Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40–70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro , had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.     

Neisseria gonorrhoeae virulence factor NG1686 is a bifunctional M23B family metallopeptidase that influences resistance to hydrogen peroxide and colony morphology

Symptomatic gonococcal infection, caused exclusively by the human-specific pathogen Neisseria gonorrhoeae (the gonococcus), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess a potent antimicrobial arsenal comprising both oxidative and non-oxidative killing mechanisms, gonococci survive this interaction, suggesting that the gonococcus has evolved many defenses against PMN killing. We previously identified the NG1686 protein as a gonococcal virulence factor that protects against both non-oxidative PMN-mediated killing and oxidative killing by hydrogen peroxide. In this work, we show that deletion of ng1686 affects gonococcal colony morphology but not cell morphology and that overexpression of ng1686 does not confer enhanced survival to hydrogen peroxide on gonococci. NG1686 contains M23B endopeptidase active sites found in proteins that cleave bacterial cell wall peptidoglycan. Strains of N. gonorrhoeae expressing mutant NG1686 proteins with substitutions in many, but not all, conserved metallopeptidase active sites recapitulated the hydrogen peroxide sensitivity and altered colony morphology of the Δng1686 mutant strain. We showed that purified NG1686 protein degrades peptidoglycan in vitro and that mutations in many conserved active site residues abolished its degradative activity. Finally, we demonstrated that NG1686 possesses both dd-carboxypeptidase and endopeptidase activities. We conclude that the NG1686 protein is a M23B peptidase with dual activities that targets the cell wall to affect colony morphology and resistance to hydrogen peroxide and PMN-mediated killing.     

Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide

The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2).     

Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils

The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniformly Opa negative (Opa⁻), whereas cultures of predominantly Opa⁻ parental Gc and an intermediate lacking the “translucent” subset of opa genes (ΔopaBEGK) stochastically gave rise to Opa-positive (Opa⁺) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa⁻ bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD⁺ Gc survived less well after exposure to PMNs compared to Opa⁻ bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology.     

Roles of the recJ and recN Genes in Homologous Recombination and DNA Repair Pathways of Neisseria gonorrhoeae

The paradigm of homologous recombination comes from Escherichia coli, where the genes involved have been segregated into pathways. In the human pathogen Neisseria gonorrhoeae (the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcal recN and recJ genes in the recombination-based processes of the gonococcus, these genes were inactivated in the N. gonorrhoeae strain FA1090. We report that both recN and recJ loss-of-function mutants show decreased DNA repair ability. In addition, the recJ mutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while the recN mutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of either recJ or recN at an irrelevant locus. These results describe the role of recJ and recN in the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.     

Neisseria gonorrhoeae suppresses the oxidative burst of human polymorphonuclear leukocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) results in a potent polymorphonuclear leukocyte (PMN)-driven inflammatory response, but the mechanisms by which Gc withstands PMN attack are poorly defined. Here we report that Gc can suppress the PMN oxidative burst, a central component of the PMN antimicrobial arsenal. Primary human PMNs remained viable after exposure to liquid-grown, exponential-phase, opacity-associated protein (Opa)-negative Gc of strains FA1090 and MS11 but did not generate reactive oxygen species (ROS), even after bacterial opsonization. Liquid-grown FA1090 Gc expressing OpaB, an Opa protein previously correlated with PMN ROS production, elicited a minor PMN oxidative burst. PMN ROS production in response to Opa(-) and OpaB+ Gc was markedly enhanced if bacteria were agar-grown or if liquid-grown bacteria were heat-killed. Liquid-grown Opa(-) Gc inhibited the PMN oxidative burst elicited by isogenic dead bacteria, formylated peptides or Staphylococcus aureus but did not inhibit PMN ROS production by OpaB+ Gc or phorbol esters. Suppression of the oxidative burst required Gc-PMN contact and bacterial protein synthesis but not phagocytosis. These results suggest that viable Gc directly inhibits PMN signalling pathways required for induction of the oxidative burst, which may contribute to gonococcal pathogenesis during inflammatory stages of gonorrhoeal disease.     

Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils

Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome‐primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome–granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule‐negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule‐negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome–granule fusion. Ectopically increasing primary granule–phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule–phagosome fusion, thus preventing formation of a degradative phagolysosome.     

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against this fungus, and several studies have shown the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, studies on polymorphonuclear neutrophils (PMNs), that are the first cells recruited to the infection sites, are scarcer. Thus, the objective of this paper was to assess whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to block the activity of IFN-gamma-activated human PMNs upon P. brasiliensis intracellular killing, in vitro. The results showed that IFN-gamma-activated PMNs have an effective fungicidal activity against the fungus. This activity was associated with the release of high levels of H(2)O(2), the metabolite involved in phagocytic cells antifungal activities. However, the concomitant incubation of these cells with IFN-gamma and IL-10 significantly blocked IFN-gamma activation. As a consequence, PMNs killing activity and H(2)O(2) release were inhibited. Together, our results show the importance of PMNs exposure to activator or suppressor cytokines in the early stages of paracoccidioidomycosis infection.     

The DNA-binding activity of the Neisseria gonorrhoeae LexA orthologue NG1427 is modulated by oxidation

Neisseria gonorrhoeae is a human-specific organism that is not usually exposed to UV light or chemicals but is likely to encounter reactive oxygen species during infection. Exposure of N. gonorrhoeae to sublethal hydrogen peroxide revealed that the ng1427 gene was upregulated sixfold. N. gonorrhoeae was thought to lack an SOS system, although NG1427 shows amino acid sequence similarity to the SOS response regulator LexA from Escherichia coli. Similar to LexA and other S24 peptidases, NG1427 undergoes autoproteolysis in vitro, which is facilitated by either the gonococcal or E. coli RecA proteins or high pH, and autoproteolysis requires the active and cleavage site residues conserved between LexA and NG1427. NG1427 controls a three gene regulon: itself; ng1428, a Neisseria-specific, putative integral membrane protein; and recN, a DNA repair gene known to be required for oxidative damage survival. Full NG1427 regulon de-repression requires RecA following methyl methanesulphonate or mitomycin C treatment, but is largely RecA-independent following hydrogen peroxide treatment. NG1427 binds specifically to the operator regions of the genes it controls, and DNA binding is abolished by oxidation of the single cysteine residue encoded in NG1427. We propose that NG1427 is inactivated independently of RecA by oxidation.     

Intracellular copper does not catalyze the formation of oxidative DNA damage in Escherichia coli

Because copper catalyzes the conversion of H(2)O(2) to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H(2)O(2) than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H(2)O(2) damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H(2)O(2) resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H(2)O(2)-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H(2)O(2) stress by inducing iron sequestration proteins, H(2)O(2)-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.     

Exposure of phytopathogenic Xanthomonas spp. to lethal concentrations of multiple oxidants affects bacterial survival in a complex manner

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1, 000-fold; conversely, treatment of cells with menadione plus H(2)O(2) resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H(2)O(2) and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H(2)O(2) and tert-butyl hydroperoxide plus H(2)O(2). These data suggest that H(2)O(2) is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H(2)O(2)-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.     

Nitroxides block DNA scission and protect cells from oxidative damage.

The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H2O2. Oxidative damage induced by H2O2 was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated the H2O2-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H2O2 cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H2O2 toxicity, without lowering H2O2 concentration. To check whether nitroxides protect against O2.(-)-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H2O2, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.     

Characterization of a novel Neisseria meningitidis Fur and iron-regulated operon required for protection from oxidative stress: utility of DNA microarray in the assignment of the biological role of hypothetical genes

