A Novel Heme Protein, the Cu,Zn-Superoxide Dismutase from Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of the genital ulcerative disease known as chancroid, is unable to synthesize heme, which it acquires from humans, its only known host. Here we provide evidence that the periplasmic Cu,Zn-superoxide dismutase from this organism is a heme-binding protein, unlike all the other known Cu,Zn-superoxide dismutases from bacterial and eukaryotic species. When the H. ducreyi enzyme was expressed in Escherichia coli cells grown in standard LB medium, it contained only limited amounts of heme covalently bound to the polypeptide but was able efficiently to bind exogenously added hemin. Resonance Raman and electronic spectra at neutral pH indicate that H. ducreyi Cu,Zn-superoxide dismutase contains a 6-coordinated low spin heme, with two histidines as the most likely axial ligands. By site-directed mutagenesis and analysis of a structural model of the enzyme, we identified as a putative axial ligand a histidine residue (His-64) that is present only in the H. ducreyi enzyme and that was located at the bottom of the dimer interface. The introduction of a histidine residue in the corresponding position of the Cu,Zn-superoxide dismutase from Haemophilus parainfluenzae was not sufficient to confer the ability to bind heme, indicating that other residues neighboring His-64 are involved in the formation of the heme-binding pocket. Our results suggest that periplasmic Cu,Zn-superoxide dismutase plays a role in heme metabolism of H. ducreyi and provide further evidence for the structural flexibility of bacterial enzymes of this class

A histidine-rich metal binding domain at the N terminus of Cu,Zn-superoxide dismutases from pathogenic bacteria: a novel strategy for metal chaperoning.

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 N-terminal 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

Studio delle proprieta molecolari e funzionali delle Cu,-ZnSOD nei batteri gram negativi

Dottorato di ricerca in scienze biochimiche e biomolecolari. 12. ciclo.Consiglio Nazionale delle Ricerche Biblioteca Centrale P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale Piazza Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Extracellular GSH decreases <i>B. cenocepacia</i> invasion. Panel A.

<p>Invasion of 9HTEo- and CFTE29o- by <i>B. cenocepacia</i> LMG 16656 was assayed in presence of 0, 0.1, 1 and 10 mM GSH. The % of invasion indicates the ratio between the number of intracellular bacteria recovered from infected cells with respect to the bacteria added to the cell monolayer. Results represent means ± SD obtained by measuring <i>B. cenocepacia</i> LMG 16656 invasion in three independent experiments. Asterisks denote statistically significant results (* p<0.05; ** p<0.01 and *** p<0.0001, respectively). White bar: untreated cells; grey bar: cells treated with 0.1 mM GSH; dotted bar: cells treated with 1 mM GSH; black bar: cells treated with 10 mM GSH. <b>Panel B.</b> 9HTEo- and CFTE29o- were pretreated with 0, 0.1, 1 and 10 mM GSH for 3 hours at 37°C, washed to remove GSH and then used in the infection assay. Results, which are expressed as the mean ± SD of intracellular bacteria isolated from cells pretreated with GSH with respect to untreated cells, are the average of three independent experiments (**p<0.01; ***p<0.0001). Bar colors are as in panel A. <b>Panel C.</b> Invasion of C38 and IB3-1 by <i>B. cenocepacia</i> LMG 16656 in presence of 0 and 10 mM GSH. Results represent means ± SD obtained by measuring <i>B. cenocepacia</i> LMG 16656 invasion ability in three independent experiments (*** p<0.0001). White bars: control cells; black bars: cells treated with 10 mM GSH. <b>Panel D.</b> Invasion of 9HTEo-, CFTE29o-, C38 and IB3-1 by <i>B. cenocepacia</i> 6L in presence of 0 and 10 mM GSH. Results are shown as % of intracellular bacteria recovered with respect to the bacteria added to the cell monolayer. The reported values are means ± SD obtained by measuring 6L invasion ability in three independent assays (** p<0.01). White bars: control cells; black bars: cells treated with 10 mM GSH.</p

Extracellular GSH compromises the interaction of <i>B. cenocepacia</i> with mucociliary-differentiated CF bronchial epithelial cells. Panel A.

