Nitric Oxide in Salmonella and Escherichia coli Infections

ANDRÉS VÁZQUEZ-TORRES^1* AND FERRIC C. FANG²

[SECTION EDITORS: FERRIC FANG AND MARTIN KAGNOFF]

November 23, 2005

Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262,¹ and Departments of Laboratory Medicine and Microbiology, University of Washington School of Medicine, Seattle, WA 98195-7242²

*Corresponding author. Phone: (303) 315-0313, Fax: (303) 315-6785, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

INTRODUCTION

The chemical reactivity of nitrogen oxides has been exploited in circumstances as varied as clinical medicine, in which these compounds have been administered for over 150 years to treat cardiovascular disorders, or the food industry, in which they have been used as popular preservatives to cure meats. Prout's discovery in 1818 that the urine of febrile patients contains abnormally high nitrite levels pointed to the existence of an endogenous biosynthetic pathway for nitrogen oxides that can be activated as a consequence of inflammation. This finding went largely unnoticed until the 1970s when Tannenbaum reported that the levels of nitrite secreted in human urine exceeded those accounted for by the nitrogen oxides ingested in food (148, 149). The existence of an endogenous nitric oxide (NO) biosynthetic pathway induced in response to inflammatory stimuli was finally demonstrated by Stuehr and Marletta, who showed that lipopolysaccharide (LPS), a major outer membrane structural component of gram-negative bacteria, and gamma interferon (IFN-γ), the prototypical cytokine of T-helper-1-type immune responses, activate murine macrophages to secrete NO (144, 145).

A family of hemoproteins known as NO synthases (NOS) catalyze the oxidation of l-arginine to generate NO and citrulline (92, 109, 142) (Fig. 1). This reaction consumes l-arginine, NADPH, and oxygen, and involves the prosthetic groups heme, tetrahydrobiopterin, calmodulin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Although enzymatic production of nitrogen oxides was first suspected in humans, many groups of investigators encountered difficulties in demonstrating high levels of NO synthesis and inducible NOS (iNOS) expression in human mononuclear phagocytes when compared with their murine counterparts (3, 134). It is now clear that differences in iNOS promoters underlie the disparity in NO output and iNOS regulation between rodent and human mononuclear phagocytes (21, 80, 112, 151). More recently, detailed analysis of different populations of inflammatory mononuclear phagocytes and novel in vitro stimulation protocols have convincingly demonstrated that human mononuclear phagocytes can express iNOS mRNA and protein, sustain abundant NO synthesis, and carry a battery of reactive nitrogen species (RNS) in their antimicrobial arsenal (21, 45, 167). Of particular interest in the context of this chapter, patients with defects in IFN-γ or interleukin 12 (IL-12)-signaling pathways, which are known stimulators of iNOS transcription, are naturally susceptible to salmonellosis (33). Furthermore, human mononuclear cells have been shown to exert enhanced NO-dependent cytotoxicity against Salmonella mutants that are unable to mount a protective response against nitrosative stress (141).

Fig. 1Enzymatic biosynthesis of NO. NOS hemoproteins oxidize the guanidine group of l-arginine to generate NO and l-citrulline. The reaction consumes molecular oxygen and requires NADPH and tetrahydrobiopterin. N^ω-Hydroxy-l-arginine is an intermediate of this enzymatic reaction. Reproduced with permission from reference 143.

Shortly after NO was found to be secreted by mammalian cells, Hibbs et al. associated NO with phagocyte tumoricidal activity (65). NO-mediated cytotoxicity is not limited to tumors but forms an integral part of the immune response to viruses, bacteria, fungi, and parasites (34, 162). Furthermore, evidence compiled from diverse disciplines in medicine have shown that endogenously produced NO participates in a variety of physiological processes that include neurotransmission and regulation of blood pressure, as well as pathological disorders as varied as inflammation and autoimmunity. This chapter discusses the role that NO and its congeners play on various stages in the pathophysiology of Escherichia coli and Salmonella infections, with special emphasis on the regulatory pathways that lead to high NO synthesis, the role of RNS in host resistance, and the bacterial molecular targets and defense mechanisms that protect enteric bacteria against the nitrosative stress encountered in diverse host anatomical sites.

REGULATION OF iNOS EXPRESSION

Enzymatic NO synthesis is catalyzed by three classes of NOS (109, 142) (Fig. 2). Endothelial and neuronal isoforms (NOS1 and NOS3) are expressed for the most part constitutively, and their activity is controlled by the availability of cytosolic calcium in the cell. In contrast, the iNOS hemoprotein (also named NOS2) is not usually expressed in resting cells but is induced in response to a variety of host factors and microbial products, of which LPS and IFN-γ are the best characterized (144, 145). TLR4 is the main receptor of LPS (120). Binding of LPS to TLR4 activates the translocation of NF-κB heterodimers to the nucleus, where they bind cognate DNA sequences in the promoters of targeted genes such as iNOS (171). In contrast to other signaling pathways elicited by gram-negative bacteria, LPS-TLR4 signaling appears to be sufficient to directly induce iNOS expression, RNS production, and anti-Salmonella activity (105, 128). The unique ability of this pattern recognition receptor to induce iNOS transcription by itself stems from its ability to trigger IFN-β production and STAT1 phosphorylation (153). Mobilization of STAT1 homodimers by TLR4 helps to rationalize the requirement for IFN/JAK/STAT1-dependent transcriptional regulator IRF1 in LPS-induced iNOS expression (74). Direct activation of iNOS expression by LPS/TLR4 signaling is of critical importance for limiting the bacterial burden early in the course of infection until an antigen-specific adaptive immune response can be generated. The indispensability of TLR4-dependent signals in innate resistance to salmonellosis is demonstrated by the inability of alternative signaling cascades to substitute for the lack of TLR4 (127, 150, 162). In addition, LPS/TLR4-stimulated NF-κB/ IRF1 transcriptional factors synergize with IFN-γ-induced IRF1signals for optimal iNOS transcription (74).

