The Brucella abortus Cu,Zn superoxide dismutase is required for optimal resistance to oxidative killing by murine macrophages and wild-type virulence in experimentally infected mice

Two-dimensional gel electrophoretic analysis of cell lysates from Brucella abortus 2308 and the isogenic hfq mutant Hfq3 revealed that the RNA binding protein Hfq (also known as host factor I or HF-I) is required for the optimal stationary phase production of the periplasmic Cu,Zn superoxide dismutase SodC. An isogenic sodC mutant, designated MEK2, was constructed from B. abortus 2308 by gene replacement, and the sodC mutant exhibited much greater susceptibility to killing by O(2)(-) generated by pyrogallol and the xanthine oxidase reaction than the parental 2308 strain supporting a role for SodC in protecting this bacterium from O(2)(-) of exogenous origin. The B. abortus sodC mutant was also found to be much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and the attenuation displayed by MEK2 in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon (IFN-gamma). The attenuation displayed by the B. abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated, however, when these host cells were treated with the NADPH oxidase inhibitor apocynin. Consistent with its increased susceptibility to killing by cultured murine macrophages, the B. abortus sodC mutant also displayed significant attenuation in experimentally infected C57BL6J mice compared to the parental strain. These experimental findings indicate that SodC protects B. abortus 2308 from the respiratory burst of host macrophages. They also suggest that reduced SodC levels may contribute to the attenuation displayed by the B. abortus hfq mutant Hfq3 in the mouse model.     

Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.     

Increased expression of periplasmic Cu,Zn superoxide dismutase enhances survival of Escherichia coli invasive strains within nonphagocytic cells

We have studied the influence of periplasmic Cu,Zn superoxide dismutase on the intracellular survival of Escherichia coli strains able to invade epithelial cells by the expression of the inv gene from Yersinia pseudotuberculosis but unable to multiply intracellularly. Intracellular viability assays, confirmed by electron microscopy observations, showed that invasive strains of E. coli engineered to increase Cu,Zn superoxide dismutase production are much more resistant to intracellular killing than strains containing only the chromosomal sodC copy. However, we have found only a slight difference in survival within HeLa cells between a sodC-null mutant and its isogenic wild-type strain. Such a small difference in survival correlates with the very low expression of this enzyme in the wild-type strain. We have also observed that acid- and oxidative stress-sensitive E. coli HB101(pRI203) is more rapidly killed in epithelial cells than E. coli GC4468(pRI203). The high mortality of E. coli HB101(pRI203), independent of the acidification of the endosome, is abolished by the overexpression of sodC. Our data suggest that oxyradicals are involved in the mechanisms of bacterial killing within epithelial cells and that high-level production of periplasmic Cu,Zn superoxide dismutase provides bacteria with an effective protection against oxidative damage. We propose that Cu,Zn superoxide dismutase could offer an important selective advantage in survival within host cells to bacteria expressing high levels of this enzyme.     

Bacterial [Cu,Zn]-cofactored superoxide dismutase protects opsonized,encapsulated Neisseria meningitidis from phagocytosis by human monocytes/macrophages

Superoxide dismutase cofactored by copper and zinc ([Cu,Zn]-SOD) contributes to the protection of opsonized serogroup B Neisseria meningitidis against phagocytosis by human monocytes/macrophages, with sodC mutant organisms being endocytosed in significantly higher numbers than are wild-type organisms. The influence of [Cu,Zn]-SOD was found to be exerted at the stage of phagocytosis, rather than at earlier (modulating surface association) or later (intracellular killing) stages.     

Physiologic oxygen tensions limit oxidant-mediated killing of schistosome eggs by inflammatory cells and isolated granulomas

