrobial activity (Fig. 1B). A dose-dependent inhibition of growth by sperNO was observed for both ∆phoP or ∆phoQ strains when inoculated in LB broth or minimal E salts medium supplemented with malic acid ( Fig. 1C and Fig. S1), which is consistent with the notion that the PhoP response regulator boosts the antinitrosative potential of Salmonella in conjunction with its cognate PhoQ sensor kinase. The growth of ∆phoQ Salmonella in E salts was completely inhibited by 250 µM sperNO, which corresponded to the dose of sperNO that resulted in significant lethality when cells were challenged in PBS ( Fig. 1 and Fig. S1). The hypersensitivity of ∆phoQ Salmonella to sperNO does not appear to be due defects in viability, as wild-type and ∆phoQ Salmonella strains grew with similar kinetics in LB and various minimal E salts media in the absence of sperNO (Fig. S2). Complementation of ∆phoQ Salmonella with a plasmid encoding a wild-type allele of phoQ (pPhoQ) restored wild-type levels of growth following sperNO treatment (Fig. 1C). Collectively, these data indicate that the PhoPQ two-component regulatory system contributes to the protection of Salmonella against the cytotoxic activity associated with RNS.
Oxygen is required for the NO-dependent killing of phoQ-deficient Salmonella. RNS 23 . To determine whether killing of ∆phoQ Salmonella by sperNO is mediated by NO itself or by a variety of RNS, Salmonella were exposed to 250 µM sperNO in the presence or absence of O 2 . To generate a hypoxic environment, PBS was flushed with N 2 for 10 min and the experiments were carried out in sealed tubes. The viability of wild-type Salmonella was not (P > 0.05) affected by sperNO in either normoxic or hypoxic conditions ( Fig. 2A). In contrast, the NO-dependent killing of ∆phoQ Salmonella was completely abrogated in hypoxic cultures (Fig. 2B). These findings suggest that the PhoPQ two-component regulatory system protects Salmonella against nitrooxidative products formed in the reaction of NO and O 2 metabolites.
The antioxidant defenses associated with PhoPQ signaling rely on the expression of a functional PmrAB two-component regulatory system and the CorA metal transporter 7,24 . The susceptibility of ∆phoQ Salmonella to NO appears, however, to be independent of pmrA (Fig. 2C), which is consistent with previous investigations that reported that a pmrA mutant is as resistant to sperNO as wild-type controls 24 . The hypersusceptibility of phoP mutants to Fe 2+ -mediated oxidative stress can be prevented by a mutation in the corA metal transporter 7 . However, a mutation in corA did not prevent the killing of ∆phoQ Salmonella by sperNO (Fig. 2C). In addition Figure 1. The PhoPQ two-component regulatory system protects Salmonella against RNS cytotoxicity. The susceptibility of wild-type (WT) and ∆phoQ Salmonella to killing by 250 µM spermine NONOate (sperNO) in PBS at 37 °C was compared after 0, 2, 4 and 6 h (A). The bactericidal capacity of 250 µM sperNO was compared to the polyamine base spermine following 6 h of incubation at 37 °C (B). Results represent the mean % survival ± SD of 4 independent observations collected from two separate experiments. *P < 0.01 compared to untreated controls. To determine effects of RNS on the growth of Salmonella, strains were cultured in LB broth in the presence or absence of 2.5 mM sperNO at 37 °C. Growth was determined by measuring the optical density (OD 600nm ) over time in 96-well microtiter plates using a Biotek Cytation 5 multi-mode plate reader (C). Data represent the mean optical density of 3 biological replicates.
to contributing to iron homeostasis, PhoPQ can activate Salmonella's antioxidant defenses through the positive regulation of SodCI expression and the stabilization of RpoS 11,12 . However, neither SodCI or RpoS appear to contribute to the increased susceptibility of ∆phoQ Salmonella under the experimental conditions tested here ( Fig. 2D and E). Interestingly, a strain of Salmonella lacking both phoQ and rpoS was even more susceptible to the RNS-dependent cytotoxicity than the phoQ mutant, suggesting that in the absence of PhoPQ the alternative sigma factor RpoS assumes a critical role in the regulation of the antinitrosative defenses of Salmonella.
