Nitric oxide evokes an adaptive response to oxidative stress by arresting respiration

Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNgamma-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H(2)O(2). NADH antagonizes the hydroxyl radical (OH(.)) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.     

Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst

Reactive nitrogen species (RNS) play an essential role in host defence against Mycobacterium tuberculosis (MTB) in the mouse model of tuberculosis (TB), as evidenced by the increased susceptibility of mice deficient in the inducible isoform of nitric oxide synthase (NOS2). In contrast, the role of reactive oxygen species (ROS) in protection against MTB is less clear, and mice defective in the ROS-generating phagocyte NADPH oxidase (Phox) are relatively resistant. This suggests that MTB might possess efficient mechanisms to evade or counter the phagocyte oxidative burst, effectively masking the impact of this host defence mechanism. In order to assess the role of ROS detoxification pathways in MTB virulence, we generated a katG null mutant of MTB, deficient in the KatG catalase-peroxidase-peroxynitritase, and evaluated the mutant's ability to replicate and persist in macrophages and mice. Although markedly attenuated in wild-type C57Bl/6 mice and NOS2(-/-) mice, the DeltakatG MTB strain was indistinguishable from wild-type MTB in its ability to replicate and persist in gp91(Phox-/-) mice lacking the gp91 subunit of NADPH oxidase. Similar observations were made with murine bone marrow macrophages infected ex vivo: growth of the DeltakatG MTB strain was impaired in macrophages from C57Bl/6 and NOS2(-/-) mice, but indistinguishable from wild-type MTB in gp91(Phox-/-) macrophages. These results indicate that the major role of KatG in MTB pathogenesis is to catabolize the peroxides generated by the phagocyte NADPH oxidase; in the absence of this host antimicrobial mechanism, KatG is apparently dispensable.     

Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.     

Role of the Siderophore Transporter SirABC in the Staphylococcus aureus Resistance to Oxidative Stress

In Staphylococcus aureus, the intracellular siderophore staphyloferrin B, which has been shown to chelate iron-bound to serum transferrin, is transported into cells by the SirABC system. In this work, we have analysed the role of the Sir transporter under stress conditions that resemble those imposed by the mammalian innate immune system. We show that exposure of S. aureus to oxidative and nitrosative stress generated by hydrogen peroxide and S-nitrosoglutathione, respectively, induced the expression of the sirA gene. The disruption of the sir operon led to a strain with lower viability and decreased resistance to oxidative stress. S. aureus sir null mutant was also analysed during infection of murine macrophages and shown to contribute to S. aureus survival inside macrophages. Altogether, our results indicate that the Sir transport system confers protection against reactive oxygen species, therefore, contributing to the virulence of S. aureus.     

Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant Tsa1

Laccases are thought to be important to the virulence of many fungal pathogens by producing melanin, a presumed oxygen radical scavenger. A laccase in Cryptococcus neoformans has been shown to synthesize melanin and contributes to the virulence and the survival in macrophages of this fungal pathogen. One C. neoformans laccase gene, LAC1, previously called CNLAC1, has been extensively studied, and we describe a homologous gene, LAC2, that is found 8 kb away from LAC1 in the genome. In this study we report a role for both laccases, in addition to the thiol peroxidase, Tsa1, in oxidative and nitrosative stress resistance mechanisms of C. neoformans. With use of real-time PCR, similar changes in expression of the two laccase genes occur in response to oxidative and nitrosative stresses, but only the regulation of the LAC2 gene during stress is influenced by Tsa1. Both laccases contribute to melanin production using L-dopa as a substrate and are differentially localized in the cell based on green fluorescent protein fusions. A single deletion of either LAC1 or LAC2 alone had no effect on sensitivity to H2O2 or nitric oxide. However, deletion of either LAC1 or LAC2 in combination with a TSA1 deletion resulted in a slight peroxide sensitivity, and a lac2Delta tsa1Delta deletion strain was sensitive to nitric oxide stress. In addition, the deletion of both laccases reduces survival of C. neoformans in primary macrophages. Based on our expression and functional analysis, we propose a novel model for the interaction of these two systems, which are both important for virulence.     

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence

Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.     

Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: insights into listeriocidal activity of oxidative and nitrosative stress

The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6)(N[N(O)NO]C3H7) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-gamma (INF-gamma) + tumor necrosis factor-alpha (TNF-alpha or INF-gamma + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections.     

