Green tea polyphenols function as prooxidants to inhibit Pseudomonas aeruginosa and induce the expression of oxidative stress-related genes

Green tea polyphenols (GTP) are widely believed to function as antioxidants and antimicrobial agents. Here we observed that GTP and epigallocatechin gallate, the most abundant catechin in GTP, could also function as prooxidants and produce hydrogen peroxide (H₂O₂) to inhibit the growth of Pseudomonas aeruginosa. pH value of the medium was the key factor that affected prooxidant versus antioxidant property of GTP. Under weakly acidic conditions (pH 5.5–6.5), GTP showed antioxidant activity by eliminating H₂O₂; whereas, under neutral and weakly alkaline conditions (pH 7.0–8.0), GTP showed prooxidant activity and inhibited the growth of P. aeruginosa. Furthermore, we studied the effects of GTP on gene expression profiles of a few oxidative stress-related genes by quantitative real-time PCR analysis. After 10 min to 1 h of exposure under weakly alkaline condition, GTP significantly up-regulated expression levels of katB, sodM, ohr, lexA, and recN gene. These findings highlight that the pH-dependent H₂O₂ production by GTP contributes to the antibacterial activity and can induce oxidative stress-related responses in P. aeruginosa.     

Response of antioxidant enzymes to high NaCl concentration in different salt-tolerant plants

The effects of NaCl on the H₂O₂ content and the activities of catalase (CAT) and superoxide dismutase (SOD) were studied in diverse group of plants, such as a unicellular alga, Chlorella sp., an aquatic macrophyte, Najas graminea, and a mangrove plant, Suaeda maritima, all showing high tolerance to NaCl. Significant accumulation of H₂O₂ was observed in all the tested plants upon their exposure to 255 mM NaCl. The activity of both CAT and SOD increased significantly in response to the NaCl treatment. Growing the plants in presence of 255 mM NaCl also resulted in the synthesis of new isoforms of both CAT and SOD.     

Pseudomonas aeruginosa MdaB and WrbA are water-soluble two-electron quinone oxidoreductases with the potential to defend against oxidative stress

Water-soluble quinone oxidoreductases capable of reducing quinone substrates via a concerted two-electron mechanism have been implicated in bacterial antioxidant defence. Twoelectron transfer avoids formation of dangerously reactive semi-quinone intermediates, moreover previous work in Pseudomonas putida indicated a direct protective effect for the quinols generated by an over-expressed oxidoreductase. Here, the Pseudomonas aeruginosa orthologs of five quinone oxidoreductases — MdaB, ChrR, WrbA, NfsB, and NQO1 — were tested for their possible role in defending P. aeruginosa against H₂O₂ challenge. In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. However, when each was individually over-expressed in P. aeruginosa no overt H₂O₂-protective phenotype was observed. It was shown that this was due to a masking effect of the P. aeruginosa catalase, KatA; in a katA mutant, H₂O₂ challenged strains over-expressing the WrbA and MdaB orthologs grew significantly better than the empty plasmid control. A growth advantage was also observed for H₂O₂ challenged P. putida strains over-expressing P. aeruginosa wrbA, mdaB or katA. Despite not conferring a growth advantage to wild type P. aeruginosa, it is possible that these quinone oxidoreductases defend against H₂O₂ toxicity at lower concentrations.     

Induction of Acid Resistance of Salmonella typhimurium by Exposure to Short-Chain Fatty Acids

Exposure to short-chain fatty acids (SCFA) is one of the stress conditions Salmonella typhimurium encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals. The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of S. typhimurium. The percent survival of S. typhimurium at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure. The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance. The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (P < 0.05). The results suggest that the virulence of S. typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.     

