TY - JOUR
AU - Skaar, Eric P.
AB - Staphylococcus aureus is a ubiquitous, versatile and dangerous pathogen. It colonizes over 30% of the human population, and is one of the leading causes of death by an infectious agent. During S. aureus colonization and invasion, leukocytes are recruited to the site of infection. To combat S. aureus, leukocytes generate an arsenal of reactive species including superoxide, hydrogen peroxide, nitric oxide and hypohalous acids that modify and inactivate cellular macromolecules, resulting in growth defects or death. When S. aureus colonization cannot be cleared by the immune system, antibiotic treatment is necessary and can be effective. Yet, this organism quickly gains resistance to each new antibiotic it encounters. Therefore, it is in the interest of human health to acquire a deeper understanding of how S. aureus evades killing by the immune system. Advances in this field will have implications for the design of future S. aureus treatments that complement and assist the host immune response. In that regard, this review focuses on how S. aureus avoids host-generated oxidative stress, and discusses the mechanisms used by S. aureus to survive oxidative damage including antioxidants, direct repair of damaged proteins, sensing oxidant stress and transcriptional changes. This review will elucidate areas for studies to identify and validate future antimicrobial targets. Staphylococcus aureus, oxidative stress, neutrophils, protein oxidation, antioxidant defenses, host–pathogen interface INTRODUCTION Staphylococcus aureus Staphylococcus aureus is a pathogen that impacts humans in both community and hospital settings. Over 30% of the human population is colonized asymptomatically by S. aureus, but this organism also causes infections ranging from minor skin infections and food poisoning to sepsis and death (Kuehnert et al.2006). While traditional antibiotic therapies can be effective against S. aureus, many resistant strains have developed, with methicillin-resistant S. aureus (MRSA) accounting for over 25% of new clinical isolates (Pendleton, Gorman and Gilmore 2013). Drug-resistant S. aureus has become a wide spread problem, and it has been named by the Centers for Disease Control and Prevention as one of the six ESKAPE pathogens (Enterococcus faecium, S. aureus, Klebseilla pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacteriaceae sp.), giving research on this organism a high priority designation (Pendleton, Gorman and Gilmore 2013). In keeping with this, it is important to understand how S. aureus evades host killing. These studies will identify and validate new antibiotic targets for therapies that work in concert with host defenses but orthogonally to current antibiotics against which S. aureus has developed resistance. Host defenses and the oxidative burst Neutrophils, macrophages and interferon gamma-activated mast cells are each able to kill S. aureus (Coady et al.2015; Swindle et al.2015). Neutrophils, the first line of defense against S. aureus, are capable of engulfing bacteria through phagocytosis, and killing bacteria primarily through the activity of NADPH oxidase and myeloperoxidase (MPO). It has also been proposed that inducible nitric oxide synthase (iNOS) contributes to the antimicrobial activity of neutrophils (Fig. 1A). In addition to phagocytosis, neutrophils release neutrophil extracellular traps (NETs) (Fig. 1B). NETs consist of neutrophil intracellular components including, but not limited to, high amounts of chromatin, the metal binding-protein calprotectin and MPO, the combined action of which can be antimicrobial towards S. aureus (Brinkmann et al.2004). Eighty percent of MPO released by neutrophils is NET bound and cannot be removed by mechanical disruption. The NET-bound MPO is active and a major component of S. aureus killing by NETs (Parker et al.2012). Interestingly, exogenous addition of hypochlorous acid (HOCl), the product of MPO activity, induces the formation of NETs. Thus, MPO and NADPH oxidase activities are necessary for the generation and release of NETs in certain contexts, suggesting that NETs can be formed in response to the initial neutrophil oxidative burst (Palmer et al.2012; Parker et al.2012). Figure 1. View largeDownload slide Neutrophils inflict cellular damage on S. aureus by (A) phagocytosis and exposure to oxidants generated by NADPH oxidase, MPO and nitric oxide synthase or by (B) releasing NETs that contain high amounts of chromatin, calprotectin and MPO. (C) The oxidative burst of neutrophils generates oxidants enzymatically and non-enzymatically that react with and damage cellular macromolecules including lipids, proteins and DNA. Figure 1. View largeDownload slide Neutrophils inflict cellular damage on S. aureus by (A) phagocytosis and exposure to oxidants generated by NADPH oxidase, MPO and nitric oxide synthase or by (B) releasing NETs that contain high amounts of chromatin, calprotectin and MPO. (C) The oxidative burst of neutrophils generates oxidants enzymatically and non-enzymatically that react with and damage cellular macromolecules including lipids, proteins and DNA. Several steps requiring multiple enzymes make up the neutrophil oxidative burst (Fig. 1C). First, NADPH oxidase produces superoxide anion through the reduction of molecular oxygen (Hampton, Kettle and Winterbourn 1998). Superoxide, while not a strong oxidant itself, is a precursor to most of the other oxidants of the oxidative burst including H2O2, hydroxyl radical, HOCl and peroxynitrite (Sawyer and Valentine 1981; Jiang, Blount and Ames 2003). Superoxide dismutase enzymes or spontaneous dismutation converts superoxide to H2O2. H2O2 is an oxidant itself, and also reacts with iron through Fenton chemistry to form hydroxyl radical (Winterbourn 1995). MPO catalyzes the reaction between H2O2 and chloride to produce HOCl (Harrison and Schultz 1976; Hampton, Kettle and Winterbourn 1998). Superoxide also reacts rapidly with nitric oxide, the product of iNOS, to generate the oxidant peroxynitrite (Beckman 1996). Peroxynitrite can decompose to hydroxyl radical and nitrogen dioxide, both potent oxidants on their own (Radi 2004). Nitric oxide and its downstream metabolites also have a vast impact on cellular physiology. One of the most highly studied oxidants derived from nitric oxide in eukaryotic cells is peroxynitrite (Fig. 1C) (Rose et al.2003; Szabo, Ischiropoulos and Radi 2007). In fact, the reaction between nitric oxide and superoxide has a greater rate constant than that of superoxide with superoxide dismutase (Beckman 1996). The effects of peroxynitrite on S. aureus have not been extensively studied, but based on the enzymes involved in the host oxidative burst, it likely contributes to S. aureus killing by leukocytes. Increased iNOS, the enzyme that generates nitric oxide, has been observed in close proximity to MPO in neutrophil granules. This increase in nitric oxide synthase is correlated with increased protein tyrosine nitration through peroxynitrite (Evans et al.1996). MPO can use nitrite, a metabolite of nitric oxide, as a substrate to generate nitrogen dioxide and subsequent tyrosine nitration (Burner et al.2000). Fluorescein nitration, a surrogate for tyrosine nitration, was not observed in neutrophil phagocytic vacuoles, complicating the role of reactive nitrogen species as