Enzymes that counteract nitrosative stress promote fungal virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.     

Oxidative and nitrosative stress in the maintenance of myocardial function

Reactive oxygen species (ROS) are generated by several different cellular sources, and their accumulation within the myocardium is widely considered to cause harmful oxidative stress. On the other hand, their role as second messengers has gradually emerged. The equilibrium of the nitroso/redox balance between reactive nitrogen species and ROS is crucial for the health of cardiomyocytes. This review provides a comprehensive overview of sources of oxidative stress in cardiac myocytes and describes the role of the nitroso/redox balance in cardiac pathophysiology. Although the exact mechanism of ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox's) is not completely understood, Nox2 and Nox4 have particularly important roles within the myocardium. Increasing evidence suggests that Nox2 produces superoxide and Nox4 generates only hydrogen peroxide. We also discuss the key role of nitric oxide synthases (NOSs) in the maintenance of the nitroso/redox balance: uncoupled endothelial NOS has been suggested to shift from nitric oxide to ROS production, contributing to increased oxidative stress within the myocardium. Furthermore, we highlight the importance of sequentially targeting and/or regulating the specific sources of oxidative and nitrosative stress to prevent and/or reverse myocardial dysfunction. Inhibition of NADPH oxidase-dependent ROS is considered to be a potential strategy for treatment of cardiomyopathy. Neither in vivo nor clinical data are available for NADPH oxidase inhibitors. Specifically targeting the mitochondria with the antioxidant MitoQ would be a very promising translation approach, because it could prevent mitochondrial permeability transition pore opening when ROS are produced during heart reperfusion. Enhancing NO signaling could also be a promising therapeutic approach against myocardial dysfunction.     

Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells

Neurodegenerative diseases are attributed to impairment of the ubiquitin–proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity.     

Adenovirus type 5 rupture of lysosomes leads to cathepsin B-dependent mitochondrial stress and production of reactive oxygen species

In response to viral infection, reactive oxygen species (ROS) mediate innate immune signaling or generate danger signals to activate immune cells. The mechanisms of virally induced ROS are poorly defined, however. We demonstrate that ROS are produced within minutes of adenovirus type 5 (Ad5) infection of macrophages and that oxidative stress supports Ad5-induced cytokine secretion. We show that short hairpin RNA (shRNA) knockdown of TLR9 has no effect on ROS production despite observed decreases in Ad-induced cytokine secretion. A major source of ROS in macrophages is NADPH oxidase. However, shRNA knockdown of the NADPH oxidase subunit NOX2 does not attenuate Ad-induced ROS. Induction of ROS is not observed in cells infected with a temperature-sensitive mutant of Ad2, ts1, which is defective in endosomal membrane penetration during cell entry. Further, Ad5, but not ts1, induces the release of lysosomal cathepsin B into the cytoplasm of infected cells. In agreement with this finding, we observe a loss of mitochondrial membrane potential upon Ad infection which requires Ad endosomal membrane penetration and cathepsin B activity. Overexpression of Bcl-2 attenuates Ad5-induced ROS, further supporting the role for mitochondrial membrane destabilization as the source of ROS in response to Ad5 infection. Together, these data suggest that ROS produced in response to Ad5 infection depends on the virally induced endosomal membrane rupture to release lysosomal cathepsins. Furthermore, the release of cathepsins leads to mitochondrial membrane disruption and thus the release of ROS from the mitochondria.     

Repressed Quorum sensing by overexpressing LsrR Hampers Salmonella evasion from oxidative killing within macrophages

Bacterial cell-to-cell communication, termed quorum sensing (QS), leads to coordinated group behavior in a cell-density-dependent fashion and controls a variety of physiological processes including virulence gene expression. The repressor of the lsr operon, LsrR, is the only known regulator of LuxS/AI-2-mediated QS in Salmonella. Although lack of lsrR did not result in noticeable differences in Salmonella survival, the down-regulation of QS as a result of lsrR overexpression decreased Salmonella survival within macrophages. We found that impaired growth of Salmonella overexpressing lsrR within macrophages was due largely to its hypersensitivity to NADPH-dependent oxidative stress. This, in turn, was a result of decreased expression of genes involved in the oxidative stress response, such as sodA, sodCI, and sodCII, when lsrR was overexpressed. These results suggest that down-regulation of QS by excess LsrR can lower Salmonella virulence by hampering Salmonella evasion from oxidative killing within macrophages.