Salmonella evasion of the NADPH phagocyte oxidase

Microbes and Infection, 3, 2001, 1313−1320 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457901014927/REV

Salmonella evasion of the NADPH phagocyte oxidase Andrés Vazquez-Torres, Ferric C. Fang* Departments of Medicine, Pathology and Microbiology, University of Colorado Health Sciences Center, 4200 E. 9th Ave, B168, Denver, CO 80262, USA

ABSTRACT – The bacteria–phagocyte interaction is of central importance in Salmonella pathogenesis. Immediately following phagocytosis, the NADPH phagocyte oxidase complex assembles in vesicles and produces highly toxic reactive oxygen species that play a major role in initial Salmonella killing by phagocytes. However, Salmonella has evolved a number of strategies to reduce the efficacy of oxygen-dependent phagocyte antimicrobial systems. Some of these strategies, such as superoxide dismutases, hydroperoxidases, oxidoreductases, scavengers and repair systems are common to most aerobic bacteria. In addition, Salmonella has acquired, by horizontal gene transfer, a type III secretory system encoded by Salmonella pathogenicity island 2 that interferes with the trafficking of vesicles containing functional NADPH phagocyte oxidase to the phagosome, thereby enhancing the survival of Salmonella within macrophages. © 2001 Éditions scientifiques et médicales Elsevier SAS Salmonella / NADPH oxidase / macrophages

1. Introduction Survival within phagocytic cells is essential for Salmonella virulence. In 1986, Fields and collaborators identified 83 Tn10 Salmonella mutants with reduced macrophage survival, and each proved to have reduced virulence in mice [1]. Macrophages are believed to play a particularly important role in the pathogenesis of Salmonella infection by providing protected sites for intracellular replication and a means of extraintestinal dissemination [2]. Nevertheless, macrophages also contribute to host resistance by limiting bacterial growth and initiating both granuloma formation and adaptive immune responses. The NADPH phagocyte oxidase is among the most effective antimicrobial weapons employed by phagocytic cells. This enzymatic complex catalyzes the univalent reduction of molecular oxygen to superoxide, a radical with modest antibacterial activity that serves as a precursor to more toxic reactive oxygen species such as hydrogen peroxide and hydroxyl radical [3]. The critical role of the NADPH phagocyte oxidase in defense against Salmonella typhimurium infections has been demonstrated in humans with chronic granulomatous disease (CGD) and in animal models with genetically engineered defects in essential oxidase components [3–6]. Herein, we review both wellknown and recently discovered mechanisms that help

*Correspondence and reprints. E-mail address: [email protected] (F.C. Fang). Microbes and Infection 2001, 1313-0

Salmonella to evade potentially lethal effects of the NADPH phagocyte oxidase.

2. Contribution of SPI2 to Salmonella pathogenesis The second Salmonella pathogenicity island (SPI2) was discovered by Holden and colleagues during the screening of a signature-tagged transposon library for the negative selection of Salmonella mutants with attenuated virulence [7]. Groisman’s laboratory independently identified the SPI2 locus while studying virulence characteristics of Salmonella strains carrying mutations within a chromosomal region originally identified by R. Fitts [8]. SPI2 encompasses a 25-kilobase-pair cluster of genes at centisome 30.7 of the Salmonella chromosome. The type III secretory systems encoded by SPI2 and the earlieridentified SPI1 region each seem to have been acquired horizontally [9]. However, these two pathogenicity islands participate in very different aspects of Salmonella pathogenesis. Whereas SPI1 contributes to the gastrointestinal manifestations of salmonellosis by facilitating invasion of M cells overlying lymphoid tissue of Peyer’s patches, SPI2 plays a role in the systemic complications of disseminated salmonellosis [9]. Specifically, the SPI2 type III secretory system is required for Salmonella survival within eukaryotic cells, including macrophages [8, 10–12]. 1313

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2.1. Regulation of SPI2 expression

