Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages (original) (raw)
Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages
Infection and immunity, 1995
Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This ...
Nitric Oxide Potentiates Hydrogen Peroxide-induced Killing of Escherichia coli
1995
Previously, we reported that nitric oxide (NO) provides significant protection to mammalian cells from the cytotoxic effects of hydrogen peroxide (H202). Murine neutrophils and activated macrophages, however, produce NO, H202, and other reactive oxygen species to kill microorganisms, which suggests a paradox. In this study, we treated bacteria (Escherichia coh~ with NO and H202 for 30 rain and found that exposure to NO resulted in minimal toxicity, but greatly potentiated (up to 1,000-fold) H202-mediated killing, as evaluated by a clonogenic assay. The combination of NO/H2O 2 induced DNA double strand breaks in the bacterial gehome, as shown by field-inverted gel electrophoresis, and this increased DNA damage may correlate with cell killing. NO was also shown to alter cellular respiration and decrease the concentration of the antioxidant glutathione to a residual level of 15-20% in bacterial cells. The iron chelator desferrioxamine did not stop the action of NO on respiration and glutathione decrease, yet it prevented the NO/H202 synergistic cytotoxicity, implicating metal ions as critical participants in the NO/H202 cytocidal mechanism. Our results suggest a possible mechanism of modulation of H202-mediated toxicity, and we propose a new key role in the antimicrobial macrophagic response for NO.
Oxidative Stress Modulates the Nitric Oxide Defense Promoted by Escherichia coli Flavorubredoxin
Journal of Bacteriology, 2012
Mammalian cells of innate immunity respond to pathogen invasion by activating proteins that generate a burst of oxidative and nitrosative stress. Pathogens defend themselves from the toxic compounds by triggering a variety of detoxifying enzymes. Escherichia coli flavorubredoxin is a nitric oxide reductase that is expressed under nitrosative stress conditions. We report that in contrast to nitrosative stress alone, exposure to both nitrosative and oxidative stresses abolishes the expression of flavorubredoxin. Electron paramagnetic resonance (EPR) experiments showed that under these conditions, the iron center of the flavorubredoxin transcription activator NorR loses the ability to bind nitric oxide. Accordingly, triggering of the NorR ATPase activity, a requisite for flavorubredoxin activation, was impaired by treatment of the protein with the double stress. Studies of macrophages revealed that the contribution of flavorubredoxin to the survival of E. coli depends on the stage of m...
Nitric Oxide-Induced Nitrative Stress Involved in Microbial Pathogenesis
Journal of Pharmacological Sciences, 2005
The pathogenic mechanism of infections is a complicated but important scientific theme that is now attracting great attention because of its association with host-derived as well as microbial factors. Recent advances in free radical research revealed that reactive oxygen and nitrogen oxide species such as superoxide (O 2 −) and nitric oxide (NO) play a leading role in the pathogenesis of infections caused by viral pathogens including influenza virus and other RNA viruses. Although NO and O 2 − have antimicrobial activity against bacteria, fungi, and parasites, in some viral infections they have an opposite effect. This exacerbation caused by NO and O 2 − is mediated by reactive nitrogen oxides, for example, peroxynitrite (ONOO −), generated by reaction of NO with O 2 −. These nitrogen oxides have strong oxidation and nitration potential and can modify biological molecules, thereby creating oxidative and nitrative stress that contributes to pathogenic processes during viral infection. Nitrative stress-mediated 8-nitroguanosine formation during influenza or Sendai virus infection has been the focus of enormous interest because it involves unique biochemical and pharmacological properties such as redox activity and mutagenic potential. In this review, we discuss the nature and impact of nitrative stress in viral infection, with emphasis on nitrative stress-mediated viral pathogenesis, which we have recently been investigating.
Journal of Experimental Medicine, 2000
The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91 phox Ϫ / Ϫ , iNOS Ϫ / Ϫ , and doubly immunodeficient phox Ϫ / Ϫ iNOS Ϫ / Ϫ mice. The respiratory burst and NO radical (NO и ) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidasedependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S . typhimurium , and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O 2 и Ϫ ), hydrogen peroxide (H 2 O 2 ), and peroxynitrite (ONOO Ϫ ) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S . typhimurium . Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon ␥ appears to augment antibacterial activity predominantly by enhancing NO и production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.