Fig. 2Differential regulation of constitutive and inducible NOS isoforms. Translated monomers of constitutive and inducible NOS (cNOS and iNOS) bind avin. The presence of l-arginine, tetrahydrobiopterin, and heme stimulate dimerization of cNOS, resulting in the pairing of oxygenase and reductase domains of opposite monomers. Elevated cytosolic calcium (white) increases the affinity of calmodulin (green) for homodimeric cNOS. Binding of calmodulin to an exposed hydrophobic site (red) of cNOS induces a conformational change in the reductase domain, which allows electron flow to the heme oxygenase domain. In contrast to cNOS, the hydrophobic site (orange) of monomeric iNOS binds calmodulin at trace intracellular calcium concentrations. The enzymatic activity of iNOS is thus controlled by the availability of l-arginine, tetrahydrobiopterin, and heme, which are normally plentiful in cells transcribing iNOS mRNA. Therefore, regulation of cNOS is controlled by cytosolic calcium levels, whereas regulation of iNOS is controlled primarily at the level of transcription. Reproduced with permission from reference 109.

Salmonellacan also engage TLR4-independent signaling pathways to stimulate the expression of iNOS mRNA in macrophages. However, in contrast to TLR4, the induction of iNOS by these alternative pattern recognition receptors requires costimulation. IFN-γ acting together with lipoprotein-, fimbria-, or porin-stimulated TLR2-signaling cascades activates iNOS transcription (4, 95, 133). In addition to IFN-γ, other host factors such as tumor necrosis factor α (TNFα) can also stimulate the transcription of iNOS in response to gram-negative enteropathogenic bacteria (1, 44), although NO synthesis can occur in the absence of TNFα/TNFRp55 signaling in Salmonella-infected macrophages (159). Furthermore, the presence of the divalent metal transporter Nramp1 increases NO production and macrophage anti-Salmonella activity via stimulation of IRF1 expression (47). The capacity of TNFα and Nramp1 to stimulate iNOS expression still probably depends on microbial stimuli because the expression of these host factors is governed by signaling cascades or cytokines triggered in response to Salmonella (55, 56).

Although most of the studies analyzing the role of iNOS on the pathogenesis of enteric bacteria have been conducted in macrophages, a multitude of eukaryotic cells have been shown to be capable of expressing iNOS. For example, a growing body of evidence indicates that epithelial cells of the intestinal mucosa can express iNOS in response to gram-negative enteropathogenic bacteria such as E. coli and Salmonella (22, 39, 71, 126). Two pathways have been invoked by which enteric bacteria can activate iNOS transcription in epithelial cells. First, binding of flagella or fimbriae to TLR2 and TLR5 pattern recognition receptors expressed on epithelial cells has been shown to activate IL-1 receptor-associated kinase (IRAK) signaling cascades, nuclear translocation of NF-κB complexes, and iNOS expression (4, 39, 105). In addition to these pathways, Salmonella can stimulate iNOS transcription by directly activating signaling cascades through the enzymatic activity of effectors of the type III secretion system encoded within the Salmonella pathogenicity island 1 (19, 20). Stimulation of iNOS expression by SPI1 effectors may be of biological significance in the epithelium and in macrophages associated with the gastrointestinal mucosa and could play several roles in the pathophysiology of gastroenteritis.

BACTERIAL TARGETS OF NO-MEDIATED CYTOTOXICITY

Biological Chemistry of NO

In general, NO can react directly with prosthetic groups containing transition metal centers, with other radicals, or with sulfhydryl groups in the presence of an electron acceptor (Fig. 3). Binding to iron complexes is probably the best characterized direct reaction of NO in biological systems. As discussed below, NO directly interacts with several E. coli and Salmonella hemoproteins and transcriptional regulators containing nonheme iron prosthetic groups. In addition, NO can indirectly mediate important biological actions via RNS generated from its interaction with molecular oxygen and superoxide. The reaction of NO with molecular oxygen follows second- and third-order kinetics for NO. This chemistry requires high NO fluxes that are likely to be sustained in activated macrophages during the course of inflammatory processes. N₂O₃ generated during the reaction of NO with oxygen is a potent S- and N-nitrosating species that promotes the formation of S-nitrosothiols and N-nitrosamines and, in addition, can act as an oxidant. Another indirect reaction of NO is via peroxynitrite, which arises from the interaction of NO and superoxide anion. Peroxynitrite is a strong oxidant capable of nitrating a variety of substrates including tyrosine residues. The presence of nitrotyrosine in infected macrophages and tissues has suggested that peroxynitrite might play a role in Salmonella pathogenesis. However, formation of nitrotyrosine is not unequivocal proof of peroxynitrite synthesis, since this modification can arise by the enzymatic action of peroxidases on nitrite, hydrogen peroxide, and tyrosine (41) or by nitrite alone at acid pH (70). This chemistry is most likely to occur in the setting of acute inflammation. In fact, nitrotyrosine in Salmonella-infected tissues has been correlated with the expression of both myeloperoxidase and iNOS (2).

