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Methicillin-resistant Staphylococcus aureus Bacterial Nitric-oxide Synthase Affects Antibiotic Sensitivity and Skin Abscess Development

Open AccessPublished:January 15, 2013DOI:https://doi.org/10.1074/jbc.M112.448738
      Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from l-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.
      Background: Methicillin-resistant Staphylococcus aureus (MRSA) generates NO through bacterial NO synthase (bNOS).
      Results: Loss of bNOS increases MRSA sensitivity to host neutrophils, cathelicidin antimicrobial peptides, and cell envelope-active antibiotics.
      Conclusion: bNOS influences MRSA disease pathology.
      Significance: Future development of bNOS-specific inhibitors could provide dual activities to reduce MRSA pathology and increase antibiotic effectiveness.

      Introduction

      Mammalian NO production is regulated by the activity of NO synthase (NOS)
      • Pant K.
      • Bilwes A.M.
      • Adak S.
      • Stuehr D.J.
      • Crane B.R.
      Structure of a nitric oxide synthase heme protein from Bacillus subtilis.
      enzymes, which catalyze the production of NO from l-arginine. The three mammalian NOS isoforms have two catalytic domains, an oxygenase domain and a reductase domain, responsible for binding the substrate arginine and co-factors, respectively. NO production has been implicated in many physiological and pathological processes, including host innate immune and inflammatory responses to pathogens (
      • Wink D.A.
      • Hines H.B.
      • Cheng R.Y.
      • Switzer C.H.
      • Flores-Santana W.
      • Vitek M.P.
      • Ridnour L.A.
      • Colton C.A.
      Nitric oxide and redox mechanisms in the immune response.
      ). Paradoxically, genome sequencing has revealed the existence of mammalian NOS homologues in a limited number of prokaryotic organisms, principally Gram-positive bacteria (
      • Gusarov I.
      • Starodubtseva M.
      • Wang Z.Q.
      • McQuade L.
      • Lippard S.J.
      • Stuehr D.J.
      • Nudler E.
      Bacterial nitric-oxide synthases operate without a dedicated redox partner.
      ). Structural analysis has shown that bacterial NOS (bNOS) enzymes are highly similar to the oxygenase domain of mammalian NOS (
      • Bird L.E.
      • Ren J.
      • Zhang J.
      • Foxwell N.
      • Hawkins A.R.
      • Charles I.G.
      • Stammers D.K.
      Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus.
      ,
      • Pant K.
      • Bilwes A.M.
      • Adak S.
      • Stuehr D.J.
      • Crane B.R.
      Structure of a nitric oxide synthase heme protein from Bacillus subtilis.
      • Pant K.
      • Crane B.R.
      Nitrosyl-heme structures of Bacillus subtilis nitric oxide synthase have implications for understanding substrate oxidation.
      ) but lack a corresponding reductase domain. Nonetheless, bNOS can efficiently generate NO from l-arginine by hijacking alternate cellular redox partners (
      • Gusarov I.
      • Starodubtseva M.
      • Wang Z.Q.
      • McQuade L.
      • Lippard S.J.
      • Stuehr D.J.
      • Nudler E.
      Bacterial nitric-oxide synthases operate without a dedicated redox partner.
      ).
      Bacterial NO production contributes to Bacillus anthracis virulence by enhancing resistance to oxidative stress (
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      • Gusarov I.
      • Avetissova E.
      • Shatalina Y.
      • McQuade L.E.
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      • Nudler E.
      Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages.
      ,
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      NO-mediated cytoprotection. Instant adaptation to oxidative stress in bacteria.
      ) and to virulence of the plant pathogen Streptomyces turgidiscabies by nitrating and activating a phytotoxin (
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      Nitration of a peptide phytotoxin by bacterial nitric oxide synthase.
      ). In addition, recent studies demonstrate a link between NO and bacterial antibiotic resistance. First, Gusarov et al. (
      • Gusarov I.
      • Shatalin K.
      • Starodubtseva M.
      • Nudler E.
      Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.
