Cysteine Homeostasis Disturbance in Escherichia coli Caused by Exposure to Ciprofloxacin.

E. coli exposure to ciprofloxacin disturbs cysteine homeostasis; an increase in the intracellular concentration of cysteine is dangerous due to its ability to enhance ROS generation. Unlike wild-type bacteria, in which the cysteine content did not exceed the control level, cells of the gshA mutant lacking glutathione are characterized by increased concentration of intracellular cysteine in proportion to the concentrations of the antibiotic, despite the intensive export of cysteine into the medium. At low concentrations of ciprofloxacin, the mutant strain formed half as many colonies as the parent strain in the survival test. These findings attest to the important role of the incorporation of excess cysteine into glutathione as one of the mechanisms of cysteine homeostasis during the stress response to antibiotic.

[Changes in the Activity of Antioxidant Systems of Escherichia coli under Phosphate Starvation].

Changes in the activity of antioxidant systems in Escherichia coli during phosphate starvation have been studied. It is shown that starvation was accompanied by a decrease in the intensity of respiration, an increase in the rate of superoxide production, and a decrease in the level of ATP. Simultaneously, there was a decrease in H2O2 in the medium and a significant increase in the expression of the katG and katE genes which encode the HPI and HPII catalases, respectively. At the same time, there was no drop in the membrane potential, which may indicate the retention of normal membrane activity in starving cells. It has been shown for the first time that the transition of E. coli to phosphate starvation is accompanied by significant changes in the status of glutathione. The most important of these are associated with a decrease in the level of reduced glutathione in the medium (GSHout) and with a simultaneous increase in its content in the cytoplasm (GSHin), as well as a shift in the GSHin to oxidized glutathione form (GSSGin) ratio towards reductive values, and GSHout/GSSGout towards oxidative values. Among the mutants used in the work, the gor trxB double mutant, which is deficient in the synthesis of glutathione reductase and thioredoxin reductase, showed the most pronounced distinctive features. Compared to the parental strain, this mutant showed a multiple higher expression of katG::lacZ, the highest level of oxidized intra- and extracellular glutathione, and, accordingly, the lowest GSH/GSSG ratio in both compartments. In general, the data we obtained indicate that during phosphate starvation the interaction of the glutathione redox-system and regulons that control protection against reactive oxygen species creates conditions that allow maintaining the concentration of ROS below the toxic level. As a result, phosphate-starved E. coli cells can maintain high viability for a long period of time, which allows them to quickly resume growth after the addition of phosphate.

ULTRASTRUCTURAL CHANGES OF WOUND MACROPHAGES UNDER THE INFLUENCE OF INTRAVENOUS OZONE THERAPY IN PATIENTS WITH DIABETES AND INFLAMMATORY PROCESSES OF SOFT TISSUES.

Investigation of ultrastructural peculiarities of morpho-functional changes of macrophages have been studied with the purpose of determining the dynamics and thrust of destructive-necrotic processes in these cells when the ischemic-gangrenous form of diabetic foot syndrome develops show what under the influence of intravenous ozone therapy stimulant effect on functional activity and beneficial effect on elimination, mainly due to genetically programmed cell death (apoptosis), playing a significant role in the regulatory mechanisms of the inflammatory process. The stimulation of macrophages functional activity under the influence of ozone, as well as the presence of destructive changes in such cells without necrotizing lesions, is explained by the inclusion of the mechanism of apoptosis as a positive factor in the regulation of local homeostasis at the completion of the inflammatory (exudative) stage of the wound process.

[Role of Thiol Redox Systems in Escherichia coli Response to Thermal and Antibiotic Stresses].

Isogenic knockout mutants of Escherichia coli deficient in components of the glutathione and thioredoxin redox systems and growing at various temperatures (20-46°C) exhibited considerable differences in growth rate and survival, as well as in expression of the antioxidant genes. In the parent strain E. coli BW25113 (wt) treated with ciprofloxacin, ampicillin, or streptomycin, dependence of survival from growth temperature was a V-shaped curve with the maximum sensitivity within the range corresponding to high growth rates (40-44°C). Significant inverse correlation was observed between log CFU at different temperatures and specific growth rate prior to antibiotic addition. This applied to most of the mutants, which exhibited higher resistance to the three antibiotics tested at nonoptimal temperatures (20 and 46°C) than at 37 and 40°C. No correlation was found between resistance to stress and antibiotics and expression of the antioxidant genes. The role of global regulators ppGpp and σ(s) in E. coli resistance to antibiotics and nonoptimal temperatures was shown.

Influence of plant polyphenols and medicinal plant extracts on antibiotic susceptibility of Escherichia coli.

