Demonstration and characterization of manganese superoxide dismutase of Providencia alcalifaciens

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N-terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA-encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens.     

Isolation of paraquat-resistant mutants of Escherichia coli: lack of correlation between resistance and the activity of superoxide dismutase

Abstract Paraquat-resistant Escherichia coli mutants were isolated. The mutants were 10- to 50-fold more resistant to paraquat than the wild type. The wild type was more responsive to the presence of paraquat by inducing higher levels of the manganese-containing superoxide dismutase (MnSOD). Thus, in minimal medium, 0.1 mM paraquat caused a 5-fold increase in MnSOD in the wild type while it had no effect on the level of MnSOD in the mutants. Yet, 50 mM paraquat exerted a dramatic induction of SOD in the mutant strains when grown in trypticase soy yeast extract (TSY) medium. In TSY medium, catalase was not significantly affected by paraquat in all the strains tested. Resistance to paraquat in these mutant strains is, therefore, unrelated to their capacity to detoxify superoxide or hydrogen peroxide.     

Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence

We studied superoxide dismutases (SODs) in the encapsulated yeast Cryptococcus neoformans (Cn) variety gattii to analyse the role of mitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 was cloned from a Cn cosmid library, sod2 mutant and sod2 + SOD2 reconstituted strains were constructed by homologous recombination, and two sod1sod2 double mutants were constructed by replacing SOD2 in the sod1 mutant with the sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids, with 36-66% identity with other fungal SOD2ps. SOD2 deletion rendered Cn highly growth-defective at 37 degrees C in 19-20% oxygen (normal air), and this defect was reversed by limiting oxygen to 1.3% as well in the presence of antioxidant, ascorbic acid. The sod2 mutant accumulated significantly more reactive oxygen species (ROS) at 37 degrees C as well at 30 degrees C in the presence of antimycin A, suggesting that SOD2p is the primary defence of Cn against the superoxide anion (O(2) (.-)) in the mitochondria. The sod2 was also highly susceptible to redox-cycling agents, high salt and nutrient limitations. The sod2 mutant was avirulent in intranasally infected mice and markedly attenuated in its virulence in intravenously infected mice. The virulence defect of sod2 mutant appeared related to its growth defects in high oxygen environment, but not resulting from increased sensitivity to oxidative killing by phagocytes. The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2 double mutants showed a marked reduction in the activities of other known Cn virulence factors; and they were more susceptible to PMN killing than was the sod2 single mutant. Previously, we reported that the attenuation of sod1 mutant in mice was resulting from enhanced susceptibility to phagocyte killing, combined with a reduction in the activities of a number of virulence factors. Thus, SOD1p and SOD2p play distinct roles in the biology and virulence of Cn var. gattii via independent modes of action.     

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.     

Functional analysis of the superoxide dismutase family in Aspergillus fumigatus

Reactive oxidant species produced by phagocytes have been reported as being involved in the killing of Aspergillus fumigatus. Fungal superoxide dismutases (SODs) that detoxify superoxide anions could be putative virulence factors for this opportunistic pathogen. Four genes encoding putative Sods have been identified in the A. fumigatus genome: a cytoplasmic Cu/ZnSOD (AfSod1p), a mitochondrial MnSOD (AfSod2p), a cytoplasmic MnSOD (AfSod3p) and AfSod4 displaying a MnSOD C‐terminal domain. During growth, AfSOD1 and AfSOD2 were highly expressed in conidia whereas AfSOD3 was only strongly expressed in mycelium. AfSOD4 was weakly expressed compared with other SODs. The deletion of AfSOD4 was lethal. Δsod1 and Δsod2 mutants showed a growth inhibition at high temperature and a hypersensitivity to menadione whereas the sod3 mutant had only a slight growth delay at high temperature. Multiple mutations had only an additive effect on the phenotype. The triple sod1/sod2/sod3 mutant was characterized by a delay in conidial germination, a reduced conidial survival during storage overtime, the highest sensitivity to menadione and an increased sensitivity to killing by alveolar macrophage of immunocompetent mice. In spite of these phenotypes, no significant virulence difference was observed between the triple mutant and parental strain in experimental murine aspergillosis models in immunocompromised animals.     