Fig. 3NO redox chemistry. Nitric oxide (NO) is generated enzymatically by NO synthases (NOS). NO and superoxide radicals react avidly to form peroxynitrite, a species that is protonated under acidic conditions, giving rise to a reactive intermediate with properties of nitrogen dioxide and hydroxyl radicals. Through its reactions with oxygen, NO is oxidized ultimately to nitrate. In the process, other reactive species such as N₂O₃ and ^•NO₂ are formed (not represented). NO can oxidize thiol groups to sulfenic acid or nitrosylate them to form nitrosothiols. Reproduced with permission from reference 108.

NO Targets

The targets of RNS are numerous. RNS can facilitate oxidative modifications including lipid peroxidation, hydroxylation, and DNA base and protein oxidation. In addition, RNS can inflict nitrosative stress through the nitrosation of amines and sulfhydryls. These modifications can result in nucleic acid deamination, enzyme inhibition (e.g., DNA ligase), zinc finger disassembly, and Fe-S cluster degradation.

Salmonellaand E. coli are exquisitely sensitive to an assortment of RNS in vitro, some of which (e.g., S-nitrosoglutathione [GSNO]) are bacteriostatic, whereas others (e.g., peroxynitrite) are directly bactericidal (2, 10, 30). In addition to their independent antimicrobial actions, RNS can potentiate the antimicrobial activity of reactive oxygen species (ROS) in vitro (10, 116, 170). The synergistic actions of NO on H₂O₂-mediated cytotoxicity do not seem to be a consequence of the modification of Fe-S clusters or depletion of the intracellular glutathione pool by NO but rather result from the reversible inactivation of cytochrome bo and bd quinol oxidases (14, 67, 116, 170). According to a model advanced by Imlay and coworkers, the inhibition of respiration by NO makes NADH available for the reduction of FAD to FADH₂. The reduced flavin regenerates the Fenton catalyst Fe²⁺. In other words, NO indirectly potentiates H₂O₂-mediated DNA damage by promoting the reduction of free iron. However, the importance of this interaction has yet to be demonstrated in the context of bacterial pathogenesis and is called into question by the temporally distinct anti-Salmonella activities of the NADPH oxidase and iNOS in both macrophages and mice (96).

E. coli /Salmonella NO Targets

Despite having an unpaired electron in the outer orbital, NO has the potential to freely diffuse across lipid membranes. In addition, low-molecular-weight S-nitrosothiols can be actively transported by enteric bacteria across the cell wall into the cytosol. The dipeptide permease encoded in the dpp operon has been shown to facilitate the cytotoxicity of GSNO for Salmonella ( 30). A γ-glutamyl transpeptidase cleaves the glutamic acid of GSNO to generate S-nitrosocysteinylglycine, which is transported by the Dpp permease into the Salmonella cytosol. S-Nitrosocysteinylglycine can then liberate NO homolytically or donate NO⁺ equivalents in transnitrosation reactions. Inside the cell, NO can modify a number of targets containing Fe-S clusters, heme groups, zinc fingers, or other radical species.

Fe-S Clusters.

Dehydratases, including aconitase, fumarase, glyceraldehyde-3-phosphate dehydrogenase, and 6-phosphogluconate dehydratase, contain Fe-S prosthetic groups that are susceptible to RNS (50, 51, 59, 75, 170). Whether the E. coli 4Fe-4S protein aconitase, an enzyme that converts citrate to isocitrate in the Krebs cycle, can be directly or indirectly modified by NO has been the subject of intense debate. Three iron molecules in the cluster are bound by cysteine residues, while the fourth ferrous atom is accessible to the substrate. There appears to be a consensus that oxidants such as peroxynitrite or superoxide react with the apical iron in the ferrous state, inactivating the enzyme irreversibly (possibly by inducing formation of disulfide bonds) (59, 75). However, whether NO can directly react with the solvent-exposed iron in the 4Fe-4S cluster is controversial (51, 59). Electron paramagnetic resonance spectroscopy has revealed the generation of dinitrosyl-iron complexes in NO-treated Fnr (4Fe-4S) and SoxR (2Fe-2S) transcriptional regulators (26, 36). These observations are consistent with the view that NO by itself is capable of modifying Fe-S clusters.

Heme-Containing Metabolic Enzymes.

Enterobacteria express several hemoproteins that are susceptible to NO inhibition. Cytochrome bo contains a low-spin heme b coordinated by two histidines, a high-spin heme o ligated by a single histidine, and a Cu_B atom coordinated by three histidines. Although cytochrome bo bears two heme groups, the Cu_B site can be the target of up to two nitrosylations (14). In addition to cytochrome bo, E. coli contains a bd-type ubiquinol oxidase that is expressed in the respiratory chain under low-oxygen tension (58). In this case, NO inhibits the two heme groups of the db binuclear center (63, 67, 170). E. coli respiratory cytochromes can be protected by induction of the Hmp flavohemoglobin, which metabolizes NO to nitrate (140). In addition to binding cytochromes of the aerobic respiratory chain, NO has been shown to inhibit the E. coli hemoprotein catalase (11) and to bind to the reduced iron of E. coli and Salmonella Hmp flavohemoglobins (49, 60).