Response of Barley Seedlings to UV-B Radiation as Affected by Proline and NaCl

Barley (Hordeum vulgare L. cv. Alfa) seedlings were treated for 4 d before UV-B irradiation with 0.05 mM proline or 150 mM NaCl. UV-B exposure induced synthesis of yellow coloured compounds with maximum absorbance at 438 nm. The content of these compounds was increased in proline-treated and decreased in NaCl-treated plants. UV-B radiation reduced chlorophyll/carotenoids ratio, oxygen evolution rate and photochemical efficiency of PS 2 as estimated by chlorophyll fluorescence and increased proline accumulation, H₂O₂ generation and lipid peroxidation. Exogenous proline had no effect on the parameters studied and did not change the response of plants to UV-B radiation. NaCl inhibited photochemical efficiency of PS 2, reduced oxygen evolution and increased H₂O₂ concentration and lipid peroxidation. The combination of NaCl and proline treatment led to lowering the inhibitory effect of NaCl in non UV-B irradiated seedlings. There was not relationship between the level of UV-B-induced compounds and UV-B tolerance of barley seedlings.     

siRNA as a tool to delineate pathway channelization in H2O2 induced apoptosis of primary Leydig cells in vitro

Using siRNA as a tool, the channelization of pathway in H₂O₂ induced apoptosis of primary Leydig cells was investigated in vitro. Exposure (4?h) to H₂O₂ (250?μM) induced maximum apoptosis but affected Leydig cell viability significantly. Therefore, expression of apoptotic marker genes, caspase-8, -9, -3 and polyadenosine ribose polymerase was subsequently investigated using the same concentration post 1?h exposure. Incubation with siRNA (20?nM) either for caspase-8 or -9, inhibited their individual expressions by 55–60?% and activity, 50–55?%. The inhibition efficiency using siRNA was comparable with post- or pre-H₂O₂ treatment of cells. Like siRNA, Eugenia jambolana (100?μg/ml) plant extract too, effectively countered over-expression of all apoptotic marker proteins. Silencing expressions of caspase 8 but not 9 through siRNA leads to a profound inhibition of caspase 3 implying that H₂O₂ induced Leydig cell apoptosis is preferably channeled through extrinsic and later extending to other pathways.     

Responses of marine macroalgae to hydrogen-peroxide stress

In this study, we determined the antioxidative potential of 15 marine macroalgae by measuring the photosynthetic efficiency under artificial oxidative stress after a 30-min exposure to a series of ascending H₂O₂ concentrations. Species exhibiting high maximum quantum yields (F_v/F_m values) were regarded as not susceptible towards H₂O₂ stress. In addition to the short-term stress experiments, the antioxidative defense systems (enzymatic and non-enzymatic) of selected algal species under longer exposure times to H₂O₂ were investigated.Species with striking photosynthetic activity under H₂O₂ stress were Chaetomorpha melagonium (Chlorophyta), showing 40% reduced F_v/F_m as compared to the control after 8 days of exposure to 20 mM H₂O₂. In Fucus distichus (Phaeophyta) F_v/F_m decreased to 50% of the control under the same exposure conditions. Polysiphonia arctica (Rhodophyta) exhibited highest F_v/F_m values with a reduction of only 25%, therefore possessing the highest antioxidative potential of the investigated species.In P. arctica the activities of the antioxidative enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as the pool size of the antioxidant ascorbic acid were investigated. When exposed to different H₂O₂ concentrations (0-2 mM) over 6 days, the intrinsic activities of SOD and GR were stimulated. In a kinetic study over 8 days, the activity of antioxidative enzymes APX and CAT as well as ascorbic acid content were recorded. APX activity was much higher in H₂O₂-treated thalli at the end of the experiment than in the control, also CAT activity increased significantly with increasing H₂O₂ stress. In parallel, ascorbic acid content was reduced under high H₂O₂ concentrations. Furthermore, by using GC-MS techniques in P. arctica bromophenolic compounds with antioxidative properties were identified.This study shows that the measurement of the in vivo fluorescence of photosystem II is a suitable tool to determine the effect of oxidative stress on macroalgae. From these studies it is obvious that different algal species have varying strategies against oxidative stress which correlate with zonation on the shore.     

Stress influenced the aerotolerance of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> hsryfm 1301

Objective

To investigate the aerotolerance of Lactobacillus rhamnosus hsryfm 1301 and its influencing factors.