bactericidal molecules (Jiang and Hurst 1997). However, the toxicity of nitrating species is well known, and may be a mechanism by which the host oxidative burst kills S. aureus (Klebanoff 1993; Hurst and Lymar 1997; Radi 2004). The oxidants generated by neutrophils react with cellular macromolecules to oxidize proteins, lipids and DNA. The functional groups of many amino acid side chains, including cysteine, methionine, lysine, histidine and tyrosine, as well as the alpha amine of all free amino acids react with HOCl, while only the sulfur-containing amino acids react with H2O2 (Fig. 2). These reactions can have deleterious effects on protein function, and can generate secondary reactive molecules that also elicit cellular changes (Gray, Wholey and Jakob 2013). HOCl has not been studied as extensively as H2O2, even though HOCl is the major product of the neutrophil oxidative burst. This is presumably due to its high reactivity as H2O2 can be bolus dosed in vitro, while HOCl has a half-life of less than a minute in amino acid-containing medium (Mutze et al.2003). These reactive components of the neutrophil oxidative burst are diffusible and can damage DNA of adjacent cells, indicating that proximity to activated neutrophils may be sufficient to cause oxidative damage to S. aureus (Shacter et al.1988). Figure 2. View largeDownload slide The oxidants HOCl and H2O2 readily react with amino acids to generate a plethora of oxidation products. (A) Cysteine reacts with both HOCl and H2O2 to generate sulfenic acid, which can either form a disulfide with an adjacent thiol or be further oxidized to sulfinic and sulfonic acids. (B) Both HOCl and H2O2 oxidize the sulfur of methionine to methionine sulfoxide. Amino acids that contain nitrogen in their side chain, (C) histidine and (D) lysine, react with HOCl to generate chloramines. Lysine chloramine can be hydrolyzed to allysine, which can form a Schiff base with an adjacent lysine to give both inter and intramolecular crosslinks. (E) HOCl reacts with tyrosine to form chlorotyrosine. This process is also facilitated by the transfer of a chlorine atom from lysine chloramine to an adjacent tyrosine. (F) Finally, chloramines can be formed on the alpha amine of free amino acids by the reaction with HOCl. These chloramines degrade to various electrophiles including glyoxal, acrolein and p-hydroxyphenylacetaldehyde that can react with and damage cellular macromolecules. Figure 2. View largeDownload slide The oxidants HOCl and H2O2 readily react with amino acids to generate a plethora of oxidation products. (A) Cysteine reacts with both HOCl and H2O2 to generate sulfenic acid, which can either form a disulfide with an adjacent thiol or be further oxidized to sulfinic and sulfonic acids. (B) Both HOCl and H2O2 oxidize the sulfur of methionine to methionine sulfoxide. Amino acids that contain nitrogen in their side chain, (C) histidine and (D) lysine, react with HOCl to generate chloramines. Lysine chloramine can be hydrolyzed to allysine, which can form a Schiff base with an adjacent lysine to give both inter and intramolecular crosslinks. (E) HOCl reacts with tyrosine to form chlorotyrosine. This process is also facilitated by the transfer of a chlorine atom from lysine chloramine to an adjacent tyrosine. (F) Finally, chloramines can be formed on the alpha amine of free amino acids by the reaction with HOCl. These chloramines degrade to various electrophiles including glyoxal, acrolein and p-hydroxyphenylacetaldehyde that can react with and damage cellular macromolecules. Oxidation of cellular proteins and bacterial killing Thiol-containing cysteine residues are reactive with species of the oxidative burst. Oxidation of cysteine incorporates sequentially higher numbers of oxygen atoms onto the sulfur atom (Fig. 2A). The addition of one atom of oxygen forms the reversible oxidation product sulfenic acid. Sulfenic acid can react with an adjacent cysteine resulting in both inter and intramolecular disulfide bonds. Further oxidation of sulfenic acid generates sulfinic and sulfonic acids sequentially. Both are irreversible cysteine oxidation products in S. aureus (Gray, Wholey and Jakob 2013; Loi, Rossius and Antelmann 2015). The sulfur of methionine can also undergo oxidation resulting in methionine sulfoxide (Fig. 2B) (Rosen et al.2009). Amino acids with nitrogen-containing side chains, including lysine and histidine, readily react with HOCl to generate chloramines, which are semi-stable intermediates capable of oxidizing proximal amino acid residues (Fig. 2C and D) (Pattison and Davies 2005). Lysine chloramines degrade to allysine, an aldehyde-containing product that can form inter and intramolecular crosslinks to adjacent lysines through a Schiff base (Hazell, van den Berg and Stocker 1994). Lysine chloramines also catalyze the chlorination of adjacent tyrosine residues (Fig. 2E). Even though HOCl can generate chlorotyrosine directly, catalysis by lysine chloramines is thought to be the major route to this product (Bergt et al.2004). HOCl-generated chloramines on the alpha amine of free amino acids decompose to advanced glycation end products (Fig. 2F), which are electrophiles capable of reacting with nucleophilic amino acid side chains (Hazen et al.1998). The neutrophil oxidative burst produces a host of primary oxidants that react with amino acids to damage proteins as well as producing secondary metabolites capable of reacting with macromolecules, further propagating the molecular damage to pathogens induced by neutrophils. Despite the reactivity of the species generated during the oxidative burst, mechanisms of bacterial killing and host protection are still not completely agreed upon. Most HOCl generated in the phagosome reacts with host proteins. However, as discussed above, chloramines generated on host proteins are oxidants that potentially deliver the antimicrobial activity to the pathogen (Winterbourn et al.2006). Using radiolabeled iodine, one study suggested that MPO activity does not play a role in bactericidal activity of the neutrophil (Reeves et al.2003). Another study quantified that 94% of the chlorotyrosine generated by neutrophils containing phagocytosed S. aureus was on host proteins (Chapman et al.2002). However, not all studies agree with these findings, and it has been shown that MPO inhibition results in better S. aureus survival (Humphreys et al.1989). Consistent with this, neutrophil killing of S. aureus is predominantly through MPO activity, and this correlates with increased tyrosine chlorination on pathogen proteins (Green, Kettle and Winterbourn 2014). A functional NADPH oxidase is also necessary for S. aureus killing in B cells (Kovacs et al.2015). Finally, in granulocytes from patients with chronic granulomatous disease, the restoration of H2O2 production through the addition of glucose oxidase rescues the bactericidal activity of the granulocyte, indicating that H2O2 and HOCl are necessary for effective bacterial killing (Gerber et al.2001). Together these studies show that both host and pathogen proteins incur extensive damage during the oxidative burst, and without the enzymes of the oxidative burst, S. aureus killing is significantly diminished. In addition to defending against oxidation, S. aureus must fight the host for access to metals as well as defending against host-generated