Zinc Fingers.

Zinc-binding motifs, also known as zinc fingers, mediate a wide variety of protein-DNA or protein-protein interactions. Although zinc-binding motifs are highly pleomorphic, free thiols are generally involved in coordination of the metal center. Recent observations have revealed that S-nitrosylation of sulfhydryls coordinating the zinc finger releases the metal, reversibly inhibiting protein function (82, 83). In Salmonella, the bacteriostasis and cell filamentation seen in response to GSNO coincides with intracellular zinc mobilization (132). Zinc fingers in the metalloproteins PriA, DnaG, and DnaJ, which are involved in DNA replication and reinitiation of collapsed replication forks, might be targets of S-nitrosylation in these experiments. In support of this hypothesis, the SOS response, which is elicited in the absence of functional PriA (115), is induced in GSNO-treated Salmonella in association with zinc mobilization, bacteriostasis, and cell filamentation (132).

Organic Radicals.

As described above for superoxide anion, molecules containing unpaired electrons are extraordinarily susceptible to attack by NO. Ribonucleotide reductase is one such target. By catalyzing the reduction of ribonucleotides to deoxyribonucleotides, ribonucleotide reductases play a critical role in DNA synthesis. In E. coli, as is the case for other class I ribonucleotide reductases, the active site of the enzyme contains a tyrosyl radical that is easily quenched by NO (57, 84).

Methionines.

Methionines can be oxidized to the sulfoxide Met-O form by ROS and RNS. This lesion can be lethal if left unrepaired as suggested by the RNS hypersusceptibility of E. coli lacking the methionine sulfoxide reductase MsrA (139). The reversibility of this oxidative lesion has prompted the idea that surface-exposed methionines may serve as repairable sinks that protect intracellular targets from oxidative damage (85). The cytotoxicity of NO donors against msrA mutant E. coli is only manifested under aerobic conditions, suggesting that RNS generated during the reaction of NO and oxygen mediate the oxidation of methionine. Peroxynitrite is a likely candidate since it has been shown to oxidize methionines (122, 165), including those present in the E. coli chaparone GroEL (77).

BACTERIAL DEFENSES AGAINST NO-MEDIATED CYTOTOXICITY

Numerous vital bacterial molecules can be targeted by NO. It is therefore not surprising that enteropathogenic bacteria are armed with a number of sensors to coordinate the protective response to nitrosative stress, along with an assortment of antinitrosative defenses that detoxify, repair, or avoid the deleterious effects of RNS encountered within the host.

Coordinated Actions Mediated by NO Sensors

Metal cofactors and free sulfhydryl groups have been coopted by transcriptional activators as sensors for nitrosative stress. The transcriptional activator SoxR was the first NO sensor to be identified in enteric bacteria (114). Oxidized SoxR has high affinity for the soxS promoter, whereas reduced SoxR binds to this promoter but it does not recruit the σ⁷⁰ RNA polymerase (35). Nitrosylation of the iron in the 2Fe-2S cluster activates SoxR (36). Activation of the soxRS operon by NO induces the expression of glucose-6-phosphate dehydrogenase, which generates NADPH-reducing equivalents for antioxidant enzymes, as well as ferrodoxin:NADPH oxidoreductase, involved in repair of oxidized Fe-S clusters (89). Nitrosative stress is also a signal that activates OxyR, a transcriptional regulator that coordinates the expression of E. coli and Salmonella antioxidant defenses against hydrogen peroxide. OxyR DNA binding is stimulated by the oxidation of a redox-active cysteine residue to sulfenic acid, which may subsequently form a disulfide bond with a vicinial cysteine residue (172). Alternatively, OxyR can directly sense NO through S-nitrosylation of one of this cysteine (62). The induction of gor by S-nitroso-OxyR maintains a pool of reduced glutathione that can help to detoxify RNS. Recent data, however, suggest that SoxR and OxyR are not primary regulators of the E. coli nitrosative protective response (106). It is possible that in the normally reduced environment of the cytoplasm (62), NO does not nitrosylate either the 2Fe-2S cluster of SoxR or the redox active cysteines of OxyR. However, depletion of glutathione in the oxidative environment of phagocytes might conceivably enable NO sensing by these regulators (113).

The iron prosthetic group of the transcriptional factor Fur (ferric uptake regulation), which is involved in maintaining iron homeostasis, has also been coopted as an NO sensor (28). Formation of a nitrosyl-iron-Fur complex derepresses transcription of Fur-regulated genes in Salmonella and E. coli (27, 28). Similarly, nitrosylation of the 4Fe-4S prosthetic group of Fnr allows expression of the E. coli hmp gene (26). More recently, the NO reductase regulator NorR has also been shown to sense various RNS (106). Although the mechanism by which NorR senses NO is not known, it is likely to involve one or more cysteine residues (105). NorR activates the expression of the NO-consuming flavorubredoxin and flavorubredoxin reductase system (50). It will be interesting to determine the contribution of NorR, which is preferentially expressed under low oxygen tension, to the virulence of E. coli and Salmonella in the microaerophilic or anaerobic environments of the phagosome, granuloma, and intestinal mucosa. There appear to be additional NO sensors, since hmp is expressed normally in E. coli lacking metR, fur, norR, soxR, or oxyR (106). The global regulatory protein ArcA is an additional potential NO sensor (87).