Explanted hepatic granulomas, eosinophils obtained from the peritoneal cavity of schistosome-infected mice, schistosome egg granuloma macrophages, alveolar macrophages, and activated peritoneal macrophages obtained from Listeria-infected mice were miracidicidal when cultured at 21% oxygen. This activity was markedly attenuated at physiologic oxygen concentrations (1-15%). Catalase and superoxide dismutase blocked the miracidicidal activity of inflammatory cells but did not prevent granuloma-mediated egg killing. However, the biomimetic superoxide dismutase, copper (II) [diisopropyl salicylate]2, inhibited granuloma-mediated egg killing in a dose-dependent, apparently nontoxic manner. Thioglycollate-elicited macrophages did not kill schistosome egg miracidia even when cultured in 21% oxygen, unless pretreated with lipopolysaccharide. Isolated schistosome eggs initiated an oxidative burst in macrophages, as measured by superoxide anion production. This burst was suppressed at reduced oxygen concentrations. Thus schistosome egg miracidia can be killed nonspecifically by macrophages through the release of cytotoxic reactive oxygen intermediates triggered by the egg. This activity is not supported by the oxygen concentrations found in most tissues, with the possible exception of the lung. Schistosoma mansoni eggs, injected intraveneously and lodged in the pulmonary vasculature of mice, were killed rapidly, with a half life of 3.5 days. Eggs, injected into the mesenteric veins and lodged in the liver, remained fully viable for several weeks. The data suggest that the high oxygen tension of the lung allows for the increased production of reactive oxygen intermediates (ROI) by local inflammatory cells, which in turn increases their miracidicidal efficiency. Conversely, the relatively hypoxic environment of the liver decreases ROI production by local inflammatory cells and decreases their miracidicidal efficiency.     

Cu,Zn]-Superoxide dismutase mutants of the swine pathogen Actinobacillus pleuropneumoniae are unattenuated in infections of the natural host

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, contains a periplasmic Cu- and Zn-cofactored superoxide dismutase ([Cu,Zn]-SOD, or SodC) which has the potential, realized in other pathogens, to promote bacterial survival during infection by dismutating host-defense-derived superoxide. Here we describe the construction of a site-specific, [Cu,Zn]-SOD-deficient A. pleuropneumoniae serotype 1 mutant and show that although the mutant is highly sensitive to the microbicidal action of superoxide in vitro, it remains fully virulent in experimental pulmonary infection in pigs.     

Superoxide dismutase influences the virulence of Cryptococcus neoformans by affecting growth within macrophages

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.     

Killing of Plasmodium yoelii by enzyme-induced products of the oxidative burst

The murine malaria parasite Plasmodium yoelii was killed in vitro when incubated with glucose and glucose oxidase, a system generating hydrogen peroxide, or with xanthine and xanthine oxidase, a system which produces the superoxide anion and subsequently other products of the oxidative burst. Catalase blocked the killing in both cases; superoxide dismutase and scavengers of hydroxyl radicals or singlet oxygen were ineffective in the xanthine oxidase system. Thus, hydrogen peroxide appears to be the main reactive oxygen species killing P. yoelii.     

Neutropenia Restores Virulence to an Attenuated Cu,Zn Superoxide Dismutase-Deficient Haemophilus ducreyi Strain in the Swine Model of Chancroid

Haemophilus ducreyi causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu, Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect H. ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in H. ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H. ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in H. ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host.     

Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.     

Inability of tumour cells to elicit the respiratory burst in cytotoxic, activated macrophages.

Activated macrophages from Corynebacterium parvum-treated mice are cytotoxic to non-antibody-coated tumour cells and have an augmented respiratory burst potential when compared to resident macrophages. We have investigated the possible involvement of the respiratory burst as an effector mechanism in this type of tumour killing. Scavengers of toxic metabolites of oxygen such as catalase, superoxide dismutase, 2,3-dihydroxybenzoate, ethanol, and cytochrome c did not inhibit macrophage cytotoxicity in this system. To investigate whether or not neoplastic cells stimulate the macrophage respiratory burst, we exposed activated macrophages to viable tumour cells and monitored macrophage superoxide anion production, chemiluminescence, and hexose monophosphate shunt activity. None of these indicators of the macrophage respiratory burst was stimulated by the tumour cells towards which the macrophages were cytotoxic. The data suggest that the macrophages burst is not utilized as an effector mechanism in the non-antibody-mediated macrophage tumour cytotoxicity reaction.     

Four superoxide dismutases contribute to Bacillus anthracis virulence and provide spores with redundant protection from oxidative stress

The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Delta sod15 Delta sodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Delta sod15 Delta sodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Delta sod15 Delta sodA1 Delta sodC Delta sodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.     

