Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora

Erwinia amylovora , the causal agent of fire blight, is an enterobacterial pathogen of Rosaceous plants including apple and pear. We have been studying the response of E. amylovora to oxidative stress because, during infection, the bacterium elicits an oxidative burst response in host plants. During the screening of a transposon mutant library for hydrogen peroxide sensitivity, we identified a mutant carrying an insertion in waaL , a gene involved in lipopolysaccharide biosynthesis, that was more sensitive to hydrogen peroxide than the parental wild-type strain. We also confirmed that a waaL mutant of Pseudomonas aeruginosa exhibited an increased sensitivity to hydrogen peroxide compared with the wild-type strain. The E. amylovora waaL mutant was also reduced in virulence, showed a decrease in twitching motility, and was more sensitive to polymyxin B than the wild type. Each of these phenotypes was complemented by the cloned waaL gene. Our results highlight the importance of the lipopolysaccharide layer to virulence in E. amylovora and the unexpected finding of an additional function of lipopolysaccharide in protection from oxidative stress in E. amylovora and P. aeruginosa .     

Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.     

Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions

Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress. We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase. The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway. Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide. Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide.     

The Campylobacter jejuni thiol peroxidases Tpx and Bcp both contribute to aerotolerance and peroxide-mediated stress resistance but have distinct substrate specificities

The microaerophilic food-borne pathogen Campylobacter jejuni experiences variable oxygen concentrations during its life cycle, especially during transitions between the external environment and the avian or mammalian gut. Single knockout mutations in either one of two related thiol peroxidase genes, tpx and bcp, resulted in normal microaerobic growth (10% [vol/vol] oxygen) but poorer growth than that of the wild type under high-aeration conditions (21% [vol/vol] oxygen). However, a tpx/bcp double mutant had a severe microaerobic growth defect and did not grow at high aeration in shake flasks. Although the single mutant strains were no more sensitive than the wild-type strains in disc diffusion assays with hydrogen peroxide, organic peroxides, superoxide, or nitrosative stress agents, in all cases the double mutant was hypersensitive. Quantitative cell viability and cellular lipid peroxidation assays indicated some increased sensitivity of the single tpx and bcp mutants to peroxide stress. Protein carbonylation studies revealed that the tpx/bcp double mutant had a higher degree of oxygen- and peroxide-induced oxidative protein damage than did either of the single mutants. An analysis of the peroxidase activity of the purified recombinant enzymes showed that, surprisingly, Tpx reduced only hydrogen peroxide as substrate, whereas Bcp also reduced organic peroxides. Immunoblotting of wild-type cell extracts with Tpx- or Bcp-specific antibodies showed increased abundance of both proteins under high aeration compared to that under microaerobic growth conditions. Taken together, the results suggest that Tpx and Bcp are partially redundant antioxidant enzymes that play an important role in protection of C. jejuni against oxygen-induced oxidative stress.     

Adipokinetic hormone counteracts oxidative stress elicited in insects by hydrogen peroxide: in vivo and in vitro study

The role of adipokinetic hormone (AKH) in counteracting oxidative stress elicited in the insect body is studied in response to exogenously applied hydrogen peroxide, an important metabolite of oxidative processes. In vivo experiments reveal that the injection of hydrogen peroxide (8 µmol) into the haemocoel of the firebug, Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae) increases the level of AKH by 2.8‐fold in the central nervous system (CNS) and by 3.8‐fold in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental insects, whereas co‐injection of hydrogen peroxide with Pyrap‐AKH (40 pmol) reduces mortality to almost control levels. Importantly, an increase in haemolymph protein carbonyl levels (i.e. an oxidative stress biomarker) elicited by hydrogen peroxide is decreased by 3.6‐fold to control levels when hydrogen peroxide is co‐injected with Pyrap‐AKH. Similar results are obtained using in vitro experiments. Oxidative stress biomarkers such as malondialdehyde and protein carbonyls are significantly enhanced upon exposure of the isolated CNS to hydrogen peroxide in vitro, whereas co‐treatment of the CNS with hydrogen peroxide and Pyrap‐AKH reduces levels significantly. Moreover, a marked decrease in catalase activity compared with controls is recorded when the CNS is incubated with hydrogen peroxide. Incubation of the CNS with hydrogen peroxide and Pyrap‐AKH together curbs the negative effect on catalase activity. Taken together, the results of the present study provide strong support for the recently published data on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting oxidative stress. The in vitro experiments should facilitate research on the mode of action of AKH in relation to oxidative stress, and could help clarify the key pathways involved in this process.     