      ) found that endogenous NO production through bNOS protects the producing bacterium against various antibiotics, either through chemical modification of the agent or by counteracting the increased levels of intracellular oxidative stress induced by bactericidal drugs. In a complementary fashion, exposure to exogenous NO protects both Gram-positive and Gram-negative bacteria from aminoglycoside toxicity by limiting drug uptake (
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      • Hoffman M.
      • Husain M.
      • Vázquez-Torres A.
      Nitric oxide protects bacteria from aminoglycosides by blocking the energy-dependent phases of drug uptake.
      ).
      Staphylococcus aureus is a Gram-positive human pathogen causing a wide spectrum of clinical disease, ranging from minor skin infections to severe invasive conditions such as pneumonia, sepsis, and endocarditis. Recently, marked increases in infections by antibiotic (methicillin)-resistant S. aureus (MRSA) have occurred in both hospital and community settings, posing a grave challenge to the public health. Epidemic strains of community-acquired MRSA (CA-MRSA) such as the USA300 clone can produce severe disease even in previously healthy individuals (
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      Community-associated meticillin-resistant Staphylococcus aureus.
      ,
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      Methicillin-resistant Staphylococcus aureus strain USA300. Origin and epidemiology.
      ), reflecting a formidable capacity of the pathogen to evade normal host innate immune clearance (
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      • Dorward D.W.
      • Gardner D.J.
      • Kreiswirth B.N.
      • Musser J.M.
      • DeLeo F.R.
      Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils.
      ).
      A key element of innate defense against invasive pathogens such as S. aureus is the function of neutrophils (
      • Mölne L.
      • Verdrengh M.
      • Tarkowski A.
      Role of neutrophil leukocytes in cutaneous infection caused by Staphylococcus aureus.
      ,
      • von Köckritz-Blickwede M.
      • Rohde M.
      • Oehmcke S.
      • Miller L.S.
      • Cheung A.L.
      • Herwald H.
      • Foster S.
      • Medina E.
      Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.
      • Robertson C.M.
      • Perrone E.E.
      • McConnell K.W.
      • Dunne W.M.
      • Boody B.
      • Brahmbhatt T.
      • Diacovo M.J.
      • Van Rooijen N.
      • Hogue L.A.
      • Cannon C.L.
      • Buchman T.G.
      • Hotchkiss R.S.
      • Coopersmith C.M.
      Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus clearance.
      ). Generation of reactive oxygen species (ROS) through respiratory burst is a central element of normal neutrophil killing activity, as evidenced by the marked susceptibility of patients with genetic defects in NADPH oxidase (chronic granulomatous disease) to recurrent severe S. aureus infections (
      • van den Berg J.M.
      • van Koppen E.
      • Ahlin A.
      • Belohradsky B.H.
      • Bernatowska E.
      • Corbeel L.
      • Español T.
      • Fischer A.
      • Kurenko-Deptuch M.
      • Mouy R.
      • Petropoulou T.
      • Roesler J.
      • Seger R.
      • Stasia M.J.
      • Valerius N.H.
      • Weening R.S.
      • Wolach B.
      • Roos D.
      • Kuijpers T.W.
      Chronic granulomatous disease. The European experience.
      ). In addition, inflammatory activation of leukocytes results in inducible NOS (iNOS) induction and release of NO, which has a direct inhibitory activity against several bacterial species, and participates in downstream reactions with different ROS to produce synergistic antibacterial effects (
      • Wink D.A.
      • Hines H.B.
      • Cheng R.Y.
      • Switzer C.H.
      • Flores-Santana W.
      • Vitek M.P.
      • Ridnour L.A.
      • Colton C.A.
      Nitric oxide and redox mechanisms in the immune response.
      ). Nonoxidant mechanisms also contribute to bacterial killing, for example cationic antimicrobial peptide (AMPs) stored in neutrophil granules that target and disrupt bacterial membrane integrity (
      • Soehnlein O.
      Direct and alternative antimicrobial mechanisms of neutrophil-derived granule proteins.
      ).
      S. aureus has evolved multifaceted resistance mechanisms to neutrophil killing that contribute to its invasive disease potential. For example, ROS activity is mitigated by the S. aureus enzyme catalase (
      • Mandell G.L.
      Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal-leukocyte interaction.
      ,
      • Park B.
      • Nizet V.
      • Liu G.Y.
      Role of Staphylococcus aureus catalase in niche competition against Streptococcus pneumoniae.
      ) and the carotenoid pigment of the bacterium (
      • Liu G.Y.
      • Essex A.
      • Buchanan J.T.
      • Datta V.
      • Hoffman H.M.
      • Bastian J.F.
      • Fierer J.
      • Nizet V.
      Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity.
      ). S. aureus can also tolerate severe nitrosative stress and high NO concentrations, because of adaptive metabolic responses including induction of the flavohemoprotein Hmp (
      • Richardson A.R.
      • Dunman P.M.
      • Fang F.C.
      The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity.
      ) and l-lactate dehydrogenase (
      • Richardson A.R.
      • Libby S.J.
      • Fang F.C.
      A nitric oxide-inducible lactate dehydrogenase enables Staphylococcus aureus to resist innate immunity.
      ). Relative insensitivity of S. aureus to cathelicidin action is promoted by cationic cell wall modifications (
      • Peschel A.
      • Otto M.
      • Jack R.W.
      • Kalbacher H.
      • Jung G.
      • Götz F.
      Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides.
      ,
      • Kristian S.A.
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      • Neumeister B.
      • Peschel A.
      • Landmann R.
      Alanylation of teichoic acids protects Staphylococcus aureus against Toll-like receptor 2-dependent host defense in a mouse tissue cage infection model.
      ) or proteolytic cleavage (
      • Jin T.
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      • Higgins J.
      • Tarkowski A.
      Staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism.
      ,
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      • McAleese F.
      • Foster T.
      • Travis J.
      • Potempa J.
      Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases.
      ).
      S. aureus expresses a bNOS enzyme, but its potential contribution to bacterial immune and antibiotic resistance has not been tested. To assess the role of MRSA bNOS in these processes, we couple targeted mutagenesis with in vitro and in vivo assays of bacterial-host interaction and antibiotic susceptibility. Our analysis was conducted using a representative isolate of the epidemic USA300 CA-MRSA strain, the most common agent of serious bacterial infections in recent epidemiologic surveillance in the United States (
      • Tenover F.C.
      • Goering R.V.
      Methicillin-resistant Staphylococcus aureus strain USA300. Origin and epidemiology.
      ).

      DISCUSSION

      In mammals, NO is an important regulator of diverse biological functions. Of the three described NOS isoforms (NOS1–3) that generate NO from l-arginine, NOS2 or iNOS is mainly responsible for functions related to immune regulation and host defense against pathogens (
      • Chakravortty D.
      • Hensel M.
      Inducible nitric oxide synthase and control of intracellular bacterial pathogens.
      ). Activation of leukocytes by inflammatory stimuli results in local release of ROS and NO as a result of iNOS induction. Studies in knock-out animals support a critical role for phagocyte oxidative burst in controlling S. aureus (
      • Pollock J.D.
      • Williams D.A.
      • Gifford M.A.
      • Li L.L.
      • Du X.
      • Fisherman J.
      • Orkin S.H.
      • Doerschuk C.M.
      • Dinauer M.C.
      Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.
      ). In contrast, studies of iNOS knock-out mice have yielded conflicting results, with one study showing increased susceptibility to S. aureus infection (
      • Sasaki S.
      • Miura T.
      • Nishikawa S.
      • Yamada K.
      • Hirasue M.
      • Nakane A.
      Protective role of nitric oxide in Staphylococcus aureus infection in mice.
      ) and a second showing no difference compared with WT animals (
      • Köhler J.
      • Breitbach K.
      • Renner C.
      • Heitsch A.K.
      • Bast A.
      • van Rooijen N.
      • Vogelgesang S.
      • Steinmetz I.
      NADPH-oxidase but not inducible nitric oxide synthase contributes to resistance in a murine Staphylococcus aureus Newman pneumonia model.
      ).
      S. aureus is among a limited number of Gram-positive bacterial species possessing an iNOS homologue (
      • Gusarov I.