proteases (Pham 2006; Amulic et al.2012; Zackular, Chazin and Skaar 2015). Staphylococcus aureus generates many molecules that contribute to its pathogenesis beyond its ability to cope with host-generated oxidative stress. These molecules include virulence factors that damage host cells and molecules that compete for transition metals necessary for cellular functions (Hood and Skaar 2012; DuMont and Torres 2014; Scherr et al.2015). This review will focus on how S. aureus avoids host killing by the reactive molecules generated during the oxidative burst. Where possible, it will focus on the molecular mechanisms that allow S. aureus to persist despite an abundant assault by highly reactive molecules including H2O2 and HOCl. PROTECTING STAPHYLOCOCCUS AUREUS FROM OXIDANTS Antioxidant enzymes Superoxide dismutase The genes that undergo transcriptional activation as a result of the oxidative burst encode for proteins important for defending Staphylococcus aureus from host killing by reactive oxidants. In this section, we will discuss how these enzymes protect S. aureus from oxidative stress, and the phenotypes observed when the protective enzymes are inactivated. Staphylococcus aureus contains two superoxide dismutases, SodA and SodM. Both enzymes convert superoxide anion to H2O2 and O2 using manganese as a cofactor. It is important to note that superoxide is not membrane permeable, and thus superoxide dismutase can only confer resistance to proximally generated superoxide (Rosen and Freeman 1984). Superoxide dismutases are important for oxidant defenses as secreted or surface-exposed superoxide dismutase makes S. aureus more resistant to oxidative killing (Chatham, Turkiewicz and Blackburn 1994; Hampton, Kettle and Winterbourn 1996). SodA was the first discovered superoxide dismutase in S. aureus, and the inactivation of sodA makes S. aureus more susceptible to superoxide stress (Clements, Watson and Foster 1999). Staphylococcal superoxide dismutase activity is increased upon phagocytosis by macrophages, resulting in bacterial survival despite increased inflammatory cytokine production and increased H2O2 production by the macrophages (Das, Saha and Bishayi 2008; Das and Bishayi 2009). SodA is upregulated early in biofilm formation, but its expression decreases across the lifespan of biofilms (Resch et al.2005). This change in expression potentially results from the biofilm becoming anoxic, and needing fewer defenses from oxygen-generated toxicants. Strains inactivated for sodA or sodM individually are more sensitive to superoxide killing compared to wild-type S. aureus, and this sensitivity is reduced by the addition of manganese. Inactivation of both sodA and sodM significantly decreases survival over strains inactivated for either superoxide dismutase individually. Adding exogenous manganese does not increase the survival of the strain inactivated for both sodA and sodM indicating that manganese alone is not acting as an antioxidant (Karavolos et al.2003). Further confirming the need for manganese in superoxide dismutase activity, inactivation of genes in the manganese transport systems mntC and mntH reduces SodM activity in metal-depleted environments. This inactivation of mnt genes reduces the survival of S. aureus when treated with the superoxide-generating compound paraquat (Kehl-Fie et al.2013). Inactivation of mntC makes S. aureus more susceptible to killing by neutrophils, but not macrophages. The addition of paraquat makes S. aureus inactivated for mntC equally vulnerable to killing by both neutrophils and macrophages. Presumably, manganese deficiency reduces superoxide dismutase activity, and S. aureus defenses are overwhelmed by the additional oxidants generated by paraquat. Small-colony variants (SCVs) of the strain inactivated for mntC were recovered from both neutrophils and macrophages regardless of the ability of the leukocyte to kill (Coady et al.2015). These findings are compounded by the fact that manganese is also needed to repair DNA damage from the oxidative burst. One interpretation of these data is that neutrophils generate an oxidative burst that requires S. aureus to acquire manganese for superoxide dismutase activity and survival. Wild-type S. aureus is better able to colonize in a mouse skin abscess model than strains lacking sodA or sodM, individually or in combination. These data indicate that loss of superoxide dismutase activity reduces pathogenesis possibly because the host is manganese depleted at sites of infection (Karavolos et al.2003; Kehl-Fie et al.2013). In another study using a systemic model of mouse infection, superoxide dismutase activity of several S. aureus strains did not show a correlation with increased mouse mortality (Mandell 1975). Inactivation of genes in the manganese transport systems, mntC and mntH, reduces liver colonization during infection models further supporting the importance of manganese and superoxide dismutase activity in S. aureus pathogenesis (Kehl-Fie et al.2013). Superoxide dismutase activity has protective effects both in vitro and in vivo, strengthening the correlation between S. aureus oxidant defenses and pathogenesis. Catalase The second step in the detoxification of superoxide is the neutralization of H2O2 by catalase, which uses a heme cofactor to convert H2O2 to H2O and O2, completely detoxifying it. A product of the S. aureus gene katA, catalase confers resistance to H2O2 stress (Mandell 1975; Martin and Chaven 1987; Cosgrove et al.2007). Catalase activity increases upon phagocytosis by macrophages, presumably due to increases in protein expression, and confers increases in bacterial survival (Das, Saha and Bishayi 2008; Das and Bishayi 2009). During biofilm formation, katA is upregulated early; however, translation wanes as the biofilm becomes more anoxic (Resch et al.2005). Reduced expression of katA in anoxic conditions potentially results from the biofilm needing fewer defenses against reactive oxygen species when oxygen is in short supply. Another study noted that katA inactivation alone does not reduce survival compared to wild type after phagocytosis by macrophages, but simultaneous inactivation of katA and beta-toxin (hlb) resulted in increased S. aureus survival. This increase in survival is presumably due to blunting the immune response by decreasing beta-toxin, and probably has little to do with KatA levels (Martinez-Pulgarin et al.2009). In a mouse model of infection, nasal colonization by a S. aureus strain inactivated for katA is reduced compared to wild type (Cosgrove et al.2007). Catalase activity of various S. aureus strains directly correlates to mouse mortality in a systemic model of infection (Mandell 1975). However, the LD50 of a strain inactivated for katA is the same as wild type when strains are injected into the mouse peritoneal cavity (Martinez-Pulgarin et al.2009). In the host-pathogen environment, S. aureus is at times competing with other bacteria as well as the host. Strains inactivated for katA are more sensitive to H2O2 when competing with other bacteria, such as Streptococcus pneumoniae, for nasal colonization. The S. aureus strain inactivated for katA has no effect on nasal colonization when the competing organism is not present (Park, Nizet and Liu 2008). Some initial experiments indicate that katA has a role in S. aureus survival both in vitro