Growth deficiency of a Xanthomonas oryzae pv. oryzae fur mutant in rice leaves is rescued by ascorbic acid supplementation

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. A mutation was isolated in the ferric uptake regulator (fur) gene of X. oryzae pv. oryzae and it was shown to result in the production of siderophores in a constitutive manner. The fur mutant is hypersensitive to the metallo-antibiotic streptonigrin, a phenotype that is indicative of intracellular free-iron overload, and also exhibits a slow growth phenotype on rich medium. The fur mutant is virulence deficient, hypersensitive to hydrogen peroxide, and exhibits reduced catalase activity. Exogenous supplementation with ascorbic acid (an antioxidant) rescues the growth deficiency of the fur mutant in rice leaves. The virulence deficiency of the X. oryzae pv. oryzae fur mutant is proposed to be due, at least in part, to an impaired ability to cope with the oxidative stress conditions that are encountered during infection.     

Deletion of all Cochliobolus heterostrophus monofunctional catalase-encoding genes reveals a role for one in sensitivity to oxidative stress but none with a role in virulence

The genome of the maize pathogen Cochliobolus heterostrophus encodes three unlinked monofunctional catalase-encoding (CAT) genes that singly or in combination could offer protection against the harmful effects of oxidative stress. Phylogenetic analysis placed the CAT2 and CAT3 proteins in a cluster with large subunit catalases (CAT3 has a secretory signal sequence and was grouped with known secreted catalases), whereas CAT1 clustered with small subunit catalases. Single, double, and triple cat mutants were created and screened for sensitivity to hydrogen peroxide and altered virulence on maize. All mutants deficient in CAT3 had enhanced sensitivity to hydrogen peroxide, as compared with wild type or with mutants deficient in CAT1, CAT2, or both. All catalase-deficient mutants had normal virulence to maize. Thus, the secreted CAT3 protein protects the fungus from oxidative stress during vegetative growth, but members of this enzyme family, alone or in combination, are not essential for virulence.     

Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis. To survive within the host, this organism must be able to protect itself from oxidative stress. Cytochrome c peroxidase (Ccp1) is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide. In the present study, we characterized the contribution of the C. neoformans Ccp1 to antioxidant defense and for virulence. Consistent with studies of Ccp1 function in Sacchromyces cerevisiae, we found that Ccp1 contributes to resistance against exogenous oxidative stress in vitro. However, the oxidative stress phenotype does not diminish the virulence of ccp1 mutant strains in a murine model of C. neoformans disease. These results suggest that Ccp1 is involved in a complex system of protection against exogenous oxidative stress and that the elimination of this component of the antioxidant defense system does not diminish the virulence of C. neoformans.     

Virulence attenuation of <Emphasis Type="Italic">Streptococcus pneumoniae clpP</Emphasis> mutant by sensitivity to oxidative stress in macrophages via an NO-mediated pathway

ClpP protease is essential for virulence and survival under stress conditions in several pathogenic bacteria. The clpP mutation in a murine infection model has demonstrated both attenuation of virulence and a sensitivity to hydrogen peroxide. However, the underlying mechanisms for these changes have not been resolved. Because macrophages play a major role in immune response and activated macrophages can kill microbes via oxygen-dependant mechanisms, we investigated the effect of the clpP mutation on its sensitivity to macrophage-mediated oxygen-dependant mechanisms. The clpP mutant derived from D39 (serotype 2) exhibited a higher sensitivity to oxidative stresses such as reactive oxygen intermediates, reactive nitrogen intermediates, and H₂O₂, but no sensitivity to osmotic stress (NaCl) and pH. Moreover, viability of the clpP mutant was significantly increased in murine macrophage cells by treatment with S-methylisothiourea sulfate, which inhibits inducible nitric oxide synthase (iNOS) activity and subsequently elicits lower level secretions of nitric oxide (NO). However, viability of wild type was unchanged. Taken together, these results indicate that ClpP is involved in the resistance to oxidative stresses after entrapment by macrophages and subsequently contributes to virulence via NO mediated pathway.     