Salmonella exposed to NO undergoes nitrooxidative stress. To determine whether wild-type and ∆phoQ Salmonella experience different degrees of nitrooxidative stress upon exposure to sperNO, we monitored the formation of N 2 O 3 (a reactive species generated upon autooxidation of NO in the presence of O 2 ) and nitrotyrosine (an oxidative signature of the reaction of ONOO − or other RNS with tyrosyl residues). Similar concentrations of N 2 O 3 were generated after treatment of wild-type or ∆phoQ Salmonella with 250 µM sperNO (Fig. 3A). Substantial nitrotyrosine formation was also detected within 30 min after Salmonella were challenged with sperNO ( Fig. 3B). Moreover, the profiles and kinetics of nitrotyrosine formation were similar in both wild-type and ∆phoQ Salmonella strains (Fig. 3B). Nitrotyrosine formation was not observed in low O 2 cultures (Fig. 3C), suggesting that ONOO − arising from the diffusion-limited reaction of exogenous NO with endogenous O 2 •− is a likely candidate for the covalent oxidation of tyrosine residues in our experiments. In addition to tyrosine residues, [4Fe-4S] clusters of dehydratases are among the most avid targets (k = 1.4 × 10 5 M −1 s −1 ) of ONOO − 25 . We therefore monitored the enzymatic activity of the [4Fe-4S] cluster-containing aconitase as a surrogate marker of ONOO − mediated oxidative stress. Wild-type and ∆phoQ Salmonella harbored comparable basal levels of aconitase activity (Fig. 3D). Moreover, the aconitase activity of both wild-type and ∆phoQ Salmonella was similarly inhibited 6 h after exposure to sperNO (Fig. 3D). Together, these findings indicate that 250 µM sperNO exert substantial nitrooxidative stress on Salmonella. However, wild-type and ∆phoQ Salmonella appear to be exposed to similar levels of nitrooxidative species under the experimental conditions tested here. This idea is further substantiated by the degree of NO-dependent genotoxicity seen in these two strains of Salmonella. DNA damage was indirectly measured by following the expression of a transcriptional lacZY fusion to the SOS response recA gene. The recA::lacZY transcriptional fusion was similarly induced in both wild-type and ∆phoQ Salmonella after exposure to 12 J/m 2 UV light or treatment with 2.5 mM sperNO (Fig. 3E).

Mutations in NADH dehydrogenases NDH-I and NDH-II protect ∆phoQ Salmonella from NO-dependent cytotoxicity and protein nitration.
We examined in more detail the mechanism underlying the cytotoxicity of RNS against ∆phoQ Salmonella. NADH dehydrogenases of the electron transport chain can be a sizable source of oxidative stress in the cell 26 . We tested whether the sperNO-mediated, O 2 -dependent killing of ∆phoQ Salmonella was the result of the synergism between exogenous NO and O 2 •− arising from the adventitious reduction of O 2 by NADH dehydrogenases of the electron transport chain. To test this hypothesis, the ∆phoQ::km mutant allele was introduced into the ∆nuo ∆ndh mutant strain AV0438 lacking both NDH-I and NDH-II NADH dehydrogenases. As noted for H 2 O 2 and ONOO − 27 , the complex I-deficient ∆nuo ∆ndh strain AV0438 was resistant to 250 µM sperNO (Fig. 4A). Strikingly, strain AV0810 harboring mutations in phoQ, nuo and ndh was also resistant to NO (Fig. 4A). Similar to the complex I-deficient isogenic strain AV0438, strain AV0810 lacking phoQ, nuo and ndh appear to be protected from ONOO − as indicated by a lack of nitrotyrosine . The contribution of the PmrA response regulator, the CorA metal transporter, the copper-zinc superoxide dismutase (SodCI) and the alternative sigma factor RpoS to the resistance of Salmonella to 250 µM sperNO is shown in (C,D and E), respectively. *P < 0.05 compared to WT. **P < 0.05 compared to the ∆phoQ strain. formation 6 h after exposure to 250 µM sperNO (Fig. 4B). Collectively, these data indicate that ONOO − dependent nitrooxidative stress engendered upon reaction of exogenous NO with O 2 •− produced by NADH dehydrogenases of the electron transport chain contributes to the NO-mediated killing of ∆phoQ Salmonella.