<p>MIP (Maximum Intensity Projection) from confocal system acquisition (Olympus IX 81 inverted microscope, software FV 1000) of monolayers infected with <i>B. cenocepacia</i> LMG 16656 for 3 hours, in absence (<b>upper</b>) or in presence (<b>bottom</b>) of 10 mM extracellular GSH. Cells were washed, fixed and permeabilized as described in Materials and Methods. Bacteria (red) and zona occludens (green) were detected using specific antibodies (R418 and anti-ZO1, respectively). Bar = 20 µm. <b>Panel B.</b> Invasion of mucociliary-differentiated CF bronchial epithelial cells by <i>B. cenocepacia</i> LMG 16656 in absence or in presence of GSH. Results are shown as % of intracellular bacteria recovered with respect to the bacteria added to the cell monolayers. The reported values represent the mean ± SD obtained by measuring LMG 16656 invasion ability from six individual cultures. (*p = 0.01; #p = 0.002; **p<0.05).</p

Effect of extracellular GSH on pro-inflammatory cytokines expression stimulated by <i>B. cenocepacia</i> LMG 16656.

<p>9HTEo-, CFTE29o- cells were incubated in presence or absence of 10 mM extracellular GSH and infected with <i>B. cenocepacia</i> LMG 16656 (10 CFU/cell) in presence of 0 and 10 mM GSH for 3 hours. The expression of IL-8, TNF-α and IL-1β was analyzed by RT-PCR. Data are from a typical experiment out of three giving qualitatively similar results. Each data point is the average of three independent measures on each sample.</p

Cytofluorimetric analysis of surface thiols.

<p>After an incubation in presence or absence of 10 mM extracellular GSH, cells were treated with 10 µM Alexa fluor C₅-maleimide to label surface free thiols and then analyzed by FACScalibur system, as described in Materials and Methods. The histograms are from a typical experiment out of three giving essentially identical results.</p

Extracellular GSH modifies <i>B. cenocepacia</i> LMG 16656 adhesion, but not intracellular replication. Panel A.

<p>Effect of extracellular GSH on <i>B. cenocepacia</i> LMG 16656 adhesion to 9HTEo- and CFTE29o- cells. Bars indicate the % of the bacteria adhering to cells with respect to the bacteria added to the cell monolayer. Results are the average ± SD of three independent experiments. White bars: control cells; black bars: cells treated with 10 mM GSH (*p<0.05; ** p<0.01). <b>Panel B.</b> Effect of 10 mM GSH on the total number (adherent + intracellular bacteria) of <i>B. cenocepacia</i> LMG 16656 recovered after 3 hours of 9HTEo- epithelial cell infection. Results are shown as % of total bacteria recovered with respect to the bacteria added to the cell monolayer. The reported values are means ± SD of three independent experiments. White bars: control cells; black bars: cells treated with 10 mM GSH (***p<0.0001). <b>Panel C. </b><i>B. cenocepacia</i> LMG 16656 replication within 9HTEo- cells. Fold replication values were determined by dividing the intracellular bacterial load at 1, 2.5 and 4 h post-infection by that determined after 30 minutes of infection. Results are the mean ± SD of three independent experiments.</p

Localized Infections with <i>P. aeruginosa</i> Strains Defective in Zinc Uptake Reveal That Zebrafish Embryos Recapitulate Nutritional Immunity Responses of Higher Eukaryotes

The innate immune responses of mammals to microbial infections include strategies based on manipulating the local concentration of metals such as iron (Fe) and zinc (Zn), commonly described as nutritional immunity. To evaluate whether these strategies are also present in zebrafish embryos, we have conducted a series of heart cavity-localized infection experiments with Pseudomonas aeruginosa strains characterized by a different ability to acquire Zn. We have found that, 48 h after infection, the bacterial strains lacking critical components of the Zn importers ZnuABC and ZrmABCD have a reduced colonization capacity compared to the wild-type strain. This observation, together with the finding of a high level of expression of Zur-regulated genes, suggests the existence of antimicrobial mechanisms based on Zn sequestration. However, we have observed that strains lacking such Zn importers have a selective advantage over the wild-type strain in the early stages of infection. Analysis of the expression of the gene that encodes for a Zn efflux pump has revealed that at short times after infection, P. aeruginosa is exposed to high concentrations of Zn. At the same time, zebrafish respond to the infection by activating the expression of the Zn transporters Slc30a1 and Slc30a4, whose mammalian homologs mediate a redistribution of Zn in phagocytes aimed at intoxicating bacteria with a metal excess. These observations indicate that teleosts share similar nutritional immunity mechanisms with higher vertebrates, and confirm the usefulness of the zebrafish model for studying host–pathogen interactions