Relationships between oxidative metabolism, macrophage activation, and antilisterial activity

Previous reports from this laboratory have revealed that macrophages obtained from 7-day Listeria-immune mice elicited 15 h before harvest with heat-killed homologous microorganisms were able to kill Listeria monocytogenes while resident or elicited cells were not [14, 16]. In the present study, experiments were conducted to determine if phagocytosis-associated oxidative metabolic activity participates in the enhanced destruction of Listeria by activated macrophages. Investigations into production of oxygen radicals by zymosan-stimulated macrophages revealed that Listeria-immune antigen-elicited (LIAE) cells produced significantly more superoxide and hydrogen peroxide than did resident, thioglycolate, or Listeria antigen-elicited macrophages. Additionally, the percentage of nitroblue tetrazolium (NBT) dye positive cells following exposure to zymosan was maximal in the immune-elicited population. Utilizing a luminol-dependent assay, a short-term chemiluminescent (CL) burst was noted in phagocytizing macrophages. This response was greatest in the LIAE population that exhibited a tenfold increase in peak chemiluminescence over other cell types. Prolonged in vitro culture of these cells diminishes their capacity to generate oxygen radicals. Additionally, LIAE macrophages cultured in excess of 38 h exhibited a significant decrease in zymosan-stimulated hydrogen peroxide release while the decline in superoxide generation was minimal. A substantial diminution in the Listeria-stimulated CL response was also noted during this time period. However, phagocytosis of Listeria by LIAE cells failed to induce the level of oxygen metabolites seen when zymosan was used as the particulate stimulant. In addition, scavengers of oxygen radicals were found to be relatively ineffective in reducing the killing of L monocytogenes by immunologically activated macrophages in culture. It therefore appears that toxic oxygen species do not play a major role in the heightened killing of Listeria by activated macrophages.     

Cloning and molecular characterization of Cu,Zn superoxide dismutase from Actinobacillus pleuropneumoniae.

Copper-zinc superoxide dismutases (Cu,Zn SODs), until recently considered very unusual in bacteria, are now being found in a wide range of gram-negative bacterial species. Here we report the cloning and characterization of sodC, encoding Cu,Zn SOD in Actinobacillus pleuropneumoniae, a major pathogen of pigs and the causative organism of porcine pleuropneumonia. sodC was shown to lie on a monocistronic operon, at the chromosomal locus between the genes asd (encoding aspartate semialdehyde dehydrogenase) and recF. The primary gene product was shown to have an N-terminal peptide extension functioning as a leader peptide, so that the mature Actinobacillus enzyme, like other bacterial examples, is directed to the periplasm, where it is appropriately located to dismutate exogenously generated superoxide. While the role of these secreted bacterial SODs is unknown, we speculate that in A. pleuropneumoniae the enzyme may confer survival advantage by accelerating dismutation of superoxide derived from neutrophils, a central host defense response in the course of porcine infection.     

High levels of catalase and glutathione peroxidase activity dampen H2O2 signaling in human alveolar macrophages

Results are presented which support the hypothesis that adequate steady-state levels of hydrogen peroxide (H2O2) are required to overcome the effects of high catalase and glutathione peroxidase (GPx) expression for p38 mitogen-activated protein (MAP) kinase activation and tumor necrosis factor (TNF)-alpha gene expression in human alveolar macrophages stimulated with asbestos. We found significant differences in the types and amounts of reactive oxygen species generated in human blood monocytes compared with human alveolar macrophages. This difference in reactive oxygen species production is related, in part, to the differences in antioxidant enzyme expression and activity. Most importantly, catalase and GPx activities were significantly increased in alveolar macrophages compared with blood monocytes. Asbestos activated the p38 MAP kinase and induced TNF-alpha gene expression only in blood monocytes. Increasing the steady-state levels of H2O2 by using polyethylene glycol superoxide dismutase, an antioxidant that crosses the cell membrane, or aminotriazole, an irreversible inhibitor of catalase, allowed the p38 MAP kinase to be activated in alveolar macrophages. In addition, asbestos-stimulated macrophages cultured with polyethylene glycol superoxide dismutase had a significant increase in gene expression mediated by the TNF-alpha promoter. These results demonstrate that high catalase and GPx activity in human alveolar macrophages limits the effectiveness of H2O2 to act as a mediator of inflammatory gene expression.     