SPI2 genes encoding structural, regulatory and effector proteins are selectively expressed by Salmonella residing inside macrophages [10, 13, 14]. Expression of these genes is controlled by the two-component regulatory system SsrA/SsrB encoded within the pathogenicity island. Acidification of the Salmonella-containing phagosome to pH 4–5 is paradoxically required for Salmonella survival and replication within mononuclear phagocytes [15]. This may be attributable to pH-dependent SPI2 gene expression or protein secretion [10]. Secretion of the putative SPI2 effector protein SseB by Salmonella grown to stationary phase can be detected upon medium acidification to pH 5 [16]. Because the expression of certain SPI2 genes such as ssaJ, sseB and spiA, but not the regulatory locus ssrA, is greatly reduced upon neutralization of pH, Cirillo and collaborators have concluded that “ssrAB appears to be under the control of additional regulatory systems [10].” Recently, the EnvZ/OmpR regulatory system of the stationary-phase acid tolerance response [17] was found to activate the transcription of ssrA/B genes [13]. SsrA additionally appears to control expression of virulence genes outside SPI2 [18–21]. 2.2. SPI2 mutants become virulent in gp91phox-deficient mice

The lethal dose of SPI2-deficient Salmonella strains is over one million times greater than that of isogenic wildtype controls [22]. SPI2-mutant Salmonella remains attenuated in IFNγ knockout mice and in mice treated with the nitric oxide synthase inhibitor aminoguanidine [12]. In contrast, all SPI2-mutant S. typhimurium strains tested (i.e. ssrA, ssaJ, ssaV, sseB) are able to cause lethal infection in mice deficient in the gp91phox subunit of the NADPH phagocyte oxidase [12], suggesting that SPI2 gene products protect Salmonella against antimicrobial actions of the NADPH phagocyte oxidase.

3. The NADPH phagocyte oxidase Macrophages can kill or limit the replication of pathogenic microorganisms by limiting the availability of essential nutrients or producing antimicrobial peptides, lysosomal enzymes, and reactive oxygen and nitrogen species. Oxygen-dependent metabolites are the most extensively studied and perhaps most efficient antimicrobial effectors produced by phagocytes. Molecular oxygen can be consumed by hemoprotein complexes of the NADPH phagocyte oxidase and inducible nitric oxide synthase for the production of reactive oxygen and nitrogen species in quantities that exert antimicrobial activity. The NADPH phagocyte oxidase was the first hemoprotein to be identified in host defense against pathogens; people lacking a functional NADPH phagocyte oxidase suffer recurrent life-threatening bacterial and fungal infections. Since its discovery, significant progress has been made in understanding the enzymology, genetics and cell biology of this complex enzyme. 3.1. Components of the NADPH phagocyte oxidase

The NADPH phagocyte oxidase uses NADPH for the univalent reduction of oxygen to superoxide. This decep1314

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tively simple reaction is catalyzed by a complex composed of membrane-bound gp91phox and p22phox subunits forming the cytochrome b558 along with cytosolic p40phox, p47phox, p67phox and Rac1 proteins [3]. Recently, the GTP-binding protein Rap1A of the Ras superfamily has been identified as an additional component of the NADPH phagocyte oxidase [23], but its function is still a matter of conjecture. 3.2. Activation of the NADPH phagocyte oxidase

Compartmentalization of membrane-bound and cytosolic components of the NADPH phagocyte oxidase insures that the production of potentially cytotoxic oxyradicals is prevented in resting cells. Soluble and particulate stimuli can signal via arachidonic acid, phospholipases A and D, and protein kinase C for the activation of cytosolic and membrane-bound components of the NADPH phagocyte oxidase. Phosphorylated p40phox, p47phox and p67phox cytosolic subunits migrate en bloc from the cytosol to the membrane, where they assemble with the cytochrome b558 heterodimer and small GTP-binding Rac cytosolic proteins to form an active enzymatic complex [24]. Rac, p47phox and p22phox are structural proteins that enhance the affinity of p67phox for gp91phox. In the current model, p67phox controls the flow of electrons from NADPH to the hemoprotein gp91phox [25], which ultimately reduces molecular oxygen to superoxide [26]. The collateral damage that oxyradicals can exert on host cells requires tight regulation of NADPH phagocyte oxidase activity. Although various components of the enzymatic complex are controlled at the transcriptional level, most regulation is post-translational. Several mechanisms have been associated with deactivation of the NADPH phagocyte oxidase. For instance, hyperphosphorylation of membrane associated p47phox leads to the disassembly of p47/p67 complexes from phagosomal membranes containing cytochrome b558 [27]. In addition, nitrosylation of p47phox by reactive nitrogen species can prevent assembly of the NADPH phagocyte oxidase. 3.3. Cellular location for NADPH phagocyte oxidase activation