and in vivo; however, results in this regard are not completely consistent and may be niche specific. Further study is needed to clarify the role of KatA in S. aureus pathogenesis. Alkyl hydroperoxide reductase In addition to H2O2, alkyl peroxides form during the oxidative burst, which differ from H2O2 in that they are peroxides attached to organic molecules. Alkyl hydroperoxide reductase consists of the protein pair, AhpC and AhpF, with reactivity to both H2O2 and alkyl hydroperoxides. These proteins function similarly to peroxiredoxins, converting alkyl hydroperoxides to the corresponding alcohol and water or H2O2 to water. AhpC confers resistance to alkyl hydroperoxide stress, and nasal colonization in mouse models is significantly reduced in the ahpC-inactivated strain (Cosgrove et al.2007). While not heavily studied in S. aureus, alkyl hydroperoxide reductase appears to play a role in pathogenesis. Small molecules involved in oxidant defenses Bacillithiol In addition to the enzymes discussed above, S. aureus generates small molecules that defend against oxidative insult. As mentioned in the introduction, the reactive species generated by leukocytes are highly reactive with thiols. Therefore, organisms generate high levels of thiols that assist in the defense against oxidants, increasing survival (Newton et al.2009; Upton et al.2012; Posada et al.2014). Bacillithiol consists of glucosamine attached to malic acid and cysteine, and is the S. aureus functional equivalent to glutathione (Fig. 3A). The cysteine of bacillithiol reacts with a toxicant, and the resulting molecule is cleaved by an amidase. After the cysteine-conjugated oxidant is released, it is acetylated and excreted from the cell. The remaining portion of bacillithiol, consisting of glucosamine conjugated to malic acid, is recycled to generate a new molecule of bacillithiol by the addition of cysteine (Newton, Fahey and Rawat 2012). In keeping with this, S. aureus strains deficient in cysteine synthesis are less viable under both H2O2 and HOCl stress (Lithgow et al.2004). Additionally, inactivation of the bacillithiol biosynthetic pathway generates mutants that are more susceptible to killing by methylglyoxal, H2O2, and other alkylating and oxidizing stressors (Rajkarnikar et al.2013). Figure 3. View largeDownload slide Staphylococcus aureus synthesizes small molecule antioxidants including (A) bacillithiol, (B) coenzyme A and (C) staphyloxanthin to protect itself from host-generated oxidants. Figure 3. View largeDownload slide Staphylococcus aureus synthesizes small molecule antioxidants including (A) bacillithiol, (B) coenzyme A and (C) staphyloxanthin to protect itself from host-generated oxidants. Another mechanism of cellular protection by small molecule thiols is protein S-thiolation. This is the process by which a protein-incorporated cysteine is disulfide bound to a thiol containing small molecule. Non-specific S-thiolation is increased in S. aureus exposed to oxidative stress, and is theorized to be a protective mechanism because these bonds are reversible, while irreversible cysteine oxidation is not. Protein S-thiolation is reversed by the enzyme thioredoxin, while a protein with extensive cysteine oxidation would need to be degraded and replaced (Pother et al.2009; Loi, Rossius and Antelmann 2015). Global bacillithiolation has not been measured in S. aureus; however, in Bacillus subtilis exposed to HOCl, protein bacilithiolation is increased. This indicates that bacilithiol can form a mixed disulfide during exposure to oxidants of the neutrophil oxidative burst (Chi et al.2011). Staphylococcus aureus strains genetically inactivated for bacillithiol production do not accumulate H2O2 as quickly as strains lacking ahpC, suggesting that bacillithiol does not have a prominent role in directly detoxifying H2O2, but potentially exerts its protective role through bacillithiolation of oxidized proteins (Rosario-Cruz et al.2015). These studies collectively show that bacillithiol plays a major role in protecting S. aureus from the oxidants it encounters during an infection through both direct detoxification and protein S-thiolation. Coenzyme A Coenzyme A is another thiol containing molecule present in S. aureus. Before the discovery of bacillithiol, it was thought that coenzyme A was the primary small molecule thiol-containing detoxicant in S. aureus (Newton et al.1996). Coenzyme A reacts with H2O2 to generate an intermolecular disulfide bond between two coenzyme A molecules. Then, coenzyme A disulfide reductase reduces the disulfide regenerating the two molecules of coenzyme A (delCardayre et al.1998). Staphyloxanthin The most unique small molecule that S. aureus synthesizes is staphyloxanthin, which gives S. aureus its golden color (Pelz et al.2005). Staphyloxanthin functions as an antioxidant, increasing survival during H2O2, superoxide, HOCl and singlet oxygen stress, and also aids in survival of S. aureus exposed to neutrophils. The mechanism by which staphyloxanthin acts has not yet been determined, but its activity is presumed to be a result of a highly conjugated isoprenyl tail reacting with oxidants (Liu et al.2005; Clauditz et al.2006). Staphyloxanthin production increases during biofilm formation (Resch et al.2005). Supplementation of staphyloxanthin to staphyloxanthin-deficient species increases survival of those species upon exposure to neutrophils. This was shown to be due to its antioxidant capabilities because host NADPH oxidase inhibition removes the survival advantage (Liu et al.2005). However, in a species related to S. aureus that is unable to produce staphyloxanthin, S. argentus, staphyloxanthin supplementation did not confer an increase in virulence, complicating the absolute role of staphyloxanthin in pathogenesis (Tong et al.2013). Staphyloxanthin is synthesized through the mevalonate pathway, sharing many steps in common with cholesterol biosynthesis in humans (Pelz et al.2005; Liu et al.2008). However, the biosynthetic enzymes differ between the two species, and inhibitors of staphyloxanthin synthesis have been developed. Blocking staphyloxanthin synthesis with such inhibitors makes bacteria more susceptible to H2O2 killing and less pathogenic in mouse infections (Liu et al.2008). Additionally, inhibiting the staphyloxanthin biosynthetic pathway makes S. aureus more susceptible to whole blood killing and reduces both pathogenesis and mouse morbidity in a systemic model of infection (Chen et al.2016). These studies confirm the necessity of staphyloxanthin to S. aureus pathogenesis and validate the staphyloxanthin biosynthetic pathway as a viable target for antimicrobial treatment. Nitric oxide Staphylococcus aureus is one of the rare species that contains a bacterial nitric oxide synthase (bNOS) (Bird et al.2002; Hong et al.2003; Vaish and Singh 2013). Strains that express bNOS are more resistant to H2O2 killing, neutrophil killing and NET killing. These strains also show increased colonization in a skin abscess mouse model of infection (van Sorge et al.2013). A single amino acid difference of valine in prokaryotes and isoleucine in eukaryotes has allowed for the development of species-specific nitric oxide inhibitors. Inhibiting bNOS makes S. aureus more susceptible to H2O2 killing, indicating that the nitric oxide or a nitric oxide-derived metabolite