Susceptibility of HRS/J mice to listeriosis: macrophage activity.

Macrophage functions, including phagocytosis and bactericidal and oxidative activities, were measured in highly susceptible Listeria monocytogenes-sensitive HRS/J homozygous and heterozygous mice. Phagocytic studies with both caseinate-elicited and L. monocytogenes-immune macrophages revealed comparable engulfment of latex particles, zymosan, and bacteria by mononuclear phagocytes obtained from all experimental mouse strains. Elicited macrophages cultivated from mutant hairless and heterozygous littermates exhibited a reduced capacity to control Listeria infection compared with cells derived from CD-1 mice. However, intracellular killing of the microorganisms by immune macrophages was comparable to that observed with the outbred controls. Studies on oxidative metabolic activities associated with the respiratory burst indicate that while intracellular nitroblue tetrazolium reduction was comparable for macrophages cultivated from all mouse strains, the liberation of superoxide anion and chemiluminescence responses were significantly diminished in caseinate-elicited HRS/J cells. Moreover, immune elicited hr/hr and hr/+ macrophages generated oxidative species at levels comparable to that observed with cells derived from resistant animals. Thus, immunologically elicited HRS/J mice are capable of responding to sublethal Listeria infection with heightened antibacterial and oxidative activities.     

Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis.

A Shigella flexneri serotype 5 strain deficient in the production of the iron-containing superoxide dismutase FeSOD (sodB) and a catalase-negative (katFG) S. flexneri serotype 5 strain were isolated. Both strains were examined for increased sensitivity to oxygen stress by using assays involving killing by mouse peritoneal macrophages and human polymorphonuclear leukocytes as well as infection of rabbit ileal loops. The sodB mutant was extremely sensitive to killing by phagocytes when compared with the wild-type parent, M90T. The catalase mutant also showed an increased sensitivity to killing, but to a much lesser extent. Upon infection of rabbit ileal loops and subsequent histopathological examination, the sodB mutant caused very little detectable damage to intestinal villi. The pattern of infection was roughly similar to that of BS176, an avirulent plasmidless derivative of M90T. The katFG mutant, on the other hand, showed a high degree of destruction, similar to that caused by M90T. This evidence suggests that the superoxide dismutase encoded by sodB may play an important role in the pathogenesis of S. flexneri. In contrast, catalases appear to make a limited contribution to virulence.     

A Mycobacterium marinum mel2 mutant is defective for growth in macrophages that produce reactive oxygen and reactive nitrogen species

Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed a Mycobacterium marinum mel2 locus mutant, which is known to affect macrophage infection. We found previously that the mel2 locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-gamma), we examined the effects of IFN-gamma on ROS and RNS production by these cells as well as the effects on growth of M. marinum in these cells. We found that an M. marinum mutant with mutation of the first gene in the mel2 locus, melF, is defective for growth in IFN-gamma-plus-lipopolysaccharide-treated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, the M. marinum melF mutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entire mel2 locus but only partially by the presence of melF alone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in the mel2 locus. These observations demonstrate that the M. marinum mel2 locus plays a role in resistance to ROS and RNS produced by activated macrophages.     

An Intact Cytoskeleton is Required for Prolonged Respiratory Burst Activity During Neutrophil Phagocytosis

The temporal relationship between phagocytosis and respiratory burst activity was investigated. Neutrophil uptake of yeast particles was synchronized and the kinetics of the oxidative burst was determined using an isoluminol/luminol amplified chemiluminescence system. The reactive oxygen species were mainly generated intracellularly (defined as the activity that remained in an luminol-enhanced system in the presence of superoxide dismutase and catalase). Following phago- cytosis, the intracellular response rapidly reached a level close to the maximum and the activity was almost constant for the first 10 to 15 min. The response then slowly declined. The presence of cytochalasin B, an inhibitor of actin polymerization, greatly reduced the respiratory burst activity, and this was true also when the inhibitor was added after completion of uptake of yeast particles. Our results thus show that there is a continuous production of oxygen metabolites long after phagocytosis is completed. There is also a requirement for an intact cytoskeleton for prolonged superoxide production inside the phagosome.