Pseudomonas aeruginosa OspR is an oxidative stress sensing regulator that affects pigment production, antibiotic resistance and dissemination during infection

Oxidative stress is one of the main challenges bacteria must cope with during infection. Here, we identify a new oxidative stress sensing and response ospR ( o xidative s tress response and p igment production R egulator) gene in Pseudomonas aeruginosa . Deletion of ospR leads to a significant induction in H₂O₂ resistance. This effect is mediated by de-repression of PA2826 , which lies immediately upstream of ospR and encodes a glutathione peroxidase. Constitutive expression of ospR alters pigment production and β-lactam resistance in P. aeruginosa via a PA2826 -independent manner. We further discovered that OspR regulates additional genes involved in quorum sensing and tyrosine metabolism. These regulatory effects are redox-mediated as addition of H₂O₂ or cumene hydroperoxide leads to the dissociation of OspR from promoter DNA. A conserved Cys residue, Cys-24, plays the major role of oxidative stress sensing in OspR. The serine substitution mutant of Cys-24 is less susceptible to oxidation in vitro and exhibits altered pigmentation and β-lactam resistance . Lastly, we show that an ospR null mutant strain displays a greater capacity for dissemination than wild-type MPAO1 strain in a murine model of acute pneumonia. Thus, OspR is a global regulator that senses oxidative stress and regulates multiple pathways to enhance the survival of P. aeruginosa inside host.     

Mutation of rpoS gene decreased resistance to environmental stresses, synthesis of extracellular products and virulence of Vibrio anguillarum

Vibrio anguillarum is a gram-negative halophilic bacterium that causes vibriosis in marine fish, freshwater fish and other aquatic animals. Bacteria have developed strategies to survive in harsh environments. The alternative σ factor, RpoS (σ^S), plays a key role in surviving under stress conditions in some gram-negative bacteria. An rpoS mutant of pathogenic V. anguillarum W-1 was constructed by homologous recombination. The sensitivity of the rpoS mutant to osmotic stress [2.4 M NaCl in artificial seawater (ASW)] did not change obviously, but the sensitivity of the rpoS mutant to high temperature (45 °C in ASW), UV-irradiation and oxidative stress (5 mM H₂O₂ in ASW) increased 33-fold, sixfold and 10-fold, respectively. The production of extracellular phospholipase, diastase, lipase, caseinase, hemolysin, catalase and protease of the rpoS mutant decreased markedly compared with those of the wild-type strain. Virulence of the rpoS mutant strain was also decreased when it was inoculated intraperitoneally into zebra fish; the lethal dose 50% of the wild type and the mutant was 8.66 × 10⁴ and 2.55 × 10⁶ CFU per fish, respectively. These results indicated that the RpoS of V. anguillarum plays important roles in bacterial adaptation to environmental stresses and its pathogenicity.     

Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase

Aims:  Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H₂O₂ concentration that is low compared to current nonenzymatic formulations.
Methods and Results:  Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus ) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l⁻¹ H₂O₂ and 100 mmol l⁻¹ Br⁻ at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme.
Conclusions:  The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions.
Significance and Impact of the Study:  For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.     

Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides

Aims:  Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H₂O₂) and stabilized H₂O₂ combination products.
Methods and Results:  The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H₂O₂ combined with silver possessed a higher bactericidal and fungicidal activity compared to native H₂O₂ with and without glycerol.
Conclusions:  Validation showed that the RMM may replace the PCCT. When applying the RMM, H₂O₂ combined with silver was clearly a more potent disinfectant compared to H₂O₂ in killing bacteria and fungi.
Significance and Impact of the Study:  The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H₂O₂ with silver may replace native H₂O₂ to increase overall disinfection efficiency.     

Fusarium response to oxidative stress by H2O2 is trichothecene chemotype-dependent

The present study aims at clarifying the impact of oxidative stress on type B trichothecene production. The responses to hydrogen peroxide (H₂O₂) of an array of Fusarium graminearum and Fusarium culmorum strains were compared, both species carrying either the chemotype deoxynivalenol (DON) or nivalenol (NIV). In both cases, levels of in vitro toxin production are greatly influenced by the oxidative parameters of the medium. A 0.5 mM H₂O₂ stress induces a two- to 50-fold enhancement of DON and acetyldeoxynivalenol production, whereas the same treatment results in a 2.4- to sevenfold decrease in NIV and fusarenone X accumulation. Different effects of oxidative stress on toxin production are the result of a variation in Fusarium 's antioxidant defence responses according to the chemotype of the isolate. Compared with DON strains, NIV isolates have a higher H₂O₂-destroying capacity, which partially results from a significant enhancement of catalase activity induced by peroxide stress. A 0.5 mM H₂O₂ treatment leads to a 1.3- to 1.7-fold increase in the catalase activity of NIV isolates. Our data, which show the higher adaptation to oxidative stress developed by NIV isolates, are consistent with the higher virulence of these Fusarium strains on maize compared with DON isolates.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Elicitation of H2O2 production in cucumber hypocotyl segments by oligo-1,4-α-D-galacturonides and an oligo-β-glucan preparation from cell walls of Phythophthora megasperma f. sp. glycinea