Biochemical and Biophysical Research Communications, 2013
Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages. However, the virulent effect of bNO in host cells has not been investigated. Here, we report that bNO contributes macrophage killing by S-nitrosylation of bioenergetic-relating proteins within mitochondria. Toxigenic Sterne induces expression of the bnos gene and produces bNO during early stage of infection. Nitroso-proteomic analysis coupled with a biotin-switch technique demonstrated that toxigenic infection induces protein S-nitrosylation in B. anthracis-susceptible RAW264.7. For each target enzyme tested (complex I, complex III and complex IV), infection by B. anthracis Sterne caused enzyme inhibition. Nx-nitro-L-arginine methyl ester, a NO synthase inhibitor, reduced S-nitrosylation and partially restored cell viability evaluated by intracellular ATP levels in macrophages. Our data suggest that bNO leads to energy depletion driven by impaired mitochondrial bioenergetic machinery that ultimately contributes to macrophage death. This novel mechanism of anthrax pathogenesis may offer specific approach to the development of therapeutics.
Nitric oxide: A cytotoxic activated macrophage effector molecule
Biochemical and Biophysical Research Communications, 1988
The experiments reported here identify nitric oxide as a molecular effector of activated macrophage induced cytotoxicity. Cytotoxic activated macrophages synthesize nitric oxide from a terminal guanidino nitrogen atom of L-arginine which is converted to L-citrulline without loss of the guanidino carbon atom. In addition, authentic nitric oxide gas causes the same pattern of cytotoxicity in LlO hepatoma cells as is induced by cytotoxic activated macrophages (iron loss as well as inhibition of DNA synthesis, mitochondrial respiration, and aconitase activity). The results suggest that nitric oxide is the precursor of nitrite/nitrate synthesized by cytotoxic activated macrophages and, via formation of iron-nitric oxide complexes and subsequent degradation of iron-sulfur prosthetic groups, an effector molecule.
Infection and Immunity, 2002
Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection by Salmonella spp. In this work we investigated the ability of attenuated strains of Salmonella enterica serovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyer's patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within ...
Pathogens
Mucosa-associated Escherichia coli are increased in Crohn’s disease (CD) and colorectal cancer (CRC). CD isolates replicate within macrophages but the specificity of this effect for CD and its mechanism are unclear. Gentamicin exclusion assay was used to assess E. coli replication within J774.A1 murine macrophages. E. coli growth was assessed following acid, low-nutrient, nitrosative, oxidative and superoxide stress, mimicking the phagolysosome. Twelve of 16 CD E. coli isolates replicated >2-fold within J774.A1 macrophages; likewise for isolates from 6/7 urinary tract infection (UTI), 8/9 from healthy subjects, compared with 2/6 ulcerative colitis, 2/7 colorectal cancer and 0/3 laboratory strains. CD mucosal E. coli were tolerant of acidic, low-nutrient, nitrosative and oxidative stress. Replication within macrophages correlated strongly with tolerance to superoxide stress (rho = 0.44, p = 0.0009). Exemplar CD E. coli HM605 and LF82 were unable to survive within Nfκb1-/- murine b...
Microbiology-sgm, 2009
Activation of macrophages by interferon gamma (IFN-c) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN-c-induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN-c-treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN-c-induced NO production, and they highlight the critical role of nirC as a virulence gene. Abbreviations: BMDM, bone-marrow-derived macrophages; FBS, fetal bovine serum; IFN-c, interferon gamma; iNOS, inducible nitric oxide synthase; JAK-STAT, Janus kinase/signal transducer and activator of transcription; L-NIL, L-N 6 -iminoethyllysine; MLN, mesenteric lymph nodes; NED, N-(naphthyl)ethylenediamine dihydrochloride; RNS, reactive nitrogen species; ROI, reactive oxygen intermediates; SOCS-3, suppressor of cytokine signalling-3; SPI2, Salmonella pathogenicity island 2. Three supplementary tables and three supplementary figures are available with the online version of this paper.