The Cu,Zn superoxide dismutase from <i>Escherichia coli</i> retains monomeric structure at high protein concentration. Evidence for altered subunit interaction in all the bacteriocupreins

Battistoni, Andrea; Folcarelli, Silvia; Gabbianelli, Roberta; Capo, Concetta; Rotilio, Giuseppe

doi:10.1042/bj3200713

Cited by 42 publications

(36 citation statements)

References 31 publications

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“…Whether this reflects a difference in plasmid copy number or an in vivo-reduced stability of Cu, ZnSOD compared to ␤-Gal or whether the measurement of Cu,ZnSOD led to an underestimation of the amount of enzyme expressed from the plasmid is unclear. However, several factors could be responsible for an underestimation: the SOD assay used (which relies on the use of an inhibitor to discriminate between the activity of Cu,ZnSOD and that of contaminating cytoplasmic SODs), protease sensitivity, the low stability of E. coli Cu,ZnSOD (2,3,5), and the requirement of adequate amounts of copper to ensure full catalytic activity to the enzyme (2,3).…”

Section: Discussionmentioning

confidence: 99%

Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

et al. 2000

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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.Until a few years ago Cu,Zn superoxide dismutase (Cu, ZnSOD) was considered almost exclusively a eukaryotic enzyme, protecting the cytosol and the extracellular environment of higher organisms from damage by oxygen free radicals (1). Recently, Cu,ZnSOD has been identified in the periplasmic space of a wide range of gram-negative bacteria, including Brucella abortus (6), Haemophilus spp., Actinobacillus spp., Pasteurella spp., Neisseria meningitidis (24-26), Escherichia coli K-12 (7), Legionella pneumophila (40), Salmonella spp. (9), and Mycobacterium tuberculosis (45). This enzyme is thought to protect bacteria from toxic oxygen-free radicals generated outside the cell or in the periplasm itself, since superoxide is unable to cross the cytoplasmic membrane (21). Therefore, Cu,ZnSOD has been proposed to be a determinant of virulence in bacteria potentially exposed to toxic free radicals produced by the host in response to bacterial infection. In vivo experiments have demonstrated the role of bacterial Cu,ZnSOD in the virulence and pathogenicity of infecting microorganisms (15,18,19,36,42,43), while in vitro models have provided conflicting data concerning Cu,ZnSOD involvement in bacterial resistance to macrophage killing (19, 42) or survival within nonprofessional phagocytes (42). However, more recent results have shown that this enzyme protects Salmonella enterica serovar Typhimurium (15) and an overproducing strain of E. coli (4) from macrophage killing and that neutropenia restores virulence to an attenuated C...

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Section: Discussionmentioning

confidence: 99%

Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

et al. 2000

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“…SodCII was purified from E. coli 71/18 cells bearing plasmid pSEsodCII, obtained by inserting the sodCII gene in plasmid pSE420 under control of the trc promoter. Expression and purification of SodCII was carried out essentially as described for the monomeric Cu,Zn-SOD from E. coli (41). The purity of the enzyme was checked by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

Ammendola

Pasquali

Pacello

et al. 2008

Journal of Biological Chemistry

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Many of the most virulent strains of Salmonella enterica produce two distinct Cu,Zn-superoxide dismutases (SodCI and SodCII). The bacteriophage-encoded SodCI enzyme makes the greater contribution to Salmonella virulence. We have performed a detailed comparison of the functional, structural, and regulatory properties of the Salmonella SodC enzymes. Here we demonstrate that SodCI and SodCII differ with regard to specific activity, protease resistance, metal affinity, and peroxidative activity, with dimeric SodCI exhibiting superior stability and activity. In particular, monomeric SodCII is unable to retain its catalytic copper ion in the absence of zinc. We have also found that SodCI and SodCII are differentially affected by oxygen, zinc availability, and the transcriptional regulator FNR. SodCII is strongly down-regulated under anaerobic conditions and dependent on the high affinity ZnuABC zinc transport system, whereas SodCI accumulation in vitro and within macrophages is FNR-dependent. We have confirmed earlier findings that SodCII accumulation in intracellular Salmonella is negligible, whereas SodCI is strongly up-regulated in macrophages. Our observations demonstrate that differences in expression, activity, and stability help to account for the unique contribution of the bacteriophage-encoded SodCI enzyme to Salmonella virulence.

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“…After digestion with NcoI, the 270-base pair NcoI-NcoI DNA fragments were purified and inserted in the NcoI site of plasmid pPHaeShort. Plasmids pPEcSODHis and pPXSODBHis, expressing Escherichia coli and Xenopus laevis Cu,Zn-SODs fused to the 22 N-terminal residues from H. ducreyi Cu,Zn-SODs, were obtained by inserting a DNA fragment obtained by amplification with HduFor1 and HduRevL into the HindIII and NcoI sites of pPEcSOD81A (28) and pPXSODB (29). The complete nucleotide sequences of all the PCR amplified DNA fragments were checked by the dideoxy chain termination method.…”

Section: Methodsmentioning

confidence: 99%

A Histidine-rich Metal Binding Domain at the N Terminus of Cu,Zn-Superoxide Dismutases from Pathogenic Bacteria

Battistoni¹,

Pacello²,

Mazzetti³

et al. 2001

Journal of Biological Chemistry

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A group of Cu,Zn-superoxide dismutases from pathogenic bacteria is characterized by histidine-rich N-terminal extensions that are in a highly exposed and mobile conformation. This feature allows these proteins to be readily purified in a single step by immobilized metal affinity chromatography. The Cu,Zn-superoxide dismutases from both Haemophilus ducreyi and Haemophilus parainfluenzae display anomalous absorption spectra in the visible region due to copper binding at the N-terminal region. Reconstitution experiments of copper-free enzymes demonstrate that, under conditions of limited copper availability, this metal ion is initially bound at the N-terminal region and subsequently transferred to an active site. Evidence is provided for intermolecular pathways of copper transfer from the N-terminal domain of an enzyme subunit to an active site located on a distinct dimeric molecule. Incubation with EDTA rapidly removes copper bound at the N terminus but is much less effective on the copper ion bound at the active site. This indicates that metal binding by the Nterminal histidines is kinetically favored, but the catalytic site binds copper with higher affinity. We suggest that the histidine-rich N-terminal region constitutes a metal binding domain involved in metal uptake under conditions of metal starvation in vivo. Particular biological importance for this domain is inferred by the observation that its presence enhances the protection offered by periplasmic Cu,Zn-superoxide dismutase toward phagocytic killing.

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The Cu,Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration. Evidence for altered subunit interaction in all the bacteriocupreins

Cited by 42 publications

References 31 publications

Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

A Histidine-rich Metal Binding Domain at the N Terminus of Cu,Zn-Superoxide Dismutases from Pathogenic Bacteria

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