BackgroundZinc is an essential element for all living cells. Recent studies have shown that the ZnuABC zinc uptake system significantly contributes to the ability of several pathogens to multiply in the infected host and cause disease, suggesting that zinc is scarcely available within different tissues of the host. To better understand the role of zinc in bacterial pathogenicity, we have undertaken a functional characterization of the role of the ZnuABC-mediated zinc uptake pathway in enterohemorrhagic Escherichia coli O157:H7.ResultsIn this work we have analyzed the expression and the role in metal uptake of ZnuA, the periplasmic component of the ZnuABC transporter, and of ZinT, another periplasmic protein which has been shown to contribute to zinc recruitment. We report that the expression of zinT and znuA, regulated by Zur, is induced in zinc-poor media, and that inactivation of either of the genes significantly decreases E. coli O157:H7 ability to grow in zinc depleted media. We also demonstrate that ZinT and ZnuA have not a redundant function in zinc homeostasis, as the role of ZinT is subordinated to the presence of ZnuA. Moreover, we have found that znuA and zinT are strongly induced in bacteria adhering to cultured epithelial cells and that lack of ZnuA affects the adhesion ability. In addition we have found that a fraction of apo-ZinT can be secreted outside the cell where the protein might sequester environmental zinc, inducing a condition of metal starvation in surrounding cells.ConclusionsThe here reported results demonstrate that ZnuABC plays a critical role in zinc uptake also in E. coli O157:H7 and that ZinT contributes to the ZnuA-mediated recruitment of zinc in the periplasmic space. Full functionality of the zinc import apparatus is required to facilitate bacterial adhesion to epithelial cells, indicating that the microbial ability to compete with the host cells for zinc binding is critical to establish successful infections. The observation that ZinT can be secreted when it is in the apo-form suggests that its presence in the extracellular environment may somehow contribute to metal uptake or facilitate bacterial colonization of the intestinal epithelia.

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calciummobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS. Key Words: Calcineurin-Familial amyotrophic lateral sclerosis-Superoxide dismutase-Neurodegeneration-Redox regulation-Reactive oxygen species. J. Neurochem. 75, 606 -613 (2000).Calcineurin (CN), also known as protein phosphatase 2B, is a heterodimeric enzyme composed of a catalytic A-subunit (molecular mass ϳ60 kDa) and a myristoylated regulatory B-subunit (molecular mass ϳ19 kDa) (Klee et al., 1988). The A-subunit has four distinct domains: the catalytic domain, the B-subunit binding domain, the Ca 2ϩ /calmodulin complex binding domain, and the auto-inhibitory domain. The B-subunit is structurally related to calmodulin, containing four EF-hand Ca 2ϩ binding loops. The A-subunit binds one zinc and one iron atom at the active site, which probably serve to position water molecules by acting as nucleophiles in the attack on the phosphoester bond (Griffith et al., 1995;Kissinger et al., 1995). The auto-inhibitory domain masks the metal ions at the active site; the activation of CN by Ca 2ϩ /calmodulin probably results from the displacement of this domain, resulting in exposure of the catalytic center. In this "open" conformation, CN is sensitive to inactivation by small (undefined) molecules (Wang et al., 1996). CN is enriched in neural tissue and comprises Ͼ1% of the total protein in the nervous system (Klee et al., 1988), where it acts as a Ca 2ϩ -and calmodulin-dependent serine/threonine protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals (Yakel, 1997). Its role in regulating neuronal excitability by controlling the activity of ion channels, the release of neurotransmitter and hormone, synaptic plasticity, and gene transcription is well documented (Yakel, 1997). A role for CN in cell cycle regulation (Davis et al., 1997) and in Ca-dependent apoptosis, mediated by interaction of CN with Bcl-2 (Reed, 1997) and by dephosphorylation of BAD (Wang...

Abstract:We have investigated the response to oxidative stress in a model system obtained by stable transfection of the human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase or a mutant of this enzyme (H46R) associated with familial amyotrophic lateral sclerosis. We report that expression of mutant H46R Cu,Zn superoxide dismutase induces a selective increase in paraquat sensitivity that is reverted by addition of D-penicillamine. Furthermore, expression of this mutant enzyme affects the activity of the endogenous wild-type enzyme both in basal conditions and in copper overloading experiments. Our data indicate that aberrant metal chemistry of this mutant enzyme is the actual mediator of oxidative stress and that concurrent impairment of the activity of wild-type endogenous enzyme compromises the cell's ability to respond to oxidative stress.

Gel-filtration chromatography experiments performed at high protein concentrations demonstrate that the Cu,Zn superoxide dismutase from Escherichia coli is monomeric irrespective of the buffer and of ionic strength. The catalytic activity of the recombinant enzyme is comparable with that of eukaryotic isoenzymes, indicating that the dimeric structure commonly found in Cu,Zn superoxide dismutases is not necessary to ensure efficient catalysis. The analysis of the amino acid sequences suggests that an altered interaction between subunits occurs in all bacterial Cu,Zn superoxide dismutases. The substitution of hydrophobic residues with charged ones at positions located at the dimer interface of all known Cu,Zn superoxide dismutases could be specifically responsible for the monomeric structure of the E. coli enzyme.