Two models, that are not mutually exclusive, have been proposed for activation of the NADPH phagocyte oxidase. 3.3.1. The plasma membrane model

The majority of cytochrome b558 is located in resting neutrophils at the membrane of specific granules [28, 29]. Activation of neutrophils with soluble stimuli leads to the translocation of the cytochrome b558 from specific granules to the plasma membrane. In this model, the plasma membrane rather than membranes of either specific or azurophil granules is targeted by translocating p47/ p67phox complexes [30–33]. Other investigators have reported that membranes of specific granules can also serve as docking targets for the translocation of p47/ p67phox complexes [34]. In addition to specific granules, it appears that the respiratory burst can be activated in alkaline phosphase-expressing rod-shaped intracellular secretory vesicles that also contain cytochrome b558 [35]. Vesicular trafficking of the superoxide-producing vesicles can result in fusion with the plasma membrane [35]. Microbes and Infection 2001, 1313-0

Salmonella evasion of the NADPH phagocyte oxidase

3.3.2. The phagosomal membrane model

The NADPH phagocyte oxidase can also be activated in phagosomal membranes [27, 36]. Cytochrome b558, p47phox and p67phox are enriched in phagosomal membranes containing serum-opsonized Neisseria meningitidis or zymosan particules [24, 27, 37]. The p47phox and p67phox components accumulate in the plasma membrane at the point of contact with the zymosan particle [37]. Production of superoxide appears to persist as long as the p47/p67 complexes are associated with the phagosomal membrane [27]. 3.4. Cytoskeleton and the NADPH phagocyte oxidase

Early studies noted that NADPH phagocyte oxidase activity is preferentially found in cellular fractions rich in plasma membrane and cytoskeletal proteins [38]. In resting neutrophils the p67phox subunit is associated with a Triton X-insoluble fraction of the cytoskeleton [39], in association with the actin-binding protein coronin [37]. Upon activation, phosphorylated p40phox and p47phox subunits relocate to the Triton-insoluble cytoskeleton [31] for association with filamentous actin [40, 41]. The ability of actin to enhance the respiratory burst in a cell-free system and the inactivation of the NADPH phagocyte oxidase by agents that depolymerize filamentous actin [42, 43] confirm the importance of the cytoskeleton in activation of the NADPH phagocyte oxidase. Filamentous actin brings the cytosolic p47phox and p67phox components of the NADPH phagocyte oxidase to the phagosomal membrane for their association with gp91phox [24]. However, cytoskeletal-dependent intracellular production of oxyradicals by the NADPH phagocyte oxidase can occur without phagosome formation [44].

4. Role of the NADPH phagocyte oxidase in Salmonella infection 4.1. Clinical correlates of NADPH phagocyte oxidase resistance to salmonellosis

Hereditary CGD resulting from recessive X-linked gp91phox or autosomal p22phox, p47phox or p67phox mutations is characterized by the inability of phagocytes to produce high concentrations of superoxide [3]. CGD patients are extremely susceptible to bacterial and fungal infections. Salmonella constitutes the second most frequent bacterial pathogen isolated from CGD patients [45], illustrating the importance of the NADPH phagocyte oxidase in innate immunity to Salmonella. 4.2. Experimental models of CGD in Salmonella infections

Mice deficient in gp91phox or p47phox provide excellent experimental models for CGD in humans. Like their human counterparts, gp91phox knockout mice do not produce a respiratory burst and are extremely susceptible to Salmonella infection [4, 5, 12, 46]. Mice deficient in gp91phox succumb to infection within 5 days of intraperitoneal injection of Salmonella. Compared to wild-type congenic mice, gp91phox knockout mice harbor 103–104fold higher bacterial burdens in spleen and liver. Several Microbes and Infection 2001, 1313-0

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populations of macrophages have been shown to efficiently kill Salmonella during the first hours of contact by a process dependent on the NADPH phagocyte oxidase [4–6, 47]. Early killing of Salmonella by macrophages correlates with production of superoxide, hydrogen peroxide and peroxynitrite [6], each of which requires a functional NADPH phagocyte oxidase for its formation.