confers oxidative stress resistance to S. aureus (Holden et al.2015). Interestingly, inhibiting iNOS reduces host survival during S. aureus infection, but does not change the bacterial burden indicating that the contribution of nitric oxide to pathogenesis is more complex than simply scavenging reactive species (Sasaki et al.1998). These differences may also result from the observation that endogenously and exogenously generated reactive species elicit very different effects on cellular processes (Wages et al.2015). The studies mentioned above indicate that nitric oxide, or potentially a downstream metabolite of nitric oxide, is responsible for the protective role of S. aureus bNOS. Nitrite can prevent bacterial killing by scavenging HOCl (Kono 1995; Marcinkiewicz et al.2000). The reaction between nitrite and HOCl produces NO2Cl, which eventually degrades to nitrate, and is less reactive than HOCl. This reaction is more favorable at low pH, such as that seen in an abscess of S. aureus undergoing fermentation (Ford et al.1989; Panasenko et al.1997; Whiteman et al.2002). Additionally, nitric oxide blocks cysteine reduction, reducing the amount of Fenton chemistry possible, and decreasing the amount of reactive oxidants generated (Park and Imlay 2003; Gusarov and Nudler 2005). Under aerobic conditions, nitric oxide and oxygen react with cysteine in a process called S-nitrosylation (Wink et al.1994). This species is reversed by flavohemoprotein, encoded by the hmp gene, and may be protective against irreversible cysteine oxidation similar to bacillithiolation (Goncalves et al.2006; Hochgrafe et al.2008). Also, nitric oxide activates catalase by an unknown mechanism, increasing H2O2 turnover (Gusarov and Nudler 2005). Staphylococcus aureus possesses an arsenal of antioxidant enzymes and small molecules that contribute to oxidant defense and pathogenesis. Future studies will more completely elucidate the mechanisms by which these defenses confer a survival advantage. REPAIRING OXIDIZED PROTEINS Thioredoxin/thioredoxin reductase Even with all of the diverse defenses Staphylococcus aureus possesses to protect its macromolecules from oxidation, it is inevitable that proteins will be oxidized. Staphylococcus aureus contains several protein repair mechanisms that allow for the regeneration of proteins instead of having to degrade and replace proteins, a time and energy-consuming process. As mentioned above, disulfide formation is a result of cysteine oxidation, and it is hypothesized to be a protective mechanism by preventing irreversible cysteine oxidation. However, a protein that has one or more cysteines blocked by a disulfide bond may not be functional, and the enzyme system consisting of thioredoxin (TrxA) and thioredoxin reductase (TrxB) repairs these proteins. TrxA reduces disulfide bonds to generate an intramolecular disulfide (Roos et al.2007). Then, TrxB recycles TrxA back to the active confirmation by reducing the intramolecular disulfide (Messens et al.1999). Disulfide bond stress, menadione and tertbutyl hydroperoxide all induce trxA and trxB, but H2O2 does not. Inactivation of trxB expression renders strains unviable, and thus is shown to be essential for growth of S. aureus (Uziel et al.2004). Methionine sulfoxide reductase Methionine is also reversibly oxidized on its sulfur atom, but to methionine sulfoxide. Methionine oxidation in S. aureus exposed to neutrophils is an MPO-dependent process indicating that HOCl is the responsible oxidant in a neutrophil, but this oxidation can also occur upon exposure to H2O2. The extent of methionine oxidation measured in this study correlated to the amount of death seen in S. aureus (Rosen et al.2009). A class of enzymes called methionine sulfoxide reductases converts methionine sulfoxide back to methionine. Staphylococcus aureus contains four genes encoding for this activity, but only two constitute most of the reductase activity, msrA1 and msrB (Singh and Moskovitz 2003; Singh and Singh 2012). Methionine sulfoxides are chiral molecules, and most bacteria possess at least two enzyme copies, one for each stereoisomer, instead of one promiscuous copy of methionine sulfoxide reductase. The active enzymes in S. aureus are MsrA1 that reduces the S-form, and MsrB that reduces the R-form of methionine sulfoxide (Moskovitz et al.2002). Inactivating msrA1 and msrB makes S. aureus slightly more susceptible to killing by H2O2 and HOCl. Interestingly, these genes are controlled by the VraRS two-component system, which regulates the S. aureus response to cell wall stress, and do not appear to be oxidant induced (Pang et al.2014). Flavohemoprotein Flavohemoprotein (Hmp) has many functions including repair of oxidized proteins and detoxification of nitric oxide. Expression of Hmp is also induced by anaerobic or microaerobic conditions, presumably to generate nitrate from nitric oxide for use as a terminal electron acceptor. In anaerobic or microaerobic conditions, Hmp demonstrates an additional activity, as a nitroxylase, reversing S-nitrosothiols (Goncalves et al.2006). Aerobically, Hmp is induced by the presence of S-nitrosothiols, the product of the reaction between cysteine and nitric oxide-derived oxidants. Hmp does not show nitroxylase activity aerobically, but converts nitric oxide to nitrate detoxifying nitric oxide and forcing S. aureus into a state of oxygen-independent respiration (Goncalves et al.2006; Hochgrafe et al.2008). Staphylococcus aureus virulence is hmp dependent as mice infected with wild type have a higher mortality than those infected by a strain with hmp inactivated. In agreement with these findings, and further demonstrating the role of host-generated nitric oxide in bacterial killing, both strains induce similar mortality in iNOS knockout mice (Richardson, Dunman and Fang 2006). While S. aureus contains several enzyme systems that repair oxidant damage of proteins, their study is far from complete, and future experiments will give a better picture of the S. aureus protein repair response during oxidative challenge. SENSING OXIDANTS PerR Staphylococcus aureus has several important proteins that are sensitive to oxidation, and act as transcriptional regulators inducing various phenotypic changes that will be discussed later in the review. PerR binds DNA, repressing the expression of the antioxidant genes katA, trxB and ahpCF. These genes are transcribed when oxidative stress, most notably H2O2, is sensed (Horsburgh et al.2001; Maalej, Dammak and Dukan 2006). However, one study showed that H2O2 alone does not induce ahpC (Armstrong-Buisseret, Cole and Stewart 1995). Unlike most redox sensors that use cysteine as the active amino acid residue, histidines of PerR are oxidized. This oxidation occurs in the presence of H2O2 and predominantly iron, but also manganese (Traore et al.2009; Ji et al.2015). Iron-bound PerR is susceptible to H2O2 signaling by histidine oxidation, and it was proposed that iron is mechanistically involved in facilitating histidine oxidation (Lee and Helmann 2006). Histidine oxidation causes the release of PerR from DNA allowing for the initiation of transcription. A zinc-binding site in PerR composed of cysteines is susceptible to oxidation, but it appears that this site is structural, and does not play a role in transcriptional regulation (Traore et al.2009; Ji et al.2015). HOCl