Exogenous Mg 2+ rescues ∆phoQ Salmonella from RNS-dependent killing. Previously, Salmonella was shown to exhibit increased susceptibility to oxidative stress following disruptions in Mg 2+ uptake through mutations of phoP or the PhoPQ-regulated Mg 2+ transporters mgtA and mgtB 7 . Therefore, we investigated whether the increased susceptibility of ∆phoQ Salmonella to RNS was due to disruptions in Mg 2+ homeostasis. The increased susceptibility of the ∆phoQ Salmonella strain to killing by 250 µM sperNO in PBS was prevented by the addition of 10 mM MgSO 4 , but had no effect on the survival of the wild-type strain or the ∆phoQ strain complemented with a pPhoQ plasmid (Fig. 5A). Moreover, the sperNO-dependent inhibition of growth of ∆phoQ Salmonella was alleviated by the addition of 10 mM MgSO 4 when cultured in LB with 2.5 mM sperNO experiments. Nitrotyrosine formation in whole-cell lysates isolated from ~ 2 × 10 8 CFU ml −1 of WT and ∆phoQ Salmonella strains exposed to 250 µM sperNO in PBS at 37 °C was measured by Western blotting (B). A cropped image of a western blot showing the effect of O 2 on the generation of nitrotyrosine in WT Salmonella exposed to 250 µM sperNO for 6 h is shown in (C). The NO-dependent damage of [Fe-S] cluster-containing dehydratases was monitored by following aconitase activity from cell lysates harvested from WT and phoQ-deficient Salmonella strains after exposure to 250 µM sperNO for 6 h at 37 °C in PBS (D). The expression of a recA::lacZY transcriptional fusion was quantified using β-galactosidase activity assays in wild-type (WT) and ∆phoQ Salmonella strains following exposure to 12 J/m 2 ultraviolet light (UV) for 30 s or to 2.5 mM sperNO (E). Data in D & E represent the mean ± S.D. of 3 biological replicates. Statistical analysis was performed using a two-way ANOVA for data. *P < 0.001 compared to untreated controls; ns, no statistically significant difference compared to untreated controls. (Fig. 5B). This protective effect of exogenous MgSO 4 was also observed in ∆phoQ Salmonella challenged with 250 µM sperNO in minimal E salts media supplemented with glucose, malic acid, or fumarate (Fig. S2). Moreover, the addition of MgCl 2 also restored the growth of ∆phoQ Salmonella challenged with sperNO, while the addition of CaCl 2 had no effect (Fig. S3). Collectively, these data suggest that the hypersensitivity of ∆phoQ Salmonella to RNS is due to disruptions in Mg 2+ homeostasis.
We hypothesized that the increased susceptibility of the ∆phoQ Salmonella strain to RNS was linked to its inability to upregulate the expression of Mg 2+ transporters encoded by mgtA and mgtB. Therefore, we compared the susceptibility of Salmonella strains lacking mgtA and/or mgtBC to killing by 250 µM sperNO. The ∆mgtA-deficient Salmonella strain showed a significantly increased susceptibility to killing by sperNO compared to both wild-type and ∆mgtBC strains (Fig. 5C). A strain harboring mutations in both mgtA and mgtBC was no more susceptible to killing by RNS as the ∆mgtA mutant strain suggesting that the PhoPQ-dependent regulation of mgtA promotes resistance to RNS. As was the case for the ∆phoQ Salmonella strain (Fig. 5A), the addition of 10 mM MgSO 4 prevented sperNO-dependent killing of the ∆mgtA Salmonella (Fig. 5D). These data suggested that the increased susceptibility of ∆phoQ Salmonella to RNS was due to its inability to upregulate the expression of mgtA, which is required for proper Mg 2+ homeostasis. To test this hypothesis, we introduced a pBAD/HisA plasmid encoding mgtA (pBmgtA) under an arabinose-inducible promoter into the ∆phoQ Salmonella strain, and compared its ability to grow in LB broth in the presence or absence of sperNO. The wild-type, ∆phoQ, and ∆phoQ pMgtA strains showed similar growth kinetics in LB broth (Fig. 5D). As described earlier, ΔphoQ Salmonella were unable to grow in LB broth in the presence of 2.5 mM sperNO (Figs 1C and 5D). In contrast, the introduction of a pMgtA plasmid to the ∆phoQ strain restored growth in LB broth in the presence of 2.5 mM sperNO, albeit with an increased lag compared to the wild-type control (Fig. 5D). This lag was eliminated by the addition of 10 mM MgSO 4 , suggesting that the expression of mgtA from the pMgtA plasmid could only partially restore Mg 2+ homeostasis in the ∆phoQ strain. Collectively, these data support the hypothesis that PhoPQ promotes resistance to nitrooxidative stress in Salmonella through the regulation of Mg 2+ homeostasis.