5. Known mechanisms of bacterial resistance to phagocyte-derived oxidants Given the cytotoxicity of oxyradicals produced by the NADPH phagcoyte oxidase, it is not surprising that many pathogenic microorganisms have developed an array of strategies to avoid or inhibit this enzymatic complex. Some pathogens such as Clostridium septicum, Neisseria, Staphylococcus aureus, Streptococcus pneumoniae and Yersinia avoid contact with phagocytes by secreting cytotoxic proteins, disrupting host cell transduction signals or producing antiphagocytic capsules. Listeria monocytogenes, Shigella and Rickettsia escape from the phagosomal vacuole to the cytosol where the NADPH phagocyte oxidase cannot be assembled. Other pathogens have devised strategies to prevent the assembly of a functional NADPH phagocyte oxidase complex. Leishmania donovani, Legionella pneumophila and Plasmodium falciparum inhibit the protein kinase C necessary for the mobilization of cytosolic NADPH phagocyte oxidase components to the membrane [48–50]. More recently, the agent of human granulocytic ehrlichiosis has been found to selectively inhibit transcription of the membrane-bound gp91phox subunit of the phagocyte oxidase [51]. In addition to these sophisticated mechanisms, pathogenic microorganisms have coapted strategies originally developed for survival in aerobic environments to resist the strong oxidative stress created by the NADPH phagocyte oxidase. 5.1. Detoxifying enzymes

In 1968, J.M. McCord and I. Fridovich made the seminal discovery of superoxide dismutase, an enzyme that catalyzes the formation of hydrogen peroxide from superoxide anion [52]. S. typhimurium carries two cytosolic manganese- and iron-cofactored superoxide dismutases (SodA and SodB, respectively) and two periplasmic copper, zinc-cofactored superoxide dismutases (SodCI and II). Although Mn-SOD does not seem to contribute substantially to macrophage survival or virulence of S. typhimurium [53], the two periplasmic Cu,Zn-SOD enzymes contribute significantly to the virulence of Salmonella [54–57]. SodCI encoded by a cryptic bacteriophage is the most potent superoxide dismutase described to date [58], which may account for its expression by the most pathogenic Salmonella strains [56]. SodCI (and presumably SodCII) enhances macrophage survival of S. typhimurium by antagonizing synergistic interactions of the NADPH phagocyte oxidase and inducible nitric oxide synthase [55]. Enzymatic or spontaneous dismutation of superoxide produces hydrogen peroxide, a membrane permeable oxidizing species capable of reacting with thiols, heme proteins, lipids and DNA. In addition, hydrogen peroxide 1315

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reacts with reduced metals to form more toxic hydroxyl radicals. Catalases detoxify hydrogen peroxide to water. Although S. typhimurium has at least two catalases, these enzymes appear to be dispensable for virulence [59]. This may be attributable to the relatively poor ability of catalases to defend bacteria from hydrogen peroxide at low cell densities [60]. 5.2. Scavengers

Several enzymes including glutathione reductase, methionine sulfoxide reductase and thioredoxin reductase use reducing equivalents from NADPH to either generate low molecular weight antioxidant species or repair oxidative lesions. It is therefore not surprising that a zwf mutant S. typhimurium strain lacking glucose 6-phosphate dehydrogenase activity required for NADPH formation is hypersusceptible to reactive oxygen and nitrogen species, as well as attenuated for virulence [61]. 5.3. Repair systems

DNA is a critical bacterial target of reactive oxygen species [62]. In contrast to catalase-deficient strains, recombination-deficient recA and recBC S. typhimurium strains are both efficiently killed by the respiratory burst of macrophages and attenuated for virulence in mice [47, 59]. These data indicate that DNA repair systems are more important than catalases for protection against products of the NADPH phagocyte oxidase. 5.4. Transcriptional regulators