      • Starodubtseva M.
      • Wang Z.Q.
      • McQuade L.
      • Lippard S.J.
      • Stuehr D.J.
      • Nudler E.
      Bacterial nitric-oxide synthases operate without a dedicated redox partner.
      ). In addition, S. aureus is relatively resistant to nitrosative stress, i.e., exogenous NO, through transcriptional adaptation that is essential for virulence (
      • Richardson A.R.
      • Dunman P.M.
      • Fang F.C.
      The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity.
      ,
      • Richardson A.R.
      • Libby S.J.
      • Fang F.C.
      A nitric oxide-inducible lactate dehydrogenase enables Staphylococcus aureus to resist innate immunity.
      ). In this paper, we describe that deletion of bNOS in a virulent strain of community-acquired MRSA increased bacterial susceptibility to oxidative stress, similar to earlier reports in Bacillus species (
      • Gusarov I.
      • Starodubtseva M.
      • Wang Z.Q.
      • McQuade L.
      • Lippard S.J.
      • Stuehr D.J.
      • Nudler E.
      Bacterial nitric-oxide synthases operate without a dedicated redox partner.
      ,
      • Shatalin K.
      • Gusarov I.
      • Avetissova E.
      • Shatalina Y.
      • McQuade L.E.
      • Lippard S.J.
      • Nudler E.
      Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages.
      ,
      • Gusarov I.
      • Nudler E.
      NO-mediated cytoprotection. Instant adaptation to oxidative stress in bacteria.
      ). Oxidant resistance of the ΔNOS mutant could be restored to WT levels by plasmid complementation of the nos gene or short term exposure of the mutant to exogenous NO. Such short term NO exposure does not involve transcriptional adaptation but rather increases activity of oxidant resistance proteins like catalase or blocking Fenton-induced cytotoxicity (
      • Gusarov I.
      • Nudler E.
      NO-mediated cytoprotection. Instant adaptation to oxidative stress in bacteria.
      ). Because NO is freely diffusible across cell membranes and S. aureus is intrinsically resistant to nitrosative stress, these results may indicate that S. aureus can co-opt host-derived NO to supplement bNOS-derived NO in mitigating ROS stress during encounters with activated phagocytes. In line with this hypothesis, it was recently shown that the pathogen Salmonella typhimurium, which lacks bNOS, utilizes macrophage-derived NO to protect itself from the activity of aminoglycosides by reducing drug uptake (
      • McCollister B.D.
      • Hoffman M.
      • Husain M.
      • Vázquez-Torres A.
      Nitric oxide protects bacteria from aminoglycosides by blocking the energy-dependent phases of drug uptake.
      ).
      Expression of bNOS increased resistance to two front-line pharmaceutical antibiotics used in treatment of serious MRSA infections: vancomycin and daptomycin. In addition, we show for the first time that bNOS can contribute to defense against so-called“natural antibiotics” such as host-derived AMP (cathelicidin). In particular, overexpression of bNOS on a plasmid significantly increased MRSA resistance to these antimicrobial agents, resulting in the generation of a daptomycin-resistant MRSA strain. This indicates that the ability to further up-regulate NO levels could aid the bacterium in dealing with antibiotic-induced (oxidative) stress. Consistent with this observation, we observed that MRSA bNOS activity increased following vancomycin exposure and subsequently boosted activity of SOD, an ROS detoxifying enzyme. How bNOS increases SOD activity is currently not known, but similar experiments in B. subtilis previously demonstrated increased bNOS-dependent Sod transcription (
      • Gusarov I.
      • Shatalin K.
      • Starodubtseva M.
      • Nudler E.
      Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.
      ). Overall, we suggest that bNOS increases MRSA resistance to antibiotics that act on the cell envelope at least in part by increasing resistance to antibiotic-induced Fenton-generated oxidative stress and through induction of oxidant neutralizing enzymes such as SOD.
      In contrast to previous observations in B. subtilis and B. anthracis (
      • Gusarov I.
      • Shatalin K.
      • Starodubtseva M.
      • Nudler E.
      Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.
      ), endogenous NO actually sensitizes MRSA to the toxic effect of aminoglycosides including gentamicin and streptomycin. Interestingly, bNOS does not affect susceptibility to linezolid, representative of the oxazolidinone class of protein synthesis inhibitors. This indicates that the spectrum of effects of S. aureus bNOS on antibiotic resistance are distinct from those observed in Bacillus (
      • Gusarov I.
      • Shatalin K.
      • Starodubtseva M.
      • Nudler E.
      Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.
      ). The underlying mechanism for this divergent effect is interesting because it may hint toward a biological function of bNOS to influence protein synthesis through the 30 S ribosomal subunit.
      Neutrophils are crucial effector cells for host containment of S. aureus infection (
      • Mölne L.
      • Verdrengh M.
      • Tarkowski A.
      Role of neutrophil leukocytes in cutaneous infection caused by Staphylococcus aureus.
      ,
      • von Köckritz-Blickwede M.
      • Rohde M.
      • Oehmcke S.
      • Miller L.S.
      • Cheung A.L.
      • Herwald H.
      • Foster S.
      • Medina E.
      Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.
      ,
      • Köhler J.
      • Breitbach K.
      • Renner C.
      • Heitsch A.K.
      • Bast A.
      • van Rooijen N.
      • Vogelgesang S.
      • Steinmetz I.
      NADPH-oxidase but not inducible nitric oxide synthase contributes to resistance in a murine Staphylococcus aureus Newman pneumonia model.
      ), deploying both ROS and cathelicidin as key components of their bactericidal arsenal. Recent studies have revealed that community-acquired MRSA strains are highly resistant to neutrophil killing compared with hospital-acquired MRSA strains (
      • Voyich J.M.
      • Braughton K.R.
      • Sturdevant D.E.
      • Whitney A.R.
      • Saïd-Salim B.
      • Porcella S.F.
      • Long R.D.
      • Dorward D.W.
      • Gardner D.J.
      • Kreiswirth B.N.
      • Musser J.M.
      • DeLeo F.R.
      Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils.
      ,
      • Palazzolo-Ballance A.M.
      • Reniere M.L.
      • Braughton K.R.
      • Sturdevant D.E.
      • Otto M.
      • Kreiswirth B.N.
      • Skaar E.P.
      • DeLeo F.R.
      Neutrophil microbicides induce a pathogen survival response in community-associated methicillin-resistant Staphylococcus aureus.
      ). We demonstrate here that endogenous NO production contributes to the CA-MRSA neutrophil resistance phenotype. Moreover, because addition of cytochalasin D did not abolish this difference, bNOS contributes as well to protection against killing by NETs. Susceptibility of the MRSA ΔNOS mutant to NET killing is likely linked to increased sensitivity to cathelicidin AMPs that are present in abundance in these structures (
      • von Köckritz-Blickwede M.
      • Nizet V.
      Innate immunity turned inside-out. Antimicrobial defense by phagocyte extracellular traps.
      ,
      • Lauth X.
      • von Köckritz-Blickwede M.
      • McNamara C.W.
      • Myskowski S.
      • Zinkernagel A.S.
      • Beall B.
      • Ghosh P.
      • Gallo R.L.
      • Nizet V.
      M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition.
      ).
      Overall, the effects of endogenous NO production on S. aureus antibiotic susceptibility and immune survival are subtler and more specific than previously reported in Bacillus species (
      • Shatalin K.
      • Gusarov I.
      • Avetissova E.
      • Shatalina Y.
      • McQuade L.E.
      • Lippard S.J.
      • Nudler E.
      Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages.
      ,
      • Gusarov I.
      • Nudler E.
      NO-mediated cytoprotection. Instant adaptation to oxidative stress in bacteria.
      ,
      • Gusarov I.
      • Shatalin K.
      • Starodubtseva M.
      • Nudler E.
      Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics.
      ). MRSA possesses a large repertoire of established ROS and AMP resistance mechanisms (
      • Palazzolo-Ballance A.M.
      • Reniere M.L.
      • Braughton K.R.
      • Sturdevant D.E.