Results

The growth rate of L. rhamnosus hsryfm 1301 weakened noticeably when the concentration of supplemented H₂O₂ reached 1 mM, and only 2% of all L. rhamnosus hsryfm 1301 cells survived in MRS broth supplemented with 2 mM H₂O₂ for 1 h. After pretreatment with 0.5 mM H₂O₂, the surviving cells of L. rhamnosus hsryfm 1301 in the presence of 5 mM H₂O₂ for 1 h increased from 3.7 to 7.8 log CFU. Acid stress, osmotic stress, and heat stress at 46 °C also enhanced its aerotolerance, while heat stress at 50 °C reduced the tolerance of L. rhamnosus hsryfm 1301 to oxidative stress. Moreover, treatment with 0.5 mM H₂O₂ increased the heat stress tolerance of L. rhamnosus hsryfm 1301 by approximately 150-fold.

Conclusions

Lactobacillus rhamnosus hsryfm 1301 possesses a stress-inducible defense system against oxidative stress, and the cross-adaptation to different stresses is a promising target to increase the stress tolerance of L. rhamnosus hsryfm 1301 during probiotic food and starter culture production.

     

Essential roles of core starvation-stress response loci in carbon-starvation-inducible cross-resistance and hydrogen peroxide-inducible adaptive resistance to oxidative challenge in Salmonella typhimurium

The starvation-stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g. C-source. A subset of SSR genes, known as core SSR genes, are required for the long-term starvation survival of the bacteria. Four core SSR loci have been identified in S. typhimuriumrpoSstiAstiB, and stiC. Here we report that in S. typhimurium C-starvation induced a greater and more sustainable cross-resistance to oxidative challenge (15 mM hydrogen peroxide (H₂O₂) for 40 min) than either N- or P-starvation. Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C-starvation-inducible cross-resistance to H₂O₂ challenge. Interestingly, (unadapted) log-phase S. typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge. Based on this, we determined if these core SSR loci were important for H₂O₂ resistance developed during a 60 min adaptive exposure to 60 μM H₂O₂ (adapted cells). Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H₂O₂ challenge. In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H₂O₂ challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild-type strain. The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C-starvation-inducible cross-resistance to oxidative challenge, and (ii) the rpoSstiA, and, in a delayed effect, stiB loci are needed for H₂O₂-inducible adaptive resistance to oxidative challenge. Moreover, we found that both stiA and stiB are induced by a 60 μM H₂O₂ exposure, but only stiA was regulated (repressed) by (reduced form) OxyR.     

Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H₂O₂) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H₂O₂ was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 μM) of H₂O₂. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.     

Influence of polyphenols on Escherichia coli resistance to oxidative stress

Among all polyphenols tested (tannic acid and flavonoids belonging to different subclasses) only tannin and quercetin significantly enhanced resistance of Escherichia coli to peroxide stress. Pretreatment of the cells with quercetin and tannin resulted in a decrease in the growth arrest duration under moderate H₂O₂ concentration (2 mM) and an increase in survival under high (10 mM) doses. The shorter growth recovery period in pretreated cells was connected with more rapid H₂O₂ elimination because of induced activity of scavenging enzymes. This effect was absent in the ΔoxyR mutant, which was unable to induce genes responding to peroxide stress. The data obtained suggest that the observed protection was a result of two overlapping effects: induction of OxyR regulon by low concentrations of H₂O₂, accumulated during extracellular autoxidation of quercetin and tannin, and protection of synthesis of OxyR-regulated antioxidant enzymes during H₂O₂ stress because of intracellular binding of iron by quercetin and tannin and suppressing Fenton chemistry.     

Exogenous Selenium Pretreatment Protects Rapeseed Seedlings from Cadmium-Induced Oxidative Stress by Upregulating Antioxidant Defense and Methylglyoxal Detoxification Systems