The SoxRS and OxyR stress regulons coordinate the transcription of genes encoding antioxidant defenses against superoxide and hydrogen peroxide. Superoxideoxidized SoxR induces the transcription of soxS, leading to expression of antioxidant genes such as zwf, sodA, and fpr (encoding ferredoxin-NADPH oxidoreductase). Recent observations suggest that hydrogen peroxide can also activate SoxRS [63]. Hydrogen peroxide activates OxyR by oxidizing cysteine 199 and promoting the reversible formation of an intramolecular disulfide bridge between cysteines 199 and 208 [64]. Oxidized OxyR binds to the promoters of genes such as katG (catalase) and gor (glutathione reductase), activating their expression. Surprisingly, although Salmonella is susceptible to oxidative stress generated by the NADPH phagocyte oxidase, neither SoxS nor OxyR are essential for virulence [65]. Other regulators including RpoS and SlyA may provide adequate intracellular activation of antioxidant defenses in the absence of SoxRS and OxyR [66, 67]. In addition, Salmonella utilizes strategies to reduce the intensity of the oxidative stress generated by the NADPH phagocyte oxidase, which may render SoxRS and OxyR dispensable.

6. SPI2-mediated resistance to the NADPH phagocyte oxidase Although a number intracellular pathogens are able to proliferate and thrive within phagosomes of professional phagocytes, the phagosome is an essentially inhospitable, 1316

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nutritionally deficient membrane-bound intracellular vacuole that acidifies and accumulates proteases and digestive enzymes to facilitate degradation of its contents. Maturing phagosomes associate with vacuolar proton ATPases that acidify the luminal space, and establish a dynamic interaction with incoming endocytic vesicles and lysosomes. Salmonella, similarly to intracellular bacterial pathogens such as Mycobacterium tuberculosis, L. pneumophila and Francisella tularensis, has the ability to replicate within remodeled phagosomes. The Salmonella-containing phagosome acidifies to pH 4–5 and is positive for Rab5, transferrin receptor, lysosomalassociated membrane protein 1 and lysosomal acid phosphatase markers, but fails to fuse with secondary lysosomes and excludes Rab7, Rab9, mannose 6-phosphate receptor and cathepsin L [15, 68–70]. We have recently shown that Salmonella further controls its vacuole by inhibiting fusion with vesicles containing the NADPH phagocyte oxidase. 6.1. SPI2 avoids contact with NADPH phagocyte oxidase-containing vesicles

Mutational inactivation of the gp91phox subunit of the NADPH phagocyte oxidase increases intracellular survival of SPI2 mutant Salmonella to levels similar to that of virulent isogenic wild-type bacteria [12]. This suggests that effector proteins secreted by SPI2 antagonize the NADPH phagocyte oxidase. However, macrophages exhibit similar oxyradical production after infection with either wild-type or SPI2-deficient bacteria. Therefore, in contrast to other bacterial pathogens such as L. pneumophila and Ehrlichia, S. typhimurium does not seem to inhibit assembly of a functional NADPH phagocyte oxidase. Electron microscopy using cerium chloride (which forms an electrodense precipitate after reacting with hydrogen peroxide) demonstrated SPI2-dependent inhibition of S. typhimurium co-localization with oxyradicals. In resting macrophages, p22phox and p47phox are segregated preferentially in the cytoplasmic membrane and cytosol, respectively. Phagocytosis of Salmonella by macrophages results in rapid mobilization of membrane and cytosolic components of the NADPH phagocyte oxidase to discrete intracytoplasmic vesicles that migrate to the proximity of phagosomes harboring SPI2-deficient S. typhimurium. The NADPH phagocyte oxidase-positive vesicles are effectively excluded from phagosomes containing virulent S. typhimurium. These observations suggest a model in which the type III secretory system encoded by SPI2 prevents trafficking or targeting of NADPH phagocyte oxidase-containing vesicles to the vicinity of the Salmonella phagosome (figure 1). In addition, these findings support the concept that assembly and trafficking of the NADPH phagocyte oxidase to the Salmonellacontaining phagosome occur as discrete events. 6.2. Possible mechanisms of SPI2 inhibition of NADPH phagocyte oxidase trafficking