also alleviates PerR repression, but also inactivates SodA, complicating the assignment of the potential causes of the observed transcriptional changes. Strains inactivated for sodA survived better than wild-type S. aureus under HOCl stress. This is presumably from the derepression of PerR-controlled genes seen in the sodA-inactivated strains, indicating that induction of PerR-repressed genes is from a lack of superoxide dismutase activity and an accumulation of oxidants, not from PerR directly detecting HOCl (Maalej, Dammak and Dukan 2006). PerR derepression of antioxidant genes plays a major role in survival during H2O2 challenge and pathogenesis of S. aureus (Traore et al.2009; Ji et al.2015). Inactivation of perR leads to increased S. aureus survival under H2O2 stress, but decreased overall growth rates (Horsburgh et al.2001). In Bacillus subtilis, the observed growth deficiency is attributed to reduced iron and heme availability due to repression of iron uptake by increased levels of the ferric uptake regulator (Fur) and the usage of high amounts of heme by catalase (Faulkner et al.2012). Staphylococcus aureus pathogenicity was decreased when perR was inactivated, while katA inactivation did not change pathogenicity in a murine skin abscess model of infection (Horsburgh et al.2001). PerR represses its own transcription as well as that of fur, but these do not appear to be peroxide driven, but likely driven by iron depletion (Fuangthong et al.2002). MgrA MgrA is another DNA-binding oxidant sensor in S. aureus. Upon oxidation of Cys12 by both H2O2 as well as alkyl peroxides to sulfenic acid, MgrA releases from DNA, and transcription of MgrA-repressed genes is initiated (Chen et al.2006). MgrA regulates S. aureus toxins, indicating that its sensitivity to oxidative stress may be a surrogate method for sensing the proximity of leukocytes. MgrA also upregulates the expression of LytS, the sensor component of the two-component system LytRS, and LrgA, while repressing CidA (Luong et al.2006). LrgA is expressed at the surface of biofilms and shown to be anti-autolysis, while CidA is expressed in the interior and shown to be pro-autolysis. Autolysis, which has been compared to apoptosis in eukaryotic cells, releases intracellular macromolecules, adding rigidity and structure to biofilms (Ranjit, Endres and Bayles 2011; Moormeier et al.2013). This interplay of controlled cell death is a necessary part of biofilm development and indicates an inhibition of biofilm formation under basal conditions, but enhancement of biofilm formation upon oxidation of MgrA (Ranjit, Endres and Bayles 2011). Strains inactivated for lytS, the two-component system that drives lrgA translation, form weaker biofilms than wild-type S. aureus strains further suggesting an anti-biofilm role for lrgA (Brunskill and Bayles 1996; Sharma-Kuinkel et al.2009). Inactivation of mgrA enhances biofilm formation, demonstrating a role of MgrA in biofilm suppression. This is consistent with the idea that MgrA positively regulates lrgA, negatively regulates cidA and that oxidative stress induces biofilm formation (Trotonda et al.2008). Lower bacterial burdens are observed in the kidneys and liver of mice infected with strains inactivated for mgrA compared to wild-type S. aureus (Chen et al.2006). The energy burden of constitutively generating these proteins and the constant autolysis of biofilm formation may incur a fitness defect. Additionally, there is an optimal time for the autolysis genes to be turned on post-colonization to generate effective biofilms in vitro, and this would be expected to also be true in vivo (Moormeier et al.2013). SarZ SarZ is a DNA-binding protein implicated in gene regulation that senses H2O2 as well as organic hydroperoxides. Oxidation of Cys13 to sulfenic acid is not sufficient to interrupt DNA binding of SarZ. However, disulfide formation at this cysteine allosterically inhibits DNA binding (Poor et al.2009). SarZ is an MgrA homolog, but inactivation of sarZ does not lead to a decrease in virulence. Inactivating both mgrA and sarZ shows a large reduction in virulence, which is even greater than that of mgrA inactivation alone. Interestingly, this virulence decrease was seen in the liver of mice, but not in the kidneys (Chen et al.2009). Using a different S. aureus background, inactivation of sarZ was less pathogenic in the spleen and kidneys during a mouse systemic infection than wild type (Kaito et al.2006). Transcription of sarZ is activated by MgrA, and a strain inactivated for sarZ has decreased transcription of mgrA, sarA and hla, but increased transcription of spa, the gene that encodes for the S. aureus virulence factor protein A. Inactivation of sarZ also increases the formation of biofilms, indicating that SarZ is important for dissemination, but induces biofilms upon oxidation, similarly to MgrA (Ballal, Ray and Manna 2009; Tamber and Cheung 2009). SarA SarA is a DNA-binding transcriptional regulator with cysteine residues more sensitive to oxidation and derepression by H2O2 and disulfide stress than alkyl hydroperoxides. SarA is more responsible for the repression of oxidative stress response genes in comparison to MgrA and SarZ (Ballal and Manna 2010). SarA represses trxB, sodM and sodA (Ballal and Manna 2009, 2010). Disulfide stress and tertbutyl hydroperoxide both induce sodA to a greater extent than sodM, which is similar to a strain inactivated for sarA. Also, sarA inactivation showed different transcription levels of sodA and sodM for different oxidants, indicating multiple control mechanisms of superoxide dismutase transcription (Ballal and Manna 2009). The role of SarA in virulence is not completely clear. In one study, SarA was required for the survival of S. aureus inside of neutrophils when compared to a strain inactivated for sarA (Gresham et al.2000). However, S. aureus survival in macrophages is not sarA dependent (Kubica et al.2008). Strains inactivated for sarA showed a reduced ability to form biofilms (Beenken, Blevins and Smeltzer 2003). In another study, SarA and MgrA appear to be antagonistic in terms of biofilm formation. Inactivation of mgrA enhances biofilm formation, while simultaneous inactivation of sarA and mgrA eliminates biofilm formation (Trotonda et al.2008). In an alternative mechanism of control, SarA, SarZ and MgrA can all be reversibly phosphorylated on their sensing cysteines that are controlled by kinase Stk1 and phosphatase Stp1. This has the same effect as if the cysteines were oxidized, releasing the repressors from DNA and activating transcription. Deletion of stp1 generates a hyper-phosphorylated state of the proteins and reduces virulence (Sun et al.2012). Spx Spx is a redox sensor that relies on intramolecular disulfide bond formation to bind DNA and initiate the transcription of trxA and trxB in B. subtilis (Nakano et al.2005). High homology exists between the B. subtilis and S. aureus proteins (Pamp et al.2006). In addition to being an oxidant sensor and transcriptional initiator, Spx expression increases upon oxidative stress (Engman et al.2012). Inactivation of spx results in no increased transcription of trxB even under oxidant stress in S. aureus. Further confirming the important role of trxB, strains inactivated for spx do not grow when challenged with oxidant stress. Inactivation of spx also results