      • Otto M.
      • Kreiswirth B.N.
      • Skaar E.P.
      • DeLeo F.R.
      Neutrophil microbicides induce a pathogen survival response in community-associated methicillin-resistant Staphylococcus aureus.
      ,
      • Ouhara K.
      • Komatsuzawa H.
      • Kawai T.
      • Nishi H.
      • Fujiwara T.
      • Fujiue Y.
      • Kuwabara M.
      • Sayama K.
      • Hashimoto K.
      • Sugai M.
      Increased resistance to cationic antimicrobial peptide LL-37 in methicillin-resistant strains of Staphylococcus aureus.
      ,
      • Nizet V.
      Understanding how leading bacterial pathogens subvert innate immunity to reveal novel therapeutic targets.
      ), and this may provide functional redundancy that allows greater compensation for loss of bNOS expression. The regulatory effects of endogenous NO are also likely to differ among species, just as differential adaptive responses of S. aureus and B. anthracis to exogenous NO have been described (
      • Richardson A.R.
      • Dunman P.M.
      • Fang F.C.
      The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity.
      ,
      • Hochgräfe F.
      • Wolf C.
      • Fuchs S.
      • Liebeke M.
      • Lalk M.
      • Engelmann S.
      • Hecker M.
      Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus.
      ). Finally, despite the overall structural similarity between S. aureus and B. anthracis bNOS and mammalian iNOS (
      • Bird L.E.
      • Ren J.
      • Zhang J.
      • Foxwell N.
      • Hawkins A.R.
      • Charles I.G.
      • Stammers D.K.
      Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus.
      ), biochemical differences have been described for the two bNOS enzymes (
      • Salard I.
      • Mercey E.
      • Rekka E.
      • Boucher J.L.
      • Nioche P.
      • Mikula I.
      • Martasek P.
      • Raman C.S.
      • Mansuy D.
      Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.
      ). This is reflected by the observation that commonly used mammalian NOS inhibitors fail to inhibit MRSA bNOS (data not shown), in contrast to B. anthracis, where mammalian NOS inhibitors can reproduce the ΔNOS mutant phenotype (
      • Shatalin K.
      • Gusarov I.
      • Avetissova E.
      • Shatalina Y.
      • McQuade L.E.
      • Lippard S.J.
      • Nudler E.
      Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages.
      ). Paradoxically, certain iNOS inhibitors have even been reported to increase S. aureus bNOS activity (
      • Choi W.S.
      • Chang M.S.
      • Han J.W.
      • Hong S.Y.
      • Lee H.W.
      Identification of nitric oxide synthase in Staphylococcus aureus.
      ), emphasizing the need for additional studies on the function and regulation of the MRSA bNOS enzyme.
      Using a mouse model of S. aureus skin infection, we demonstrate that MRSA bNOS contributes to lesion development and bacterial survival in vivo. Enhanced survival of the WT versus ΔNOS mutant in a co-infection experiment suggests that increased resistance to killing by host defenses (e.g., AMPs, ROS, and neutrophils) is a contributor to the bNOS-associated virulence phenotype. However, future work could also explore whether bNOS regulates expression of MRSA cytotoxins or induces increased host inflammatory response pathways that also contribute to larger lesion sizes.
      As an alternative to traditional antibiotics in an era of increasing bacterial resistance, new attention has focused upon development of agents that target bacterial virulence factors, effectively disarming the pathogen to allow clearance by the host (
      • Nizet V.
      Understanding how leading bacterial pathogens subvert innate immunity to reveal novel therapeutic targets.
      ,
      • Escaich S.
      Novel agents to inhibit microbial virulence and pathogenicity.
      ,
      • Clatworthy A.E.
      • Pierson E.
      • Hung D.T.
      Targeting virulence. A new paradigm for antimicrobial therapy.
      ). Our observations suggest that S. aureus bNOS deserves consideration as a therapeutic target, particularly because inhibition of bNOS would not only reduce MRSA pathology but may in specific cases may have the dual benefit of increasing bacterial susceptibility to commonly used antibiotics including vancomycin and daptomycin.

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