The mechanisms by which SPI2 inhibits both trafficking of NADPH phagocyte oxidase-containing vesicles and fusion of phagosomes with lysosomes may be related. The spiC gene product encoded within SPI2 is secreted into the Microbes and Infection 2001, 1313-0

Salmonella evasion of the NADPH phagocyte oxidase

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NADPH phagocyte oxidase-containing Salmonella-containing phagosomes.

vesicles

to

6.3. Host factors that target NADPH phagocyte oxidasecontaining vesicles to the vicinity of the phagosome

Tumor necrosis factor receptor p55 (TNFR) knockout mice succumb within 3 days of Salmonella challenge during a phase of infection strongly influenced by the antimicrobial actions of the NADPH phagocyte oxidase [46, 71]. The increased susceptibility of TNFR knockout mice to salmonellosis is correlated with defective killing of Salmonella by macrophages [72]. Macrophages lacking TNFR p55 are able to sustain synthesis of reactive oxygen and nitrogen species, but do not target NADPH phagocyte oxidase-containing vesicles to phagosomes harboring SPI2 deficient S. typhimurium. These data indicate an important role for TNFα signaling in delivering oxyradicals to the Salmonella phagosome and suggest that TNFα signaling pathways represent yet another potential target for SPI2-secreted effector proteins.

7. Conclusions Figure 1. SPI2-dependent evasion of vesicles containing the NADPH phagocyte oxidase. In the resting state, the components of the NADPH phagocyte oxidase are segregated into cytosolic and membrane fractions. Upon phagocytosis of Salmonella, the p67phox, p47phox and p40phox cytosolic components translocate via the cytoskeleton for association with gp91phox, p22phox and Rap1A membrane-bound subunits together with cytosolic Rac proteins. Effector proteins secreted by the type III secretory system encoded by SPI2 prevent trafficking of NADPH oxidasecontaining endosomes to the vicinity of the Salmonella phagosome. Avoidance of the NADPH oxidase by SPI2 in combination with enzymes and scavengers that detoxify reactive oxygen species (ROS) provides a selective advantage for survival of Salmonella within the hostile environment of the phagocyte. macrophage cytosol [71]. A functional SpiC protein is required for prevention of lysosomal or endosomal fusion with Salmonella-containing phagosomes, and purified SpiC can inhibit fusion between endosomes in vitro. It is possible that SpiC and other SPI2-secreted effector proteins block fusion of vesicles harboring active NADPH phagocyte oxidase with the phagosomal membrane. An additional mechanism may relate to effects of SPI2 on the cytoskeleton. Actin polymerization is transiently required for the invagination of the plasma membrane and formation of phagosomes. However, actin fibers depolymerize shortly after particle internalization to allow contact of phagosomes with the endocytic pathway. Salmonella has been found to promote associations of phagosomal membranes with actin aggregates (D. Holden, personal communication) by a SPI2-dependent process. This actin web could hinder the targeting of incoming Microbes and Infection 2001, 1313-0

Adaptation to the macrophage intracellular environment offers Salmonella and other intracellular pathogens unique opportunities for replication in a site protected from many humoral and cellular host defenses. However, the host cell extorts a high price from its resident since Salmonella must withstand or avoid the antimicrobial arsenal normally delivered to the phagosomal lumen. The cytotoxic actions of the NADPH phagocyte oxidase complex are one of the most important challenges that Salmonella has to surmount inside phagocytes. Effector proteins secreted into the cytosol by the type III secretory system encoded by SPI2 prevent targeting of NADPH oxidasecontaining vesicles to the Salmonella phagosome. Avoidance of NADPH oxidase trafficking to the vicinity of the phagosome in combination with enzymes and scavengers that detoxify oxyradicals dramatically enhances the pathogenic potential of Salmonella. A better understanding of the interaction between Salmonella and its host cell will not only increase our knowledge of microbial pathogenesis and macrophage cell biology but may shed light onto novel approaches for treatment and prevention of infectious diseases caused by intracellular microbes.

Acknowledgments We thank J. Jones-Carson for reviewing the manuscript. This work was supported by an NIH postdoctoral fellowship, NIH grants AI39557 and AI44486 and the James Biundo Foundation. 1317

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