in decreased icaR expression, the negative regulator of the ica operon. Increased icaA expression increases biofilm formation, indicating that Spx is potentially a repressor of biofilm formation by increasing icaR transcription (Pamp et al.2006). QsrR QsrR is a quinone sensing transcriptional regulator that binds DNA and represses the synthesis of enzymes that are potentially involved in the detoxification of reactive electrophiles, NADH-dependent flavin mononucleotide reductase, nitroreductase and glyoxalase. Menadione, the precursor to menaquinone, covalently binds to Cys5, generating a large conformational change and derepression of the QsrR-controlled genes (Ji et al.2013). Oxidative stress from heme toxicity pushes the menaquinone pool to the quinone form, which reacts with QsrR (Wakeman et al.2012). It is not unreasonable to think that exogenous oxidative stress can similarly affect menaquinone pools triggering QsrR derepression (Ji et al.2013). However, the isoprenyl tail of menaquinone may sterically inhibit the bond between menaquinone and QsrR. Regardless, the identification of this protein as an electrophile sensor is quite intriguing because electrophiles are abundant at sites of inflammation including the host–pathogen interface (Schopfer, Cipollina and Freeman 2011). Strains inactivated for qsrR were not killed by macrophages, and the extent of phagocytosis was decreased greatly, presumably due to the constitutive expression of genes involved in survival of quinone stress (Ji et al.2013). Other physiologically relevant electrophiles that S. aureus will encounter at the host–pathogen interface, like methylglyoxal and lipid electrophiles, may also bind to QsrR. However, at this point, electrophile stress in S. aureus has not been extensively studied. CELLULAR CONSEQUENCES OF PROTEIN OXIDATION Transcriptional changes induced by oxidation Host-generated oxidants induce a wide range of effects in Staphylococcus aureus, including transcriptional changes of genes encoding for enzymes used in oxidant defense. Copper stress, used to simulate an oxidative burst, has revealed that katA, trxA, trxB, sodA, sodM, ahpC and ahpF are all upregulated during oxidative stress in S. aureus. Inactivation of each of these genes makes S. aureus less able to survive the oxidative burst from copper stress (Baker et al.2010). H2O2, superoxide and disulfide stress induce ahpC, while trxB is induced by superoxide and disulfide stress, but not H2O2 (Wolf et al.2008). Exposure of various S. aureus strains to neutrophils induces katA, sodA, sodM, trxA, trxB, ahpC and ahpF (Voyich et al.2005). Induction of the iron-regulated surface determinant (isd) genes only occurs in H2O2-treated cells, and not cells treated with HOCl or azurophilic granule proteins. H2O2 reacts with iron through Fenton chemistry, changing the oxidation state of iron. HOCl has a rate constant three orders of magnitude larger for the reaction with thiol containing amino acids than with ferrous iron, and HOCl is the major product generated by azurophilic granule proteins as well (Folkes, Candeias and Wardman 1995; Palazzolo-Ballance et al.2008). Therefore, it is possible that the measured isd induction is a result of a perceived iron deficiency and not due to oxidative signaling. Inactivation of genes encoding the Isd system makes S. aureus less resistant to neutrophil killing presumably from the role iron plays as a cofactor in many of the S. aureus antioxidant enzymes. Transcription of trxA, trxB, ahpC and sodA were all upregulated in response to H2O2, HOCl and exposure to azurophilic granule proteins (Palazzolo-Ballance et al.2008). In addition to antioxidant enzymes and enzymes involved in protein damage repair, there is also an induction of DNA repair machinery that occurs following treatment with H2O2. These genes are induced in response to a growth defect generated by H2O2, indicating extensive macromolecule damage during oxidative challenge (Chang et al.2006; Wolf et al.2008). The transcriptional changes noted in these studies show that S. aureus is particularly suited to respond to and neutralize the oxidative challenges it encounters during colonization. Protein modification by the oxidative burst The most well studied molecular effect of highly concentrated doses of H2O2 is cysteine oxidation resulting in various oxidation products shown in Fig. 2A. Treatment with 10–100 mM H2O2 oxidizes the cysteines of AhpC and AhpF. The isoelectric point shift of AhpC indicates that the oxidation is irreversible. Spx, TrxA and TrxB are all reversibly oxidized by H2O2 (Wolf et al.2008). Cys151 on glyceraldehyde-3-phosphate dehydrogenase is oxidized to sulfonic acid by 100 mM H2O2. This study also noted the oxidation of AhpC by a shift in isoelectric point, but the site of oxidation was not determined. The oxidation of these proteins is not repaired, but each protein is replaced with new protein during growth arrest (Weber et al.2004). In addition to H2O2, HOCl also oxidizes cysteines, and has been shown to impair superoxide dismutase activity (Maalej, Dammak and Dukan 2006). HOCl treatment resulted in increased alkaline phosphatase activity and decreased acid phosphatase activity (Abid, Maalej and Rouis 2004). The qualitative oxidation of S. aureus proteins important for withstanding oxidative stress and generating cellular energy may have detrimental cellular consequences. However, in-depth mechanistic studies need to be performed to determine if oxidation directly changes enzyme activity or is correlated with changes in enzyme activity, and to identify whether oxidation of specific residues is responsible for activity changes. In addition to the primary oxidants detailed above, the oxidative burst from leukocytes generates many secondary oxidation products and reactive electrophiles that react with amino acid side chains (Fig. 2F). HOCl can react with serine to generate glyoxal and glycoaldehyde, while its reaction with free threonine generates acrolein and 2-hydroxypropanal (Anderson et al.1997, 1999). Free tyrosine reacts with HOCl to generate p-hydroxyphenylacetaldehyde (Hazen, Hsu and Heinecke 1996). These aldehydes all react with the epsilon amine of lysine, and increases in protein carbonyl levels are dependent upon NADPH oxidase and MPO activity (Wilkie-Grantham et al.2015). Methylglyoxal is antibacterial against S. aureus in vitro, but methylglyoxal toxicity and the glyoxalase detoxification system, which is present in S. aureus, have been more extensively studied in Gram-negative bacteria (Booth et al.2003; Kilty et al.2011). It will be interesting to see if the secondary oxidation products mentioned here play a role in the host mediation of S. aureus pathogenesis. Taken together, these studies suggest that S. aureus must be prepared to neutralize and repair molecular damage from oxidants of the oxidative burst, and from a series of secondary metabolites generated by the primary oxidants reacting with both host and pathogen macromolecules. Phenotypic changes in response to oxidants Many of the cellular changes discussed above induce SCV phenotypes and biofilms, both of which are thought to be protective mechanisms used by S. aureus to survive stressful situations. Generation of SCVs and biofilms is accompanied by metabolic changes (Proctor et al.2014). For example, respiration-deficient SCVs