The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum

In this study, we analyzed the whcA gene from Corynebacterium glutamicum , which codes for a homologue of the WhiB-family of proteins. Deletion of the gene did not affect the growth of the mutant cells, indicating that the whcA gene was not essential under ordinary growth conditions. However, cells overexpressing the protein not only showed retarded growth as compared with the wild-type or the Δ whcA mutant cells but also showed increased sensitivity to a variety of oxidants, such as diamide, menadione, and hydrogen peroxide. Thioredoxin reductase activity was repressed in the whcA -overexpressing cells, whereas its activity in the Δ whcA mutant strain was derepressed regardless of the presence of oxidative stress. The whcA gene was constitutively expressed throughout the growth phase and its expression level was not affected by oxidative stress. A set of proteins under the control of whcA were identified by two-dimensional polyacrylamide gel electrophoresis and they were annotated as NADH oxidase, alcohol dehydrogenase, quinone reductase, and cysteine desulfurase. The corresponding genes encoding the identified proteins were not transcribed in Δ sigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH -mediated stress response pathway.     

Staphylococcus aureus CymR is a new thiol-based oxidation-sensing regulator of stress resistance and oxidative response

As a human pathogen, Staphylococcus aureus must cope with oxidative stress generated by the human immune system. Here, we report that CymR utilizes its sole Cys-25 to sense oxidative stress. Oxidation followed by thiolation of this cysteine residue leads to dissociation of CymR from its cognate promoter DNA. In contrast, the DNA binding of the CymRC25S mutant was insensitive to oxidation and thiolation, suggesting that CymR senses oxidative stress through oxidation of its sole cysteine to form a mixed disulfide with low molecular weight thiols. The determined crystal structures of the reduced and oxidized forms of CymR revealed that Cys-25 is oxidized to Cys-25-SOH in the presence of H(2)O(2). Deletion of cymR reduced the resistance of S. aureus to oxidative stresses, and the resistance was restored by expressing a C25S mutant copy of cymR. In a C25S substitution mutant, the expression of two genes, tcyP and mccB, was constitutively repressed and did not respond to hydrogen peroxide stress, whereas the expression of the genes were highly induced under oxidative stress in a wild-type strain, indicating the critical role of Cys-25 in redox signaling in vivo. Thus, CymR is another master regulator that senses oxidative stress and connects stress responses to virulence regulation in S. aureus.     

Role of the Siderophore Transporter SirABC in the Staphylococcus aureus Resistance to Oxidative Stress

In Staphylococcus aureus, the intracellular siderophore staphyloferrin B, which has been shown to chelate iron-bound to serum transferrin, is transported into cells by the SirABC system. In this work, we have analysed the role of the Sir transporter under stress conditions that resemble those imposed by the mammalian innate immune system. We show that exposure of S. aureus to oxidative and nitrosative stress generated by hydrogen peroxide and S-nitrosoglutathione, respectively, induced the expression of the sirA gene. The disruption of the sir operon led to a strain with lower viability and decreased resistance to oxidative stress. S. aureus sir null mutant was also analysed during infection of murine macrophages and shown to contribute to S. aureus survival inside macrophages. Altogether, our results indicate that the Sir transport system confers protection against reactive oxygen species, therefore, contributing to the virulence of S. aureus.     