Enzymatic Detoxification

The field of oxygen radical biology followed in the wake of the discovery of the superoxide-consuming activity of superoxide dismutase. Therefore, the long interval between the discovery of endogenous NO synthesis and the first identification of RNS-metabolizing enzymes is somewhat surprising. A variety of enzymes with different catalytic sites and various prosthetic groups are now known to detoxify NO and other RNS.

Detoxification of NO.

E. coliand Salmonella express distinct flavohemoproteins that oxidize NO to NO₃^– or reduce NO to N₂O. Flavohemoglobins contribute to the inducible nitrosative protective response of bacteria as evolutionarily diverse as Mycobacterium tuberculosis and gram-negative enteric bacteria. Hmp flavohemoglobins convert NO to NO₃^– in a reaction that requires oxygen, NADPH, and FAD (25, 52, 61, 78, 101). Although the dioxygenase activity of Hmp is conserved in E. coli and Salmonella, divergent mechanisms seem to regulate hmp expression in these enteric bacteria. In Salmonella, for example, NO-Fe-Fur derepresses transcription of hmp (24), whereas in E. coli hmp is derepressed by NO-Fe-Fnr and induced in response to intracellular nitrosylated homocysteine levels and MetR binding to the glyA-hmp intergenic region (26, 102, 121). MetR-independent regulation also seems to operate in E. coli (106). The induction of Hmp has been shown to protect E. coli and Salmonella against cytotoxicity associated with several NO congeners (25, 52, 61, 101) and to defend Salmonella against NO-mediated killing by human macrophages (141).

NO is an intermediate of the nitrogen cycle, in which nitrate/nitrite are reduced to molecular N₂. In denitrifying bacteria, NO reductases contain a heme b₃-Fe_B binuclear center that converts NO to N₂O (173). It has recently been discovered that the E. coli and Salmonella type A flavoprotein flavorubredoxin represents another class of NO reductase (7, 98, 118). Flavorubredoxin contains a nonheme di-iron site that catalyzes the reduction of NO to N₂O (49, 54). This dinuclear metal center is reminiscent of cytochrome c oxidase and cytochrome bo, but with iron instead of a copper as the nonheme metal (131, 158). In E. coli, NorR is a positive regulator of the flavorubredoxin encoded by the flrd gene, whereas Fnr downregulates its expression. In contrast to Hmp, expression of flavorubredoxin occurs preferentially under anaerobic conditions (29, 50). The role of flavorubredoxin in pathogenesis awaits investigation, although flavorubredoxin has been shown to protect the Krebs cycle aconitase and the Entner–Doudoroff pathway 6-phosphogluconate dehydrogenase from NO-related cytotoxicity (50).

Detoxification of S-Nitrosothiols.

Ascorbate, thiols, and copper ions detoxify S-nitrosothiols in vivo (135, 138). More recently a number of enzymes, including the thioredoxin system, glutathione peroxidase, γ-glutamyl transpeptidase, xanthine oxidase, and glutathione-dependent formaldehyde dehydrogenase, have been shown to have GSNO-consuming activity (66, 68, 72, 110, 154). E. coli glutathione-dependent formaldehyde dehydrogenase is a GSNO reductase that detoxifies GSNO to oxidized glutathione (GSSG) and NH₃ (86). In addition, GSNO can be consumed by the thioredoxin system in a reaction that generates glutathione, NO, and superoxide (110)

Detoxification of Peroxynitrite.

Peroxynitrite (ONOO ⁻) and its conjugated acid peroxynitrous acid (ONOOH) are products of the reaction of NO and superoxide. Peroxynitrite is a potent oxidizing and nitrating agent that can modify lipids, amino acids (cysteine, methionine, tyrosine, and tryptophan), DNA, and the metal prosthetic groups of cytochromes and Fe-S-containing dehydratases (124). These reactions are mediated by peroxynitrite itself or by peroxynitrous acid via caged hydroxyl and nitrogen dioxide radicals (OH^•-NO₂^•) (122). Despite its high reactivity, peroxynitrite is a relatively stable species (81), a property that increases the number of potential targets and necessitates its enzymatic detoxification. Enteric bacteria can enzymatically detoxify peroxynitrite by two independent strategies. The first was revealed by the observation that Salmonella lacking the periplasmic Cu/Zn superoxide dismutase SodCI are hypersusceptible to the synergistic cytotoxicity of superoxide and NO produced either chemically in vitro or by macrophages in vivo (31, 130). The superoxide-consuming activity of periplasmic SodC prevents the formation of peroxynitrite in situ. SodC can effectively compete with NO for superoxide because the rate constants for the reaction of superoxide with NO and Cu/Zn SOD are approximately 6.7 × 10⁹ and 1.4 × 10⁹ M^–1 s^–1, respectively (46, 130). Alternatively, the peroxyredoxin-alkylhydroperoxide reductase AhpC can directly detoxify peroxynitrite to nitrite (12, 17). The role of ahpC in pathogenesis remains to be established, and one group has reported that ahpC is dispensable for Salmonella virulence (152). Temporally dissociated ROS and RNS production in vivo, limited lipid diffusion of peroxynitrite anion, and strategically localized SodC enzymes may all contribute to the apparent dispensability of ahpC in Salmonella virulence.