The protective effect of selenium (Se) on antioxidant defense and methylglyoxal (MG) detoxification systems was investigated in leaves of rapeseed (Brassica napus cv. BINA sharisha 3) seedlings under cadmium (Cd)-induced oxidative stress. Two sets of 11-day-old seedlings were pretreated with both 50 and 100???M Se (Na₂SeO₄, sodium selenate) for 24?h. Two concentrations of CdCl₂ (0.5 and 1.0?mM) were imposed separately or on the Se-pretreated seedlings, which were grown for another 48?h. Cadmium stress at any levels resulted in the substantial increase in malondialdehyde and H₂O₂ levels. The ascorbate (AsA) content of the seedlings decreased significantly upon exposure to Cd stress. The amount of reduced glutathione (GSH) increased only at 0.5?mM CdCl₂, while glutathione disulfide (GSSG) increased at any level of Cd, with concomitant decrease in GSH/GSSG ratio. The activities of ascorbate peroxidase (APX) and glutathione S-transferase (GST) increased significantly with increased concentration of Cd (both at 0.5 and 1.0?mM CdCl₂), while the activities of glutathione reductase (GR) and glutathione peroxidase (GPX) increased only at moderate stress (0.5?mM CdCl₂) and then decreased at 1.0?mM severe stress (1.0?mM CdCl₂). Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon exposure to any levels of Cd. Selenium pretreatment had little effect on the nonenzymatic and enzymatic components of seedlings grown under normal conditions; i.e., they slightly increased the GSH content and the activities of APX, GR, GST, and GPX. On the other hand, Se pretreatment of seedlings under Cd-induced stress showed a synergistic effect; it increased the AsA and GSH contents, the GSH/GSSG ratio, and the activities of APX, MDHAR, DHAR, GR, GPX, CAT, Gly I, and Gly II which ultimately reduced the MDA and H₂O₂ levels. However, in most cases, pretreatment with 50???M Se showed better results compared to pretreatment with 100???M Se. The results indicate that the exogenous application of Se at low concentrations increases the tolerance of plants to Cd-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Stability of crude ligninases: Effect of different hydrogen peroxide concentrations in the presence of aromatic alcohols

Summary In absence of veratryl alcohol (VA),Phanerochaete chrysosporium ligninases were extensively inactivated by H₂O₂ concentrations as low as 5.0 μM (1 hr exposure time, pH 4.5, 38°C). In the presence of 2.5 mM VA (but not 2.5 mM benzyl alcohol), protection occurred below 500 μM H₂O₂.     

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M?=?0.6?±?0.2 and 1.8?±?0.5?mM H₂O₂, respectively), with similar turnover numbers (k_cat?=?19?±?2 and 13?±?2?s^?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.     

Prothrombin and fibrinogen carbonylation: How that can affect the blood clotting

Objectives: The aim of the work was the development of a simple method for measuring the plasma prothrombin carbonylation and the study the impact of prothrombin and fibrinogen oxidation on the rate of plasma clotting.

Methods: A new method was based on the ability of prothrombin to be adsorbed by the barium sulfate. It consists of four steps: prothrombin mixing with the water suspension of BaSO₄; reaction of 2,4-dinitrophenylhydrazine with the BaSO₄-bound prothrombin; desorption of prothrombin-2,4-dinitrophenylhydrazone complex from BaSO₄ in an alkaline medium; neutralization and reading of the optical absorbance of the complex (λ?=?370?nm). The prothrombin/fibrinogen carbonylation and plasma clotting rate in vitro in the presence of reactive oxygen species (ROS)-generating agents (0.05–0.8?mM Fe²⁺/H₂O₂) were monitored.

Results: The plasma volume required for measurement of carbonylated prothrombin was 0.4?ml. High level of linearity and reproducibility was observed (r?=?0.9995, P?=?0.0005 – for the protein; r?=?0.9971, P?=?0.0029 – for carbonyls). In the intact rats, the concentration of blood plasma prothrombin was 0.355?±?0.009?mg/ml, and that of carbonyls was 4.94?±?0.09?nmol/mg.

Discussion: Prothrombin and plasma clotting rate was not affected by low concentrations of ROS (0.05–0.2?mM Fe²⁺/H₂O₂). The fibrinogen was susceptible to ROS-related effect over all the used range of concentration (0.05–0.8?mM Fe²⁺/H₂O₂). Carbonylation of fibrinogen did not affect the plasma clotting activity at low ROS concentration (0.05–0.2?mM Fe²⁺/H₂O₂), however it retarded the clotting at higher ROS (0.2–0.8?mM Fe²⁺/H₂O₂).