Molecular analysis of the psa permease complex of Streptococcus pneumoniae

The psaBCA locus of Streptococcus pneumoniae encodes a putative ABC Mn2+-permease complex. Downstream of the operon is psaD, which may be co-transcribed and encodes a thiol peroxidase. Previously, there has been discordance concerning the phenotypic impact of mutations in the psa locus, resolution of which has been complicated by differences in mutant construction and the possibility of polar effects. Here, we constructed unmarked, in frame deletion mutants DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaD, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD in S. pneumoniae D39 to examine the role of each gene within the locus in Mn2+ uptake, susceptibility to oxidative stress, virulence, nasopharyngeal colonization and chain morphology. The requirement for Mn2+ for growth and transformation was also investigated for all mutants. Inductively coupled plasma mass spectrometry (ICP-MS) analysis provided the first direct evidence that PsaBCA is indeed a Mn2+ transporter. However, this study did not substantiate previous reports that the locus plays a role in choline-binding protein pro-duction or chain morphology. We also confirmed the importance of the Psa permease in systemic virulence and resistance to superoxide and hydrogen peroxide, as well as demonstrating a role in nasopharyngeal colonization for the first time. Further evi-dence is provided to support the requirement for Mn2+ supplementation for growth and transformation of DeltapsaB, DeltapsaC, DeltapsaA, DeltapsaBC, DeltapsaBCA and DeltapsaBCAD mutants. However, transformation, as well as growth, of the DeltapsaD mutant was not dependent upon Mn2+ supplementation. We also show that, apart from sensitivity to hydrogen peroxide, the DeltapsaD mutant exhibited essentially similar phenotypes to those of the wild type. Western blot analysis with a PsaD antiserum showed that deleting any of the genes upstream of psaD did not affect its expression. However, we found that deleting psaB resulted in decreased expression of PsaA relative to that in D39, whereas deleting both psaB and psaC resulted in at least wild-type levels of PsaA.     

Effects of the overexpression of the small heat shock protein,HSP27, on the sensitivity of human fibroblast cells exposed to oxidative stress

The role of the human small heat shock protein (HSP27) in oxidative stress was examined using stable transformants of an immortalized human fibroblast cell line (KMST-6) isolated by transfection of HSP27 expression vectors. Several stable transformants that expressed high or low levels of HSP27 protein were obtained. Clones expressing high levels of HSP27 were more sensitive to growth inhibition by a low dose of hydrogen peroxide (0.1 mM) than those expressing low levels. Clones expressing high levels of HSP27 did not acquire obvious resistance to hyperthermy and cytotoxic agents, except for one (#13), in which resistance to cytotoxic agents was increased. The level of phosphorylated HSP27 in clones expressing high levels of this protein increased at 30 min and was sustained even 4 hours after exposing the cells to 0.1 mM of hydrogen peroxide. On the other hand, the levels in clones expressing low levels of HSP27 were reduced within 4 hours after exposure to hydrogen peroxide. Furthermore, overexpression of nonphosphorylatable mutant HSP27 did not affect sensitivity to oxidative stress. These results suggested that constitutively high expression of HSP27 in KMST-6 cells make them susceptible to oxidative stress resulting in growth arrest, and this mechanism could involve the phosphorylation of HSP27. © 1995 Wiley-Liss, Inc.     

Influence of autocrine factor deficit in culture on survival and energy metabolism of CTLL-2 cells under oxidative stress

A lot of data has shown recently that survival of mammalian cells is under a control of growth factors and autocrine survival factors (AF). We studied the influence of AF deficit on survival, intracellular ATP content, and transmembrane potential of mitochondria of IL-2-dependent CTLL-2 cells under oxidative stress. CTLL-2 cells cultivated under deficit of AF have been shown to be more susceptible to oxidative injury in comparison with the cells cultivated without deficit of AF (control); they died at smaller concentrations of H2O2 than control cells did. The ATP content in CTLL-2 cells was decreased under AF deficit conditions even without any stress and treatment of the cells by hydrogen peroxide resulted in additional large decrease of it. ATP depression was accompanied by disruption of cell membrane (blebbing) and drop of mitochondrial potential. Cell death under oxidative stress in the presence of AF deficit has been shown to proceed by both apoptosis and necrosis.