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.     

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.     

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.     

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.     

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.     

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.     

Identification of GtgE,a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.     

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.     

The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli

The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram-negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. However, its presence in some non-pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions. We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS. Surprisingly, the entry of wild-type E. coli into stationary phase is accompanied by a several-hour-long period of acute sensitivity to hydrogen peroxide. Induction of the RpoS regulon helps to diminish that sensitivity. While mutants of E. coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase. This sensitivity required prior growth in air. Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide. The molecular details await elucidation.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Periplasmic copper–zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide

Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H . ducreyi gene products involved in virulence and pathogenesis, we created random Tn phoA insertion mutations in an H . ducreyi 35 000 library cloned in Escherichia coli . Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu–Zn superoxide dismutase (SOD) enzyme. The Cu–Zn SOD was periplasmic in H . ducreyi and accounted for most of the detectable SOD activity in whole-cell lysates of H . ducreyi grown in vitro . To investigate the function of the Cu–Zn SOD, we created a Cu–Zn SOD-deficient H . ducreyi strain by inserting a cat cassette into the sodC gene. The wild-type and Cu–Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu–Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu–Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H . ducreyi Cu–Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.     

Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.     

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.     

Identification of sodC encoding periplasmic [Cu,Zn]-superoxide dismutase in Salmonella

Abstract sodC , encoding [Cu,Zn]-cofactored Superoxide dismutase, once thought to be virtually confined to eukaryotes, has now been described in many Gram-negative pathogens that have their primary niche of colonization in the upper respiratory tract. Their role in host-parasite interactive biology is unknown. We here show that members of the major human and animal enteric pathogenic species Salmonella harbour a version of sodC most closely resembling that found in Brucella abortus . The enzyme it encodes is a novel candidate determinant of virulence in Salmonella , an intracellular pathogen potentially exposed to toxic oxygen free radicals within its intracellular niche.     

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.