Amedeo Albano

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH.) generated by H2O2 and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H2O2 lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxamine, did not affect the survival of cells exposed to 2.5 mM H2O2. In addition cell vulnerability to the same H2O2 concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H2O2 higher than 10 mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content. It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.

Morphological Changes in Escherichia coli Cells Exposed to Low or High Concentrations of Hydrogen Peroxide

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (<2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times (< 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (>10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mM H2O2 resulted in a time‐dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.

Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 microM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems. In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 microM and was statistically significant only for cystamine. Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.

Differential effect of the amino acid cystine in cultured mammalian and bacterial cells exposed to oxidative stress

The effect of cystine in the cytotoxic response of cultured Chinese hamster ovary and Escherichia coli cells to challenge with hydrogen peroxide has been investigated. It was found that this amino acid could either protect or sensitize cells, depending on the cellular system. In fact, although a reduction in the growth-inhibitory effect of hydrogen peroxide was observed in mammalian cells, a marked increase in the susceptibility to oxidative stress was induced by cystine in bacteria. None of the amino acid precursors of glutathione, e.g., glutamate, glycine or cysteine, afforded protection in the mammalian cell system, whereas cysteine, but not glycine or glutamate, markedly sensitized bacteria to hydrogen peroxide-induced cell killing. In mammalian cells, methionine, an amino acid which is converted to cysteine, was also unable to modify the oxidative response. The results presented indicate that cystine displays differential effects in oxidatively injured mammalian or bacterial cells and suggest that the mechanism whereby the amino acid modulates the lethal action of hydrogen peroxide differs in the two cellular systems.

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli).

The toxicity of H2O2 in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H2O2 (i.e. mode one killing, which is produced by concentrations of H2O2 lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H2O2 higher than 10 mM). Although the data obtained do not clarify the molecular basis of H2O2 toxicity and/or do not explain the specific function of superoxide ions in H2O2-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H2O2 lethality. A mechanism of H2O2 toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O2-. generating system. This enzyme should be active at low concentrations of H2O2 (less than 5 mM) and high concentrations of the oxidant (greater than 5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H2O2 concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H2O2 in the range 5-10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H2O2 (greater than 10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe2+ since superoxide ions may also reduce trivalent iron to the divalent form.

Superoxide anions are required for the inactivation ofEscherichia coli induced by high concentrations of hydrogen peroxide

The wild-type strain and mutants ofEscherichia coli lacking Mn-superoxide dismutase (Sod A) or Fe-superoxide dismutase (Sod B) are compared for their sensitivity to the H2O2 insult (exposure for 15 min at 37°C, in M9 salts). Whereas mode one killing is similar in superoxide dismutase mutants and wild-type cells, the latter strain appears to be more resistant than the former ones to mode two lethality. Furthermore, Sod B cells, as well as wild-type cells but unlike Sod A cells, are capable of reversing the toxicity of the oxidant (even in the presence of chloramphenicol), this effect being observed by gradually increasing the H2O2 concentration from 2.5 to 10 mM. It is concluded that (a) superoxide ions may not be involved in the production of mode one killing by H2O2, although further experiments are needed to validate or modify this hypothesis; (b) superoxide ions mediate mode two killing by H2O2, possibly by reducing trivalent iron to the divalent form; and (c) the intervening zone of partial resistance observed in wild-type and Sod B cells exposed to intermediate H2O2 concentrations is not a consequence of Mn-superoxide dismutase induction; it would appear, however, that cells lacking this superoxide dismutase isoenzyme are not proficient in this acquired response.

The effect of K2Cr2O7 on the growth and morphology ofEscherichia coli

Treatment with 16 μM K2Cr2O7 results is a time-dependent loss of the clonogenicity ofE. coli cells. Under the same experimental conditions, optical density values do not change significantly, since cells have the ability to replicate but not to septate. In fact, microscopic analysis of samples taken over 240 min of exposure to the toxin has revealed the formation of filaments. The filamentous response appears similar to that generated by mitomycin C.