Discussion
The PhoP regulon controls the antioxidant defenses of Salmonella, Yersinia pestis and Enterococcus faecalis 12,28,29 , and work from our laboratory indicates that this two-component regulatory system also is contributes to the antinitrosative defenses of Salmonella 13 . Elegant investigations by Dr. Groisman's group have elucidated that the  . The data are represented as mean % survival ± SD from 8 replicates collected from 2 separate experiments. *P < 0.01 compared to sperNO-treated WT controls. The OD 600nm was measured over time as described in Fig. 1 to determine the growth of Salmonella strains cultured in LB + 2.5 mM sperNO or LB + 2.5 mM sperNO + 10 mM MgSO 4 at 37 °C (D). Data represent the mean OD 600nm of 3 biological replicates.
Scientific REPORTS | 7: 15083 | DOI:10.1038/s41598-017-15445-y formed adventitiously at the flavin or quinone-binding sites of NADH dehydrogenases of the electron transport chain and its production requires O 2 . The ONOO − produced in the reaction of endogenous O 2 •− and exogenous NO is a powerful nitrating and oxidizing agent that could explain the formation of nitrotyrosine residues in cytoplasmic proteins and the oxidation of the [4Fe-4S] clusters of dehydratases. The protection afforded by mutations in NADH dehydrogenases against ONOOdependent cytotoxicity could be explained by three independent and complementary mechanisms. First, the accumulation of NADH in ∆ndh ∆nuo Salmonella effectively scavenges NO 2 • and OH • radicals caged in peroxynitrous acid (ONO-OH), which is the dominant ONOO − congener at the neutral pH of the bacterial cytoplasm 27 . Second, NADH fuels the enzymatic detoxification of ONOO − by the AhpCF alkylhydroperoxidase 27,30 . And third, a lack of NADH dehydrogenases diminishes ONOO − synthesis by limiting the flow of electrons through the respiratory chain that is required for the generation of O 2 •− . Our investigations suggest that ONOO − is necessary but not sufficient for the NO-mediated antimicrobial activity, because wild-type Salmonella and the phoQ mutant bacteria suffer a similar degree of nitrotyrosine formation and inactivation of aconitase upon exposure to sperNO, but are differently killed by the oxidative congeners of this diatomic radical.
[ •− react with a second order rate constant of 10 9 M −1 sec −1 to form ONOO − 32 . However, high concentrations of NO readily consume ONOO − . Therefore, generation of ONOO − in the Salmonella cytoplasm is most likely to be maximal at the low rates of NO synthesis supported during the innate immune response. In the presence of a functional PhoPQ two-component regulatory system the ONOO − produced endogenously appears to be tolerated by Salmonella.
The Hmp-mediated and cytochrome bd-mediated detoxification of NO, the stringent response, low-molecular weight thiols, along with DNA repair systems minimize the cytotoxicity of NO produced in the innate response to Salmonella 23,33-35 . Our investigations identify PhoPQ-dependent regulation of Mg 2+ homeostasis as an additional antinitrosative defense that shields Salmonella from the cytotoxicity of low NO fluxes. While the hypersensitivity of ∆phoQ Salmonella to RNS is tied to disrupted Mg 2+ homeostasis, this phenotype appears to be independent of both PmrAB-dependent and CorA-dependent resistance Fe 2+ toxicity. This conclusion is supported by the fact that 1) pmrA or corA single mutant strains do not exhibit increased sensitivity to RNS compared to wild-type strains, and 2) phoQ pmrA or phoQ corA double mutant Salmonella strains were as sensitive to killing by sperNO as ∆phoQ Salmonella. Cellular concentrations of Mg 2+ in Salmonella are capable of reaching 100 mM 36 , with the majority of Mg 2+ bound to ribosomes and nucleotide triphosphates 37 . Therefore, the reduced cytoplasmic Mg 2+ concentrations in ∆phoQ Salmonella may increase the susceptibility to killing by RNS due to several factors including 1) reduced protein synthesis resulting from the dissociation of Mg 2+ from ribosomes, and 2) leaching of Mg 2+ from nucleotide triphosphates preventing their use as substrates for enzymatic reactions necessary to repair RNS-induced cellular damage. This is supported by the fact that the addition of 10 mM MgSO 4 rescued both ∆phoQ and ∆mgtA Salmonella strains from RNS-dependent killing.