AIMS: To investigate the influence of polyphenols and plant extracts on the susceptibility of Escherichia coli to antibiotics. METHODS AND RESULTS: Susceptibility of E. coli to antibiotics in the presence of extracts and polyphenols was estimated by the determination of the minimum inhibitory concentrations (MICs). To study gene expression, we used strains of E. coli carrying fusions between promoters of genes katG, sodA, iucC and structural ß-galactosidase gene. Treatment with polyphenols and some plant extracts significantly decreased the antibacterial effects of antibiotics, to a larger extent, ciprofloxacin. The most remarkable protective effect was observed for the extracts of Chamerion (Epilobium) angustifolium, Filipendula vulgaris, Tanacetum vulgare and Serratula coronata. These extracts increased the MICs of ciprofloxacin by four and more times. In case of kanamycin, extracts of Artemisia austriaca and Artemisia pontica increased MICs by four and eight times, respectively. Polyphenol quercetin also caused protective effect against ciprofloxacin, increasing the MIC by four times. A positive correlation was found between protective effects of polyphenols and extracts and their antioxidant activity. CONCLUSION: Medicinal plant extracts and polyphenols may protect cells of E. coli against antibiotic toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study may be used to enhance the efficiency of antibacterial therapies.

Assessment of anti-oxidant activity of plant extracts using microbial test systems.

AIMS: To evaluate the anti-oxidant properties of extracts from 20 medicinal herbs growing in western Siberia using microbial test systems and different in vitro methods. METHODS AND RESULTS: In vivo anti-oxidant activity of extracts was evaluated for their capacity to protect bacteria, Escherichia coli, against bacteriostatic and bactericidal effects of H(2)O(2) and menadione, and action on anti-oxidant gene expression. In vitro anti-oxidant activity has been examined by a number of methods including: the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(*))-scavenging assay, chelating activity and capacity to protect plasmid DNA against oxidative damage. In addition, total polyphenol content was determined. The extracts of Fragaria vesca, Rosa majalis, Pentaphylloides fruticosa, Alchemilla vulgaris and Pulmonaria mollis possessed the highest levels of anti-oxidant activity in vivo and in vitro. The protective properties were more closely related to the DPPH(*) radical-scavenging activity, tannin content and action on anti-oxidant gene expression than to other parameters. CONCLUSION: The extracts of medicinal plants may have anti-oxidant effects on bacteria simultaneously through several different pathways, including direct inhibition of reactive oxygen species, iron chelation and anti-oxidant genes induction. SIGNIFICANCE AND IMPACT OF THE STUDY: Using microbial test systems, we revealed herbs that may be used as potential sources of natural anti-oxidants.

[The antioxidant characteristics of medicinal plant extracts from Western Siberia].

An antioxidant activity of the water-alcohol extracts of leaves of ten herbs from Western Siberia was studied. In vivo the capability of extracts to protect cells of Escherichia coli against the bacteriostatic action of H2O2 and the influence of the extracts on the expression of the antioxidant gene katG coding catalase-hydroperoxidase I were investigated. In vitro the radical-binding activity with DPhPG (1,1-diphenyl-2-picrylhydrazyl radical), the chelating capability with ferrozine, and total composition of flavonoids and tannins were determined. The extracts of Filipendula stepposa and Limonium gmelinii were characterized by the highest antioxidant activity. According to data, the test extracts could have an antioxidant effect on bacteria in different ways at once including the direct inhibition of ROS (reactive oxygen species), iron ion chelation and antioxidant gene induction.

[The role of thiol redox systems in the peroxide stress response of Escherichia coli].

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H2O2, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.

Effects of cystine and hydrogen peroxide on glutathione status and expression of antioxidant genes in Escherichia coli.

Cysteine or cystine was earlier shown to multiply enhance the toxic effect of hydrogen peroxide on Escherichia coli cells. In the present work, the treatment of E. coli with H2O2 in the presence of cystine increased fivefold the level of extracellular oxidized glutathione (GSSG(out)) and decreased fivefold the GSH/GSSG(out) ratio (from 16.8 to 3.6). The same treatment of cells with deficiency in glutathione oxidoreductase (GOR) resulted in even more severe oxidation of GSH(out), so that the level of oxidized glutathione exceeded that of reduced glutathione and the GSH/GSSG(out) ratio decreased to 0.4. Addition of cystine to the GOR deficient cells resulted in significant oxidation of extracellular glutathione even in the absence of oxidant and in tenfold increase in intracellular oxidized glutathione along with a decrease in the GSH/GSSG(out) ratio from 282 to 26. However, in the cytoplasm of wild type cells, the level of oxidized glutathione (GSSG(in)) was changed insignificantly and the GSH/GSSG(in) ratio increased by 26% (from 330 to 415). Data on glutathione status and cystine reduction in the E. coli gsh and gor mutants suggested that exogenous cystine at first should be reduced with extracellular GSH outside the cells and then imported into them. The high toxicity of H2O2 in the presence of cystine resulted in disorders of membrane functions and inhibition of the expression of genes including those responsible for neutralization of oxidants and DNA repair.

Role of glutathione in regulation of hydroperoxidase I in growing Escherichia coli.

To examine role of glutathione in regulation of catalases in growing Escherichia coli, katG::lacZ and katE::lacZ fusions were transformed into a glutathione-deficient Escherichia coli strain and wild-type parent. In the absence of H2O2 and in the presence of the low H2O2 concentrations (0.1-3 mM), the gshA mutation stimulated katG::lacZ expression and the total catalase activity in exponential phase. In the absence of H2O2, the mutation in gshA also stimulated katE::lacZ expression. At higher H2O2 concentrations, the gshA mutation suppressed katG::lacZ expression and catalase activity. In stationary and mid-exponential phases, the intracellular concentrations of H2O2 in the gshA mutant were markedly increased compared to those in the wild type. These results suggest that glutathione may be involved in regulation of catalases.

Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli.

Menadione (MD) and H2O2 caused distinct effects on glutathione status in growing Escherichia coli. Treatment of E. coli AB1157 with 1-25 mM H2O2 did not result in an appreciable decrease in intracellular total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]). Only when cells were treated with 25 mM H2O2 an increase in GSSG and a decrease in the GSH:GSSG ratio were observed. In cells deficient in catalase HPI, such effect was observed even at 10 mM H2O2. The exposure of E. coli AB1157 to MD caused a dose-dependent decrease in intracellular total glutathione, an increase in GSSG, and a decrease in the ratio of GSH:GSSG. In E. coli deficient in cytosolic superoxide dismutase activity, a decrease in total glutathione after incubation with 0.2 mM MD was not accompanied by an increase in GSSGin, and the ratio of GSHin:GSSGin was three times higher than in the wild-type cells. The changes in the redox status of extracellular glutathione under the action of both oxidants were similar. Although the catalase activity increased several times after exposure to both oxidants, there were little or no changes in the activity of enzymes related to glutathione metabolism. A possible role of changes in redox status of glutathione under oxidative stress is discussed.

The role of antioxidant enzymes in response of Escherichia coli to osmotic upshift.

Aerobic growth of Escherichia coli sodAsodB and katE mutants lacking cytosolic superoxide dismutases and catalase hydroperoxidase II was inhibited by osmotic upshift to a greater extent than of their wild-type parent strains. The fur mutation leading to an intracellular overload of iron also increased sensitivity of growing E. coli cells to osmotic upshift. Using lacZ fusions, it was shown that expression of antioxidant genes soxS and katE was stimulated by an increase in osmolarity. These data suggest that in aerobically growing E. coli cells, moderate osmotic upshift causes activation of certain antioxidant systems.

Oxidative stress resistance of Escherichia coli strains deficient in glutathione biosynthesis.

Sensitivity to various oxidants was determined for Escherichia coli strains JTG10 and 821 deficient in biosynthesis of glutathione (gsh-) and their common parental strain AB1157 (gsh+). The three strains showed identical sensitivity to H2O2. E. coli 821 was more resistant than AB1157 and JTG10 to menadione, cumene hydroperoxide, and N-ethylmaleimide. This resistance was not related to the gsh mutation because the other gsh- mutant and the parental strain showed similar sensitivity to these oxidants. The measured activities of NADPH:menadione diaphorase and glucose-6-phosphate dehydrogenase and the extracellular level of menadione suggested that the enhanced resistance of E. coli 821 to menadione might be due to decreased diaphorase activity, but not to a lowered rate of menadione uptake.

[Hydrogen peroxide modulates intracellular levels of thiols and potassium in Escherichia coli cells]. / Perekis' vodoroda moduliruet vnutrikletochnye urovni tiolov i kaliia v kletkakh Escherichia coli.

Exposure of growing Escherichia coli K12 cells to 2.0-11.0 mM H2O2 resulted in an increase in the intracellular level of low-molecular-weight thiols (LMWT), whereas exposure to 25 mM H2O2 resulted in its decrease. An inverse correlation between levels of LMWT and potassium was revealed. The treatment of E. coli delta oxyR cells, which are incapable of the adaptive response to H2O2, with 10 mM H2O2 caused a decrease in the LMWT level. In E. coli oxyR2 cells, which constitutively express oxyR-controlled proteins, the same treatment caused a 20% increase in the LMWT level. In response to treatment with the oxidant, delta oxyR mutants lost two times more potassium than wild-type cells (oxyR+). A time course study of the levels of LMWT and potassium in mutants with an affected katG gene, which encodes the HPI catalase and is under the control of oxyR, showed that oxyR may regulate LMWT and potassium levels indirectly, through the regulation of catalase activity. A relationship between catalase activity and the LMWT level was revealed in hydrogen peroxide-treated E. coli cells.

Effects of penetrating and non-penetrating oxidants on Escherichia coli.

Treatment of Escherichia coli K-12 cells aerobically grown in M9 glucose salt medium (H2O2) and non-penetrating (ferricyanide) oxidants resulted in similar inhibition of growth and decrease in intracellular K+ pool by 15%. Only H2O2 inhibited growth of auxotrophic strains grown in M9 medium supplemented with protein hydrolysate. Ferricyanide reduction was associated with decrease in low-molecular-weight thiols, whereas the treatment of cells with H2O2 increased their level. Pretreatment of cells with ferricyanide enhanced the H2O2-induced expression of katG gene encoding for catalase HPI; this gene is a member of the gene family controlled by the oxyR gene. Pretreatment with ferricyanide inhibited H2O2-induced expression of the sfiA gene which is the member of the gene family controlled by the recA and lexA genes. Glutathione is the major low-molecular-weight thiol in E. coli, and it can play different roles in cellular responses to H2O2 and ferricyanide.