are better able to avoid host killing by inducing a more blunted immune response and are able to persist intracellularly. They are also more resistant to antibiotic killing allowing for their persistence (Tuchscherr et al.2010). H2O2 induces genes responsible for anaerobic metabolism, and also causes growth defects. This metabolic shift eliminates the need for oxygen, reducing further damage from reactive oxygen species generated during aerobic respiration (Chang et al.2006). In agreement with previous studies investigating the cellular effects of H2O2 stress, H2O2 induces an initial upregulation of S. aureus metabolic genes that are eventually downregulated, while protein synthesis, DNA synthesis and cell division genes show the opposite trend (Voyich et al.2005). Oxidation of glyceraldehyde-3-phosphate dehydrogenase by H2O2 results in lower ATP generation and correlates with growth arrest, which is resolved when active enzyme is synthesized (Weber et al.2004). All of these studies show that S. aureus undergoes metabolic changes either in response to, or because of, oxidants generated by the host response to the bacteria. H2O2 stress induces SCVs that have increased catalase synthesis to help eliminate this stress. An S. aureus strain deficient in DNA repair is more sensitive to H2O2, and SOS response induction promotes SCV reversion indicating that reversion of the phenotype occurs only after oxidative damage has been repaired (Painter et al.2015). Many SCV phenotypes revert when the stressor is removed indicating a role for metabolic changes in the formation of the phenotype. Phenotype switching under antibiotic stress has been proposed as a mechanism of survival, resulting in similar metabolic changes as those discussed above (Edwards 2012). HOCl treatment of S. aureus generates not only SCVs, but also a smaller cell size. The cells shrink from 700 to 280 nm upon exposure to HOCl (Abid, Maalej and Rouis 2004). Reactive aldehydes including methylglyoxal, glycoaldehyde and acrolein all generate SCVs as well as biofilms in clinical isolates of S. aureus. Interestingly, these phenotypes are stronger in MRSA strains than they are in methicillin-sensitive S. aureus strains (Bui, Turnidge and Kidd 2015b). Staphylococcus aureus LAC USA300, an MRSA strain, can replicate after being phagocytosed by macrophages. Not all S. aureus strains are able to survive phagocytosis, and it is currently unknown how this strain of S. aureus is able to survive the oxidative burst and harsh conditions it encounters, but an SCV phenotype is one possible explanation (Flannagan, Heit and Heinrichs 2015). Stable SCVs generated under constant methylglyoxal stress have downregulated virulence factors and enhanced propensity to form biofilms. High cell heterogeneity was observed under these conditions, a further indicator that a biofilm-forming phenotype was elicited (Bui, Turnidge and Kidd 2015a). Understanding the mechanistic relationship between pathogenesis and the formation of biofilms during the oxidative burst is a significant challenge. Therefore, biofilm generation as a defense against oxidative stress in S. aureus has not been extensively studied, but based on many of the cellular changes discussed in this review, it appears that biofilm formation plays a significant role in S. aureus defense against host oxidants. CONCLUSION/OUTLOOK Staphylococcus aureus is a versatile and adaptable pathogen that has many defenses allowing it to avoid killing by leukocytes. Here, we have focused on and discussed those defenses that protect against oxidative stress mediated by the immune response (Fig. 4). Staphylococcus aureus possesses a plethora of antioxidant enzymes including the superoxide dismutases, catalase and alkyl hydroperoxide reductase. In addition to these proteins, S. aureus generates the small molecules bacillithiol, coenzyme A, staphyloxanthin and nitric oxide to protect its cellular macromolecules from oxidative damage. When these defenses fail or are overwhelmed by a robust oxidative burst, several enzymes are expressed to repair damage incurred on S. aureus proteins, including thioredoxin/thioredoxin reductase, methionine sulfoxide reductases and flavohemoprotein. Finally, S. aureus has several oxidant sensors that induce transcription of antioxidant enzymes, oxidation repair enzymes and biofilm-promoting genes. These expression changes induce physiological changes that allow S. aureus to survive in the face of leukocyte-generated oxidative stress. Figure 4. View largeDownload slide Staphylococcus aureus uses multiple mechanisms to survive the host oxidative burst including antioxidant proteins and small molecules to neutralize oxidants, enzymes that repair protein damage and oxidant sensing transcriptional regulators. Figure 4. View largeDownload slide Staphylococcus aureus uses multiple mechanisms to survive the host oxidative burst including antioxidant proteins and small molecules to neutralize oxidants, enzymes that repair protein damage and oxidant sensing transcriptional regulators. Moving forward with S. aureus oxidative stress research, we feel there are three areas in which this research should be expanded. First, research should have a focus on HOCl as an oxidant due to the prominent role of neutrophils interacting with S. aureus at the host–pathogen interface. Studies discussed here show that HOCl is the most relevant oxidant in this setting; yet, little is understood about the effects of HOCl on S. aureus (Humphreys et al.1989; Green, Kettle and Winterbourn 2014). Second, many enzymes have been reportedly inactivated by oxidative stress. Now that we have the ability to rapidly profile protein post-translational modifications, the mechanism of inactivation must also be elucidated to validate and expand potential targets for future antimicrobials. Finally, more of this work needs to be done in S. aureus or other members of the ESKAPE pathogens. The Centers for Disease Control and Prevention has designated these pathogens as having the highest priority for developing future antimicrobials, and performing research in this area provides the greatest opportunity for breakthroughs that impact human health. Additionally, the strain of S. aureus should be carefully selected so that discoveries will have the highest potential for translation to clinical practice. Studies using lab-adapted strains that facilitate ease of use have greatly expanded the research into how S. aureus evades oxidative stress. However, future S. aureus oxidative stress research should be conducted in a setting most relevant and impactful to generating advancements that contribute to improving human health. We thank the members of the Skaar research group for critical review of this manuscript. FUNDING The writing of this manuscript was supported by the National Institutes of Health through fellowships to WNB (T32 AI00747421) and research grants to EPS (R01 AI069233 and R01 AI73843). Conflict of interest. None declared. 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TI - Neutrophil-generated oxidative stress and protein damage in Staphylococcus aureus
JF - Pathogens and Disease
DO - 10.1093/femspd/ftw060
DA - 2016-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/neutrophil-generated-oxidative-stress-and-protein-damage-in-9cpXOCXzaY
SP - ftw060
VL - 74
IS - 6
DP - DeepDyve
ER -