The virulence of Vibrio vulnificus is affected by the cellular level of superoxide dismutase activity

The virulence of superoxide dismutase (SOD) mutants of Vibrio vulnificus, as tested by intraperitoneal injection into mice, decreases in the order of sodC mutant, sodA mutant, and sodB mutant lacking CuZnSOD, MnSOD, and FeSOD, respectively. The survival of SOD mutants under superoxide stress also decreases in the same order. The virulence of soxR mutant, which is unable to induce MnSOD in response to superoxide, is similar to that of the sodA mutant, as the survival of the soxR mutant under superoxide stress is similar to that of the sodA mutant. Consistently, the lowered survival of the soxR mutant is complemented not only with soxR but also with sodA. Thus, the virulence of V. vulnificus is significantly affected by the cellular level of SOD activity, and an increase in SOD level through MnSOD induction by SoxR under superoxide stress is essential for virulence.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants

An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.     

Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against hydrogen peroxide toxicity

In living organisms, exposure to oxygen provokes oxidative stress. A widespread mechanism for protection against oxidative stress is provided by the antioxidant enzymes: superoxide dismutases (SODs) and hydroperoxidases. Generally, these enzymes are not present in Lactobacillus spp. In this study, we examined the potential advantages of providing a heterologous SOD to some of the intestinal lactobacilli. Thus, the gene encoding the manganese-containing SOD (sodA) was cloned from Streptococcus thermophilus AO54 and expressed in four intestinal lactobacilli. A 1.2-kb PCR product containing the sodA gene was cloned into the shuttle vector pTRK563, to yield pSodA, which was functionally expressed and complemented an Escherichia coli strain deficient in Mn and FeSODs. The plasmid, pSodA, was subsequently introduced and expressed in Lactobacillus gasseri NCK334, Lactobacillus johnsonii NCK89, Lactobacillus acidophilus NCK56, and Lactobacillus reuteri NCK932. Molecular and biochemical analyses confirmed the presence of the gene (sodA) and the expression of an active gene product (MnSOD) in these strains of lactobacilli. The specific activities of MnSOD were 6.7, 3.8, 5.8, and 60.7 U/mg of protein for L. gasseri, L. johnsonii, L. acidophilus, and L. reuteri, respectively. The expression of S. thermophilus MnSOD in L. gasseri and L. acidophilus provided protection against hydrogen peroxide stress. The data show that MnSOD protects cells against hydrogen peroxide by removing O(2)(.-) and preventing the redox cycling of iron. To our best knowledge, this is the first report of a sodA from S. thermophilus being expressed in other lactic acid bacteria.     

Managing odds in stem cells: insights into the role of mitochondrial antioxidant enzyme MnSOD

Reactive oxygen species (ROS) have been poised at a straddled state of being beneficiary as well detrimental depending on its threshold levels. Maintaining the homeostasis of ROS is imperative for normal cellular physiology, wherein physiological concentrations of ROS are involved in cell signaling and elevated ROS contribute to the development of various diseases. Superoxide dismutases (SODs), enzymes involved in dismutation of superoxide anion to hydrogen peroxide, arrive as a first line of defense when there is perturbation in the homeostasis of ROS. As mitochondria are the main site of superoxide production, among SODs, mitochondrial manganese SOD (MnSOD) is the primary antioxidant enzyme that protects cells from ROS. Most importantly, knockout of MnSOD leads to postnatal lethality and tissue-specific conditional knockout in brain resulted in death of mice, conclusively portraying the essential role of MnSOD in development. Although MnSOD has been extensively discussed with the purview of tumor biology and aging, understanding the crucial role of MnSOD in stem cell physiology is still at its infant stage. Ever increasing progress in stem cell research has recently unveiled the essential role of MnSOD in self-renewal and differentiation of stem cells. In this review, we will conglomerate the current aspects by which MnSOD can contribute to embryonic stem cells’ and adult stem cells’ functions and interpret the necessity of understanding MnSOD for further stem cell mediated applications.     