We have previously shown that in the human pathogen Neisseria meningitidis group B (MenB) more than 200 genes are regulated in response to growth with iron. Among the Fur-dependent, upregulated genes identified by microarray analysis was a putative operon constituted by three genes, annotated as NMB1436, NMB1437 and NMB1438 and encoding proteins with so far unknown function. The operon was remarkably upregulated in the presence of iron and, on the basis of gel retardation analysis, its regulation was Fur dependent. In this study, we have further characterized the role of iron and Fur in the regulation of the NMB1436-38 operon and we have mapped the promoter and the Fur binding site. We also demonstrate by mutant analysis that the NMB1436-38 operon is required for protection of MenB to hydrogen peroxide-mediated killing. By using both microarray analysis and S1 mapping, we demonstrate that the operon is not regulated by oxidative stress signals. We also show that the deletion of the NMB1436-38 operon results in an impaired capacity of MenB to survive in the blood of mice using an adult mouse model of MenB infection. Finally, we show that the NMB1436-38 deletion mutant exhibits increased susceptibility to the killing activity of polymorphonuclears (PMNs), suggesting that the 'attenuated' phenotype is mediated in part by the increased sensitivity to reactive oxygen species-producing cells. This study represents one of the first examples of the use of DNA microarray to assign a biological role to hypothetical genes in bacteria.     

Adaptive response to oxidative stress in the filamentous fungus Aspergillus niger B1-D

In the present study, we used a recombinant filamentous fungus strain, Aspergillus niger B1-D, as a model system, and investigated the antioxidant defences in this organism. Our findings indicate that pretreatment with low concentrations of H(2)O(2) completely prevents killing by this oxidant at high concentrations. It shows that A. niger adapts to exposure to H(2)O(2) by reducing growth and inducing a number of antioxidant enzyme activities, including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, of which the induction of catalase is the most pronounced. Moreover the decline of these antioxidant enzymes activities after H(2)O(2) detoxification, coincides with recommencement of growth. Results from monitoring the extracellular H(2)O(2) concentration clearly indicate a very rapid detoxification rate for H(2)O(2) in adapted A. niger cultures. A mathematical model predicts only very low concentrations of intracellular H(2)O(2) accumulating in such cultures. Our results also show that glutathione plays a role in the oxidative defence against H(2)O(2) in A. niger. On addition of H(2)O(2), the intracellular pool of glutathione increases while the redox state of glutathione becomes more oxidized.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

Protective effects of peoniflorin against hydrogen peroxide-induced oxidative stress in human umbilical vein endothelial cells

Peoniflorin (PF), extracted from the root of Paeonia lactiflora Pall., has been reported to have anti-inflammation and antioxidant effects in several animal models. Herein, we investigated the protective effects of PF against hydrogen peroxide (H(2)O(2))-induced oxidative damage in human umbilical vein endothelial cells (HUVECs). HUVECs were treated by H(2)O(2) (240?μmol/L) with or without PF. PF significantly increased the percent cell viability of HUVECs injured by H(2)O(2) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. By flow cytometric analysis, PF markedly attenuated H(2)O(2)-induced apoptosis and intracellular reactive oxygen species production. In addition, PF also displayed a dose-dependent reduction of lactate dehydrogenase leakage, malondialdehyde formation, and caspase-3 proteolytic activities in H(2)O(2)-treated cells, which was accompanied with a restoration of the activities of endogenous antioxidants, including total superoxide dismutase and glutathione peroxidase. Finally, Western blot data revealed that H(2)O(2) upregulated phosphorylation of extracellular signal-regulated kinase 1/2 in HUVECs, which was almost completely reversed by PF. Taken together, our data provide the first evidence that PF has a protective ability against oxidative damage in HUVECs. PF may be a candidate medicine for the treatment of vascular diseases associated with oxidative stress.     

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells. Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire L-glutamate through its GltT uptake system. We demonstrate that the uptake of available L-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst. In the meningococcus, L-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide. We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.     

Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans

The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defences for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H(2) O(2) ), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the Galleria mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared with the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism.     

Molecular cloning and oxidative stress response of a sigma-class glutathione S-transferase of the bumblebee Bombus ignitus

Glutathione S-transferases (EC 2.5.1.18; GSTs) are multifunctional enzymes that are mainly involved in xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTS gene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H(2)O(2) overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H(2)O(2) overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3h after H(2)O(2) exposure, whereas BiGSTS and BiGSTD are highly induced at 9h after H(2)O(2) exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitus are differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.