Nonenzymatic Detoxification/Scavengers

Small molecules containing thiol groups provide a potent means to remove radicals. Two endogenous thiol-containing compounds that antagonize nitrosative stress have been identified in enteric bacteria. Homocysteine is an intermediate of the biosynthesis of methionine that has been associated with Salmonella virulence and resistance to an assortment of S-nitrosothiols (32). In addition, the tripeptide glutathione has been shown to reduce E. coli nitrosative stress, possibly by serving as a transnitrosation sink (62).

Repair

ROS and RNS produced by phagocytes in response to infection can oxidize purines and pyrimidines. The endonucleases Xth and Nfo protect Salmonella against the cytotoxicity of the NADPH oxidase and iNOS expressed by macrophages, and by doing so these enzymes increase Salmonella virulence in a murine experimental model (146). Evidently Salmonella base-excision DNA-repair systems repair oxidative damage produced by macrophage-derived ROS and RNS.

Exclusion of iNOS-Containing Vesicles

In addition to the enzymatic and nonenzymatic detoxification of RNS, Salmonella exploits its capacity to remodel the phagosomal vacuole to avoid contact with vesicles containing NADPH oxidase or iNOS complexes (16, 48, 159, 163). Avoidance of vesicles harboring oxygen-dependent antimicrobial host defenses is mediated by effectors secreted through the type III secretion system encoded within the Salmonella pathogenicity island 2. This mechanism may not be as important for evading NO (which can cross membranes) as for avoiding ROS actions and ROS-RNS synergy. Furthermore, this mechanism might be limited to mononuclear phagocytes in specific stages of activation and/or differentiation because iNOS remains associated with the cortical actin cytoskeleton in some populations of Salmonella-infected macrophages (166).

Hypostimulatory LPS Modifications

Salmonellalipid A acyl chain and inner LPS core carbohydrates signal through TLR4 to induce iNOS expression (107, 147). Repeated Salmonella passage in macrophage cultures in vitro selects for LPS modifications that stimulate suboptimal iNOS expression (42). The strategy of expressing LPS molecules that are hypostimulatory for iNOS has been exploited by gram-negative pathogens such as Francisella and Burkholderia as well (23, 156).

NO IN MUCOSAL HOST DEFENSE

NO serves diverse functions in the gastrointestinal tract (Fig. 4). Nitrogen oxides generated in the acid environment of stomach are among the first defenses encountered by enteropathogenic bacteria in the host. Enzymatic synthesis of nitrogen oxides by epithelial cells contributes to host defense and mediates some of the typical clinical signs of gastrointestinal infection.

Fig. 4Functions of nitrogen oxides in the gastric mucosa. Saliva accumulates NO₃^– from the diet and endogenous sources. Nitrate reductases (red) expressed by bacteria colonizing the posterior dorsal surface of the tongue generate NO₂^– from NO₃^–. Nitrites are reduced to NO in the stomach by its low pH or in the presence of vitamin C. NO serves as precursor of an array of reactive nitrogen species (RNS) including N₂O₃ and RSNO (S-nitrosothiols). RNS are beneficial in the gastric mucosa and have been associated with cytotoxicity against bacteria that include several members of the Enterobacteriaceae, as well as the stimulation of mucus secretion and vasodilation. On the other hand, N-nitroso compounds (NOC) generated from RNS or by the actions of nitrate reductases expressed by overgrowing bacteria may be carcinogenic. Reproduced with permission from reference 90.

Exogenous, Nonenzymatic Sources of Nitrogen Oxides in the Gastrointestinal Tract

The extreme acidity of the stomach poses an insurmountable barrier for most microorganisms. However, enteropathogenic bacteria are innately capable of resisting the low gastric pH for short periods and can further adapt to this harsh environment by mounting an acid tolerance response. In addition to its direct antimicrobial activity, gastric acidity promotes the generation of a battery of toxic RNS. Dietary nitrate is an important source of nitrogen oxides in the alimentary tract. Nitrate ingested in food and water undergoes enterosalivary circulation, whereby it is estimated that about 25% of the dietary nitrate is secreted in saliva back into the mouth (reviewed in reference 99). Salivary nitrate is reduced to nitrite by facultative anaerobes such as Staphylococcus sciuri and S. intermedius populating the dorsal surface of the tongue (37). Nitrite is protonated in the acidic environment of the stomach, decomposing spontaneously to generate various nitrogen oxides such as NO, N₂O₃, NO₂^•, and S-nitrosothiols. It is estimated that chemical reactions in the upper intestine produce about 10,000 times the amount generated in tissues by enzymatic synthesis (100). The capacity of enteric bacteria to resist RNS is inversely correlated with their infective oral dose. For example, resistance of S. enteritidis to acidified nitrite is correlated with increased virulence of the microorganism via the oral route (88).