Independent of the regulation of antioxidant defenses or targets of nitrooxidative stress, a functional PhoPQ two-component regulatory system is likely to promote antinitrosative defenses through the activation of SPI2 transcription [38][39][40] , because the SPI2 type III secretion system has been shown to minimize fusion of Salmonella-containing vacuoles with vesicles harboring iNOS 41 . In contrast to the concerted and rich repertoire of antinitrosative defenses that protect Salmonella against the low NO fluxes produced in the innate response, no antinitrosative defenses are known to protect this facultative intracellular pathogen against the massive nitrosative stress unleashed in IFNγ-activated macrophages. Paradoxically, N 2 O 3 and other high oxidation NO congeners generated by IFNγ-primed macrophages exert profound anti-Salmonella activity by repressing SPI2 transcription and PhoPQ signaling 13,16,17 . In turn, RNS-dependent repression of PhoPQ signaling and SPI2 transcription promotes the maturation of the Salmonella phagosome along the degradative pathway for fusion with lysosomes.
In summary, this study has revealed that in addition to its known roles in protecting Salmonella from acid pH, bile salts, antimicrobial peptides, and oxidative stress 1,4-10 , the PhoPQ two-component regulatory system contributes to the resistance of Salmonella against the nitrooxidative stress generated in the reaction of exogenous NO and endogenously produced O 2 •− by maintaining Mg 2+ homeostasis (Fig. 6).

Methods
Bacterial Strains. Salmonella enterica serovar Typhimurium strain ATCC 14028 s was used throughout this study as wild-type, and as a background for the construction of mutations and a recA::lacZY transcriptional fusion ( Table 1). The mutations were generated following the one-step, λ-Red-mediated gene replacement method of Datsenko and Wanner 42 . Briefly, primers encoding 40-42 nucleotides homologous to the target gene followed by 20 nucleotides homologous to the pKD13 template plasmid were used for the PCR amplification of the Flp recombinant target (FRT)-flanked kanamycin resistance cassette. The resulting PCR products were DpnI digested and electroporated into S. Typhimurium strain TT22236 carrying the pTP2223 plasmid that expresses the λ-Red recombinase under Ptac control. Mutations were moved between strains by P22-mediated transduction and pseudolysogens eliminated by streaking on Evans blue uranine agar plates. Nonpolar deletions were generated by recombining the two FRT sites flanking the kanamycin resistance cassette with the Flp recombinase encoded by the pCP20 plasmid 43 . The mutations were confirmed by PCR analysis. A recA::lacZY transcriptional fusion was constructed by the pCP20-mediated integration of pCE36 encoding a promoterless lacZY operon into the unique FRT scar engineered immediately downstream of the recA stop codon. The pMgtA plasmid was generated by cloning a wild-type copy of the magnesium transporter mgtA into the pBAD/HisA vector using the primers listed in Table 2. The pMgtA plasmid was electroporated into Salmonella strain AV0475 carrying a ∆phoQ::FRT allele to produce the TJB1301 strain. in Salmonella strains exposed to 250 μM spermine NONOate was determined indirectly by following the formation of the N-nitrosonapthalen derivative of 2,3-diaminonaphthalen (Sigma-Aldrich) as described 45 . A 100 mM stock of 2,3-diaminonaphthalen prepared in dimethylformamide was used at a final concentration of 200 μM in PBS. Accumulation of N-nitrosonapthalen was recorded for 30 min following treatment of Salmonella strains with spermine NONOate. Fluorescence was measured on a Synergy HT fluorometer (BioTek) set at λ ex = 375 nm and λ em = 460 nm.

Detection of nitrotyrosine formation by Western blot analysis. Salmonella strains grown in
Luria-Bertani (LB) broth with shaking at 325 r.p.m. at 37 °C for 20 h were pelleted by centrifugation, and resuspended in PBS to an OD 600 of 0.5. Bacteria were incubated at 37 °C in the presence or absence of 250 μM spermine