The production of H₂O₂ by cucumber hypocotyl segments ( Cucumis sativus L. cv. Wisconsin SMR 58) in response to α-1,4-linked oligomers of galacturonic acid and oligo-β-glucans from the cell walls of Phytophthora megasperma f. sp. glycinea was studied. Oligogalacturonides with degrees of polymerization of 9 to 13 elicited H₂O₂ production, the most effective being the deca-, undeca- and dodecamers. A similar relationship between size and effect was previously obtained when oligogalacturonides were tested for their ability to elicit lignification in cucumber hypocotyls. The oligogalacturonide-induced increase in H₂O₂ concentration was detected after 4 h, reaching a maximum after 10 h of incubation. The glucan elicitor induced lignification at a 100-fold lower concentration than the oligogalacturonides, but yielded only 10% of the maximum H₂O₂ accumulation seen with oligogalacturonides. The glucan elicitor-induced H₂O₂ production was detectable after 2 h, and reached a maximum after 4 to 6 h. Catalase abolished the elicitation of both phenol red oxidation and lignification in cucumber hypocotyls. At least part of the oligogalacturonide-induced H₂O₂ production appeared to be dependent upon de novo protein synthesis.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Catalase inhibition alters suberization and wound healing in potato (Solanum tuberosum) tubers

In response to wounding, potato ( Solanum tuberosum L.) tubers generate hydrogen peroxide (H₂O₂) in association with suberization, a critical phase of the wound-healing process. In the present study, the effect of aminotriazole (AT), a catalase (CAT, EC 1.11.1.6) inhibitor, on cut tubers was investigated using fresh weight (FW) loss and pathogen attack symptoms as indicators of wound-healing efficiency. Seven days after treatment, AT-treated tuber halves lost more FW and developed infection signs compared with the controls. Thiourea, another CAT inhibitor, as well as exogenous H₂O₂ treatments induced the same effects as AT suggesting that the alteration of the wound healing may be caused by CAT inhibition and the resulting accumulation of H₂O₂. Using transgenic tubers, FW losses 1 week after wounding were either higher (CAT repression) or lower (CAT overexpression) than those of the wild-type. When tuber halves were allowed to wound heal for different periods before treatment, AT had no effect on the progress of their wound healing if wound-healed for at least 3 days. This implies that AT may affect early wound-healing-related events, especially those occurring before or during suberization. A time-course analysis of the effects of AT treatment on wounded tuber tissues revealed that AT prevented the deposition of the polyphenolic domain of suberin in association with CAT inhibition and H₂O₂ accumulation. These data are important in identifying factors that may be required to regulate suberization and contribute to a better understanding of this critical process to hasten its rate and limit wound-related losses in stored potato tubers.     

Infection biology and defence responses in sorghum against Colletotrichum sublineolum

Aims:  To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes.
Methods and Results:  Infection biology and defence responses were studied quantitatively by light microscopy, H₂O₂ accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H₂O₂, HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H₂O₂ accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration.
Conclusions:  The early accumulation of H₂O₂ and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis.
Significance and Impact of the Study:  The fact that there is a significant correlation between induced accumulation of H₂O₂, papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.     

Antioxidative protection in a drought-resistant maize strain during leaf senescence

Leaves of 7- and 18-day-old plants of two maize strains, one resistant (LIZA) and one sensitive (LG11) to water stress, were floated in 1 m M paraquat and 1 m M H₂O₂ for 12 h in light and in darkness. The aim of this work was to analyse the effects of these substances on the activities of enzymes involved in the scavenging of active oxygen species during senescence. Three senescence parameters; chlorophyll loss, lipid peroxidation and conductivity; showed a general cell damage caused by both oxidative treatments and revealed a higher tolerance of LIZA than LG11 to paraquat and H₂O₂ both in light and in darkness. Activities of antioxidative enzymes increased by the effect of oxidative treatments in young and senescent leaves of the drought-resistant maize strain LIZA. These increases were about 3-to 6-fold in glutathione reductase. 3-to 4-fold in superoxide dismutase and 2-fold in ascorbate peroxidase activities. The possible correlation between water stress resistance. senescence and the potential of antioxidant enzymes was analysed.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Effect of carbohydrate and nitrogen on hydrogen peroxide formation by wood decay fungi in solid medium

Abstract Hydrogen peroxide (H₂O₂) has been implicated in degradation of wood by both brown-rot and white-rot fungi. This study found that low concentrations of nitrogem and carbohydrates (cellobiose, glucose, xylose and mannose) in an agar medium had little effect on H₂O₂ production by white-rot fungi. However, low concentrations of nitrogen and carbohydrates stimulated H₂O₂ production by brown-rot fungi. Use of the chromogen 2,2'-azino-di(3-ethyl benzthiazoline-6-sulphonic acid) (ABTS) with horseradish peroxidase to detect H₂O₂ by the fungi was slightly better than detection by the chromogen o -dianisidine with horseradish peroxidase. An auxiliary test to check the role of H₂O₂ in wood decay found that hydrogen peroxide-negative isolates of the white-rot fungi Pharnerochaete chrysosporium and Ganoderma applanatum were unable to decay sweetgum and southern pine.     

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H₂O₂ in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H₂O₂ production started after a lag time of 30–40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H₂O₂ did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H₂O₂ (0.1–1 m M ). These results suggest that H₂O₂ production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H₂O₂ is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.