Endogenous, Enzymatic Sources of Nitrogen Oxides in the Intestinal Mucosa

In addition to the chemical generation of nitrogen oxides from dietary nitrates, NO can be synthesized in the gastrointestinal mucosa in situ in response to enteropathogenic bacteria. This was first evidenced by elevated urinary NO_x in humans with gastrointestinal infection (38, 123), and subsequently reaffirmed when elevated NO levels were found in the rectal lumen and plasma of patients with gastroenteritis (64). Colonic and ileal epithelial cells can express iNOS in response to invasive Shigella dysenteriae, S. flexneri, and S. enteritidis (15, 53, 71). Infection of human colon epithelial cells in vitro with the enteropathogens E. coli, Salmonella, or Shigella suggests that activation of iNOS and NO synthesis requires cell invasion (39, 169). However, this view has been challenged in a mouse model of enteropathogenic E. coli (EPEC) infection, in which expression of iNOS is induced in colonic epithelial cells in response to noninvasive Citrobacter rodentium (157). A paradigm in which signaling pathways initiated by effector proteins inoculated into the cytosol via type III secretion systems synergize with signals engendered through binding of pattern recognition receptors with cognate microbial molecules is gradually emerging to explain the activation of iNOS expression in enterocytes in response to enteropathogenic bacteria (20, 22, 39, 104, 105, 157).

NO PRODUCTION BY PHAGOCYTES

The expression of iNOS in Salmonella-infected mice occurs predominantly within macrophages in discrete infected foci (76), although iNOS can also be found in neutrophils shortly after infection (2). Macrophages are capable of producing a variety of RNS, including NO, nitrosothiols, peroxynitrite, dinitrosyl iron complexes, nitrogen dioxide, and dinitrogen trioxide. Some RNS have been shown to exert potent anti-Salmonella activity in vitro. However, there has been controversy about whether Salmonella is susceptible to the nitrosative stress encountered in macrophages. Despite noting the expression of iNOS and the generation of RNS, several groups of investigators initially reported that macrophages do not exploit RNS in their anti-Salmonella arsenal (129, 137). However, although mononuclear phagocytes can use NO-independent mechanisms to inhibit Salmonella, recent reports have unequivocally shown that iNOS contributes to the anti-Salmonella actions of a variety of mononuclear phagocytes of different cell lineages and host species (16, 31, 141, 161, 166). Two factors are important determinants of NO-mediated anti-Salmonella activity of macrophages. First, as a consequence of the time required for de novo iNOS synthesis, the contribution of RNS to anti-Salmonella activity of macrophages increases at later time points. Second, IFN-γ treatment accelerates iNOS expression and increases NO output.

Intestinal lamina propria dendritic cells can sample the intestinal lumen in an M-cell-independent manner and are capable of migrating to draining mesenteric lymph nodes and systemic circulation (125, 160). These dendritic cells therefore have the potential to extraintestinally disseminate pathogenic microorganisms that are capable of surviving in the intracellular environment of professional phagocytes (94, 160). Dendritic cells must be endowed with potent antimicrobial defenses to reduce the likelihood of their exploitation by microbes, and these defenses include iNOS (8, 43, 73, 136). NO produced by murine dendritic cells appears to exert bactericidal activity against Salmonella, whereas NO produced by macrophages is primarily bacteriostatic (43, 161).

The specific NO congeners responsible for anti-Salmonella actions of activated macrophages have not been identified. Biochemical assays and the presence of the nitrotyrosine suggest that peroxynitrite is produced by Salmonella-infected macrophages (16, 163). However, lack of inhibition by the peroxynitrite scavenger uric acid and the absence of bacterial nitration suggest that peroxynitrite does not contribute to the iNOS-dependent, anti-Salmonella activity of macrophages. Several factors may account for this apparent paradox. First, production of ROS/RNS for the most part is segregated in time, in which a brief oxidative phase dependent on the NADPH oxidase is followed by a prolonged nitrosative phase that relies on the activity of iNOS. Second, negatively charged peroxynitrite formed in NADPH oxidase-containing vesicles cannot cross lipid membranes and may therefore be excluded from the Salmonella phagosome by the actions of SPI2 effectors (16) unless protonated to form peroxynitrous acid. Third, Cu/Zn superoxide dismutases may limit the amount of peroxynitrite formed in the periplasm in situ (31).

Cell Biology of iNOS in Macrophages

Vodovotz et al. have reported that about 50% of cytosolic iNOS is loaded onto vesicles after posttranslational modifications facilitate partitioning of the hemoprotein into lipid membranes (164). This may have important implications for the antimicrobial activity associated with this enzymatic complex, since docking of iNOS-containing vesicles to the phagosomal membrane could allows host cells to deliver high NO fluxes to intraphagosomal bacteria. Analogous to its interactions with lysosomes and NADPH oxidase-containing vesicles, the Salmonella SPI2 type III secretion system thwarts trafficking of iNOS-harboring vacuoles (16). This activity limits Salmonella exposure to RNS and therefore increases Salmonella's prospect for survival. However, some populations of macrophages that are unable to mobilize iNOS from the vicinity of the plasma membrane to the Salmonella phagosome still possess NO-dependent anti-Salmonella activity (166), indicating that the intracellular localization of iNOS is not as critical for RNS to exert antimicrobial activity as for NADPH oxidase-derived ROS (163), presumably because NO is freely diffusible.

