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.     

Extracellular hydrogen peroxide,produced through a respiratory burst oxidase/superoxide dismutase pathway,directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons

The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.     

Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

Direct ROS Scavenging Activity of CueP from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu, Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.     

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.     

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.     

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress in Saccharomyces cerevisiae

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 μmol quanta m^− 2 s^− 1 it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

Impact of copper on reactive oxygen species,lipid peroxidation and antioxidants in <Emphasis Type="Italic">Lemna minor</Emphasis>

Lemna minor L. treated with 20, 50, or 100 μM CuSO₄ accumulated Cu and reactive oxygen species (hydrogen peroxide and superoxide radical) in frond and root cells. The time-course analysis of lipid peroxidation showed high increment in malondialdehyde production only after 12 and 48 h of Cu treatment. Guaiacol peroxidase and superoxide dismutase activities decreased after 48 h while glutathione reductase activity enhanced 48 h after Cu-treatment. Ascorbate and glutathione contents increased with the increasing Cu stress.     

A bentazone-resistant mutant of cyanobacterium,Synechococcus elongatus PCC7942 adapts different strategies to counteract on bromoxynil- and salt-mediated oxidative stress

A bentazone-resistant mutant of Synechococcus elongatus PCC7942, called Mu2, tolerated elevated NaCl concentrations. As bentazone and bromoxynil exhibit similar mechanism of action, we investigated whether the mutant also toleratedbromoxynil and found it to be true. The line of investigation was then whether the acclimation strategy for the three stressors, bentazone, bromoxynil and NaCl was same or different. The cellular contents of malondialdehyde, hydrogen peroxide and superoxide increased in wild type strain following all the treatments suggesting their toxicities due to oxidative response. Notwithstanding, there were apparently different anti-oxidative measures pertaining to the herbicide and salinity stress. Glutathione contents and activities of superoxide dismutase, catalase-peroxidase, glutathione S-transferase and glutathione reductase decreased under NaCl, whereas bromoxynil affected only glutathione S-transferase reductase. Moreover, in-gel assays revealed that bromoxynil promoted appearance of isozymes of catalase-peroxidase, while NaCl induced such response only for superoxide dismutase. On the other hand, in Mu2, glutathione peroxidase-reductase and glutathione showed upward trend after bromoxynil exposure, whereas NaCl raised peroxidase and superoxide dismutase. Proteome comparison revealed peroxiredoxin Q to be highly expressed in wild type strain under bromoxynil, whereas NaCl favoured flavodoxin over-expression. Their amounts were already high in Mu2. We suggest that Mu2 acclimatized to bromoxynil in a manner similar to bentazone by upgrading peroxiredoxin Q and glutathione peroxidase-reductase. Conversely, for NaCl it devised another mechanism involving peroxidase and superoxide dismutase, and flavodoxin.     

Thermal stability and redox properties of M. tuberculosis CuSOD

The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases. We have also observed that, unlike the zinc-free eukaryotic or bacterial enzymes, the active site copper of the mycobacterial enzyme is not reduced by ascorbate, confirming that its redox properties are comparable to those typical of the enzymes containing zinc in the active site. Our findings highlight the role of zinc in conferring stability to Cu,Zn superoxide dismutases and indicate that the structural rearrangements observed in M. tuberculosis Cu,SOD compensate for the absence of zinc in achieving a fully active enzyme.     

Inducible prophages contribute to Salmonella virulence in mice

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H₂O₂) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H₂O₂ stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.     

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC , encoding [Cu,Zn]-SOD, from Salmonella typhimurium . The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC , in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S . typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S . typhimurium , S . choleraesuis and S . dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro , perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella , the most efficient overwhelming the proposed protective effect of periplasmic SOD.     

Differential accumulation of Salmonella[Cu,Zn] superoxide dismutases SodCI and SodCII in intracellular bacteria: correlation with their relative contribution to pathogenicity

Most Salmonella enterica strains have two peri-plasmic [Cu, Zn] superoxide dismutases, SodCI and SodCII, encoded by prophage and chromosomal genes respectively. Both enzymes are thought to play a role in Salmonella pathogenicity by intercepting reactive oxygen species produced by the host's innate immune response. To examine the apparent redundancy, we have compared the levels of epitope-tagged SodCI and SodCII proteins in bacteria growing in vitro, as well as inside tissue culture cells and in mouse tissues. Concomitantly, we have measured the abilities of mutants of either or both sodC genes to proliferate in infected mice in competition assays. Our results show a striking variation in the relative abundance of the two proteins in different environments. In vitro, both proteins accumulate when bacteria enter stationary phase; however, the increase is much sharper and conspicuous for SodCII than for SodCI. In contrast, SodCI vastly predominates in intracellular bacteria where SodCII levels are negligible. In agreement with these findings, most, if not all, of the contribution of [Cu, Zn] superoxide dismutase activity to murine salmonellosis can be ascribed to the SodCI protein. Overall the results of this work suggest that the duplicate sodC genes of Salmonella have evolved to respond to different sets of conditions encountered by bacteria inside the host and in the environment.     

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobocterium leiognothi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide disrnutases shows 21 invariant residues in key positions related to maintenance of the β-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobocterium Cu/Zn superoxide dismutase. Copper/zinc superoxide disrnutase from the trematode Schisiosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide. as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase. including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of β-barrel fold and active site structure for the extracellular enzyme.     

Cu,Zn-superoxide dismutase is required for cell wall structure and for tolerance to cell wall-perturbing agents in Saccharomyces cerevisiae

Here we report that deletion of SOD1, the Cu,Zn-superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall-perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N-acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.