A biologically effective fullerene (C60) derivative with superoxide dismutase mimetic properties

Superoxide, a potentially toxic by-product of cellular metabolism, may contribute to tissue injury in many types of human disease. Here we show that a tris-malonic acid derivative of the fullerene C60 molecule (C3) is capable of removing the biologically important superoxide radical with a rate constant (k(C3)) of 2 x 10(6) mol(-1) s(-1), approximately 100-fold slower than the superoxide dismutases (SOD), a family of enzymes responsible for endogenous dismutation of superoxide. This rate constant is within the range of values reported for several manganese-containing SOD mimetic compounds. The reaction between C3 and superoxide was not via stoichiometric "scavenging," as expected, but through catalytic dismutation of superoxide, indicated by lack of structural modifications to C3, regeneration of oxygen, production of hydrogen peroxide, and absence of EPR-active (paramagnetic) products, all consistent with a catalytic mechanism. A model is proposed in which electron-deficient regions on the C60 sphere work in concert with malonyl groups attached to C3 to electrostatically guide and stabilize superoxide, promoting dismutation. We also found that C3 treatment of Sod2(-/-) mice, which lack expression of mitochondrial manganese superoxide dismutase (MnSOD), increased their life span by 300%. These data, coupled with evidence that C3 localizes to mitochondria, suggest that C3 functionally replaces MnSOD, acting as a biologically effective SOD mimetic.     

Does Manganese Protect Cultured Human Skin Fibroblasts Against Oxidative Injury by Uva, Dithranol and Hydrogen Peroxide?

Reactive oxygen species (ROS) are involved in the mechanism of photoaging and carcinogenesis. Skin is endowed with antioxidant enzymes including superoxide dismutases (SOD): cytosolic copper zinc SOD and mitochondrial manganese SOD. The aim of our study was to estimate the protective effect of manganese against oxidative injury on cultured human skin fibroblasts. Dithranol, hydrogen peroxide and UV-A radiation (375 nm) were employed as oxidative stressors. The supply of manganese chloride produced an increase in cellular content of this element up to 24 fold without concomitant elevation of MnSOD activity. Nevertheless, manganese protects cells against two of the three ROS generating systems assessed, namely hydrogen peroxyde and UV-A. This protective effect depends on the concentration of manganese in the medium, 0.1 mM and 0.2 mM protect against UVA cytotoxicity, only 0.2 mM protects against H₂O₂ cytotoxicity.     

Induction of superoxide dismutases in Escherichia coli B by metal chelators

The effects of metal salts, chelating agents, and paraquat on the superoxide dismutases (SODs) of Escherichia coli B were explored. Mn(II) increased manganese-containing SOD (MnSOD), whereas Fe(II) increased iron-containing SOD (FeSOD). Chelating agents induced MnSOD but decreased FeSOD and markedly increased the degree of induction seen with Mn(II). Paraquat also exerted a synergistic effect with Mn(II). High levels of MnSOD were achieved in the combined presence of Mn(II), chelating agent, and paraquat. All of these effects were dependent on the presence of oxygen. MnSOD, not ordinarily present in anaerobically grown E. coli cells, was present when the cells were grown anaerobically in the presence of chelating agents. These results are accommodated by a scheme which incorporates autogenous repression by the apoSODs and competition between Fe(II) and Mn(II) for the metal-binding sites of the apoSODs. It is further supposed that oxygenation and intracellular O2- production favor MnSOD production because O2- oxidizes Mn(II) to Mn(III), which competes favorably with Fe(II) for the apoSODs.     

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.     

Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dismutase

There are two types of intracellular superoxide dismutases: the mitochondrial manganese SOD (MnSOD) and the cytoplasmic copper/zinc SOD (CuZnSOD). Mutant mice that lack MnSOD die shortly after birth because of cardiomyopathy and mitochondrial injury. In order to verify if CuZnSOD could compensate for MnSOD deficiency, a new mutant mouse that overexpresses CuZnSOD but is deficient in MnSOD was generated by crossing MnSOD knockout mice with CuZnSOD transgenic mice. CuZnSOD activity was significantly increased in the blood, brain, liver, and heart of MnSOD knockout, CuZnSOD transgenic mice when compared with nontransgenic mice. However, overexpression of CuZnSOD did not prevent neonatal lethality in mice that lack MnSOD, nor did it prevent oxidative aconitase inactivation, nor did it rescue MnSOD-deficient astrocytes in culture. Based on our findings, which emphasize the strong enzymatic compartmentalization of CuZnSOD and MnSOD, therapeutic antioxidant strategies should consider the final intracellular localization of the antioxidant used, especially when those strategies are directed against mitochondrial diseases.     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.