IMPORTANCE OF NO IN SALMONELLA AND E. COLI INFECTIONS

NO produced endogenously by iNOS exerts a variety of effects in the complex pathophysiology of enteric infections. The antimicrobial activity associated with different RNS directly contributes to the elimination of enteropathogens. NO can exert beneficial effects on host cells by regulating immune responses (40, 91) and by acting as a cytoprotectant (168), but NO overproduction can also be detrimental by promoting bacterial cell invasion, dissemination, and tissue damage.

Contribution of NO to the Pathophysiology of Mucosal Infections

Circumstantial evidence suggests that dietary nitrates and nitrites play important roles in gastric host defenses (90). In addition, experimental evidence strongly implicates endogenously produced NO in resistance to pathogens at the mucosal surface. For example, the high NO output of iNOS plays a role in resistance to oral salmonellosis. This has been nicely demonstrated by the observation that iNOS-deficient mice infected orally with Salmonella carry greater bacterial loads, suffer increased liver damage and hepatocellular apoptosis, and have higher septicemia and mortality rates than wild-type controls (2). Salmonella-infected iNOS-deficient mice exhibit increased hepatocellular apoptosis, while iNOS-related apoptosis is observed in renal epithelial cells exposed to uropathogenic E. coli (18) or Peyer's patches isolated from mice infected with S. enteritidis (15). Notably apoptosis in the latter case is correlated with the development of protective immunity.

Expression of iNOS is important in maintaining the homeostasis of the intestinal mucosa and, as described above, in protection against enteropathogenic bacteria. However, the overproduction of iNOS-derived NO has been implicated in a variety of intestinal disorders (22, 53, 97, 126). Overexpression of iNOS in response to S. enteritidis can inflict collateral damage to the intestinal epithelium (53), and high NO output stimulates guanylyl cyclase-mediated cGMP production that in turn causes hypersecretion and diarrhea (22). Coexpression of prostaglandin E2-producing COX2 can compound the negative effects of NO on chloride secretion and barrier function (22, 126). Therefore, NO can contribute along with other factors to diarrhea associated with enteropathogenic bacteria. In addition, iNOS has been associated with loss of the intestinal barrier and consequent increased bacterial translocation (69, 103), although the possibility also exists that NO decreases bacterial entry by stimulating mucus and fluid secretion (9).

NO has been implicated in the pathogenesis of inflammatory tissue damage in E. coli urinary tract infections (119) and seizures complicating shigellosis (5). In the former case, iNOS does not seem to be required to eliminate E. coli from the bladder but is involved in tissue tyrosine nitration (119). The NO-mediated tissue damage seen in this model contrasts with the observation that NO production is inversely correlated with the severity of experimental pyelonephritis (111).

Systemic NO Effects in Enteric Bacterial Infections

Increased nitrite in plasma and iNOS mRNA can be detected as early as three days after infection, concomitantly with increased NO-DETC-Fe²⁺ ESR signals (2, 155). The expression of iNOS results in increased resistance to systemic Salmonella infections (96), as demonstrated by the fact that iNOS-deficient mice have a 1,000-fold reduction in LD₅₀ compared with wild-type controls (2). Studies with mice carrying defined immunodeficiencies in iNOS are consistent with studies using pharmacological inhibitors of NO synthesis (32, 91, 155). However, high NO production does not necessarily correlate with enhanced resistance if other components of host defense such as macrophage migration inhibitory factor are lacking (79).

As described for mucosal immunity, NO can also be associated with pathological effects in certain systemic infections. NO may play a role in enhancing blood–brain-barrier permeability (13), and NO derived from nNOS, but not iNOS, seems to mediate inflammatory injury in a model of E. coli neonatal meningitis (117). In general, attempts to therapeutically block NO production have not improved the outcome of septic shock, although iNOS-immunodeficient mice are hyperresistant to LPS-induced endotoxemia (93). These studies suggest that the hypotension associated with LPS-induced septic shock principally results from massive production of NO by the iNOS isoform (6). Overall, these studies call for evaluation of inhibitors selective for individual NOS isoforms in the treatment of specific pathological conditions.

CONCLUSIONS

NO and NO-derived RNS play important roles in innate immunity to Salmonella and E. coli. Enzymatic NO production by NO synthases can be enhanced by microbial and other inflammatory stimuli and it exerts direct antimicrobial actions as well as immunomodulatory and vasoregulatory effects. Nonenzymatic NO production from ingested nitrate may be particularly important in enhancing the gastric acid barrier to foodborne pathogens. The antibacterial actions of NO largely result from targeting of thiols and metal centers and include blockade of respiration, inhibition of DNA replication, and promotion of oxidative macromolecular modifications. These actions in turn are opposed by diverse resistance mechanisms including enzymatic detoxification, scavenging, repair, expression of stress regulons, and modulation of NO delivery or production. Although most evidence supports a protective role of NO in Salmonella and other enteric bacterial infections, NO overproduction may also be deleterious to the host by promoting bacterial invasion or dissemination, tissue injury, and vascular collapse.

ACKNOWLEDGMENTS

Support of this work was provided by the National Institutes of Health and the Schweppe Foundation.