Toxic Mechanisms Of Soft Metals Against Escherichia Coli

Metal toxicity mechanism has gaining increasing focus due to severer metal pollution and higher people conscious with health and environmental safety. Many metals are toxic to humans. Overload can result from genetic defects that abrogate homeostatic controls, or from excessive environmental exposure. The mechanisms by which most metals poison cells are unclear, due in part to the breadth of their chemical activities. A lot of toxic metals share one characteristic:thiophilicicy. Accordingly, those metals are named soft metal, generally speaking, Cu(Ⅱ), Ag(Ⅰ), Hg(Ⅱ), Cd(Ⅱ), Zn(Ⅱ), Pb(Ⅱ), Ni(Ⅱ), Co(Ⅱ) and Mn(Ⅱ) all have substantial affinity to sulfur and are all included. Also, their toxicity roughly parallels their thiophilicity, suggesting that their primary biological targets are likely to be enzymes that contain key sulfhydryl moieties. In this research, soft metal toxicity and their mechanism were investigated using E. coli as the model organism.Iron has been a particular focus of study, because it is naturally abundant inside cells. Current data indicate that high levels of intracellular iron are toxic primarily due to its tendency to react with endogenous hydrogen peroxide and generate hydroxyl radicals and then damage cells. Copper shares with iron its ability to react with hydrogen peroxide, and so it has been widely suspected that its toxicity involve a similar mechanism. However, our laboratory has found that intracellular copper directly damaged enzymes that contain iron-sulfur clusters.Toxicity of Cu(Ⅱ) against E. coli under weak acidic conditions both with and without L-AscA were discussed. Comparison were made between Cu(II), Fe(Ⅲ) and Cr(Ⅲ) which were Fenton and Haber-Weiss reaction relevant. The toxic mechanism, especially for Cu(Ⅱ), regarding to oxidative stress and copper thiolphilicity were investigated and discussed.E. coli viability were tested while exposed to varied metal systems and the toxic parameters were determined; L-AscA degradation under varied conditions were tested; the ability of Cu/L-AscA system to disinfect strains isolated from natural water were investigated; ESR. was used to detect and quantify the OH produced; copper toxicity was systematically discussed regarding both oxidative stress and damaging Fe-S cluster enzymes. Results showed that L-AscA obviously enhanced the toxicity of Cu(II) under pH4.0, and the toxicity was copper concentration dependent; E. coli viability dropped to zero in2h after exposed to200μM,20μM,2μM,0.2μM with0.01%L-AscA; the coexistence system also showed high potency to kill other strains isolated; L-AA enhanced Cu(Ⅱ) but not Fe(Ⅲ) and Cr(Ⅲ) bactericidal efficiency at pH4, under which all cell death was shown in30minutes after full contact with200μM,20μM Cu(II) and2hours with2μM,0.2μM Cu(Ⅱ). Cr(Ⅲ) alone, which is widely recognized as innocuous and stable, at0.2mM, pH4, also showed surprisingly high disinfection capability.ESR results showed that L-AscA's addition doubled OH concentration of200μM Cu(Ⅱ) system. However, some hydroxyl radical was detected by0.2mM Cu(II) alone but not by0.02mM Cu(Ⅱ) and0.01%L-AscA which contradictory showed higher cell killing ability. No OH were detected with Cr(Ⅲ)/Fe(Ⅲ) and L-AscA system.A synergism effect existed between Cu(Ⅱ) and L-AscA, and Cu(Ⅱ) palyed the major role for causing cell toxicity. In the presence of L-AscA, high copper concentration under aerobic conditions produced high amount or OH and showed relationship with the cell death; however, under lower cooper concentration which didn't has OH detected, Cu(Ⅱ)'s ability to damage Fe-S cluster enzymes made the major contribution. Whereas, the toxicity of Cr(Ⅲ) was complicated and remains to be elucidated.The other soft metals'(including Ag(Ⅰ), Hg(Il), Cd(Ⅱ), Zn(Ⅱ),Pb(Ⅱ), Ni(Ⅱ), Co(Ⅱ), Mn(Ⅱ)) toxicity against E. coli and their ability to damage Fe-S containing enzymes.Toxicity of each soft metal against Wild type E. coli MG1655were investigated under low OD (0.005). As a representative, Ag(1)'s ability of cell growth was discussed in detail. Parameters discussed including oxygen conditions, medium types, medium components and branched chain amino acid supplementation. Resistance of copA, a p-type ATPase, over Ag(I) and Cu(Il) were analyzed and compared. Anaerobic purification of fumA was conducted ordered as follow:protamine sulfate treatment, DEAE Sepharose column,(NH4)2SO4treatment, Phenyl Sepharose treatment, concentrating, Superdex200treatment and final concentrating. Furthermore, Soft metal ability to attack exposed iron-sulfur containing enzymes was investigated systematically both in vitro and in vivo.In vitro analysis was mainly conducted with purified fumA. Those included destruction, substrate protection, damaged enzyme reactivation and rebuild, EPR and Fe loss analysis of damaged Fe-S cluster structure.In vivo experiment were as follow:threshold dose of Ag(Ⅰ), Hg(Ⅱ), Cd(Ⅱ) and Zn(Ⅱ) were tested under OD6oo0.2; then cells were then harvested after exposure to the metal concentrations that initial toxicity and [4Fe-4S] dehydratase enzymes (fumA, Edd, IPM1) activity were measured afterwards; As negative control, enzymes that don't contain the cluster(β-gal, MDH) or have shielded clusters(Ndh I) were also analyzed; metal effect against Fe-S build and assembly Isc system were conducted with β- gal assay of suf::lacZ fusion strain SJ253;△zntA724::kan mutant (named FX10) was made by P-1transduction from MG1655and then used to analyze Zn(II) resistance.In vitro experiments revealed that low-micromolar concentrations of Ag(Ⅰ) and Hg(Ⅱ) directly inactivated purified fumarase A, a member of the dehydratase family. The enzyme was also poisoned by higher levels of Cd(II) and Zn(II), but it was unaffected by even millimolar Mn(Ⅱ), Co(Ⅱ), Ni(Ⅱ), and Pb(II). EPR analysis and measurements of released iron confirmed that damage was associated with destruction of the [4Fe-4S] cluster, and indeed the reconstruction of the cluster fully restored activity. Growth studies were then performed to test whether dehydratase damage might underlie toxicity in vivo. Barely toxic doses of Ag(Ⅰ), Hg(Ⅱ), Cd(Ⅱ), and Zn(Ⅱ) inactivated all tested members of the [4Fe-4S] dehydratase family. Again, activity was recovered when the clusters were rebuilt. The metals did not diminish the activities of other sampled enzymes, including NADH dehydrogenase I, an iron-sulfur protein whose clusters are shielded by polypeptide. Thus the data indicate that dehydratases are damaged by the concentrations of metals that initiate bacteriostasis.