![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, 9000 Ghent, Belgium
Received 6 February 2003/ Accepted 27 June 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
E. coli mainly expresses two systems to destroy H2O2. The first system is embodied by two types of catalase enzymes, a bifunctional catalase/peroxidase (HPI), encoded by katG (39), and a monofunctional catalase (HPII), encoded by katE (45), which are both heme-containing enzymes involved in the dismutation of H2O2 into O2 and H2O. Clear evidence shows that the two catalase genes are regulated differently in terms of growth phase and responsiveness to oxidative stress (reviewed in reference 22). HPI is transcriptionally induced during logarithmic growth in response to low micromolar concentrations of H2O2. This induction requires the positive transcriptional activator OxyR, which directly senses oxidative stress. On the other hand, HPII is not peroxide inducible and its gene, katE, is transcribed at the transition from the exponential growth phase to the stationary growth phase by RNA polymerase containing the alternative sigma subunit 
s, the product of the rpoS (or katF) gene.
Next to these catalases, E. coli expresses a two-enzyme H2O2-scavenging system, the alkyl hydroperoxide reductase (AhpR) system, which was initially characterized as rapidly reducing diverse organic hydroperoxides such as cumene- and t-butylhydroperoxide (18). Later, Niimura et al. (26) showed that the specific activities of the Salmonella enterica serovar Typhimurium AhpR homolog for the reduction of cumene hydroperoxide and H2O2 are within the same order of magnitude. One component of the AhpR system, AhpF, is a flavoprotein that shuttles reducing equivalents from NAD(P)H (with a strong preference for NADH) to AhpC, the actual peroxidase and a member of the peroxiredoxin family of thiol peroxidases. The ahpC and ahpF genes are organized in a two-gene operon, and transcription is OxyR controlled (38).
In E. coli, AhpR and HPI have discrete roles in scavenging H2O2. AhpR is the primary scavenger of endogenous H2O2. HPI contributes little when H2O2 concentrations are low, but it becomes the more effective scavenger when H2O2 concentrations are high or, presumably, when the absence of a carbon source depletes the cell of the NADH necessary for AhpR activity (36).
Analogously, catalases are believed to be indispensable in eukaryotic cells to protect compartments subjected to high H2O2 concentrations, e.g., the peroxisomal matrix and membrane (6), whereas the H2O2 resulting from the cellular metabolism of molecular oxygen is primarily scavenged by thiol-dependent peroxidases (13, 29). In animals, these peroxidases are fueled by either the glutathione or the thioredoxin redox cycle. The tripeptide-reduced glutathione (GSH; 
-L-glutamyl-L-cysteinyl glycine) is an abundant and ubiquitous low-molecular-weight thiol with a presumed role in many cellular processes, ranging from oxidative stress defense to cysteine storage (reviewed in reference 31). During these processes, GSH is often converted into its symmetrical disulfide form, oxidized glutathione (GSSG), and regeneration occurs via the NADPH-dependent action of glutathione reductase. Glutathione deficiency in higher eukaryotes is at the basis of several diseases caused by the degradation of mitochondria (8, 19), an observation which allowed us to conclude that H2O2 generated through respiration is scavenged inside the mitochondria by GSH peroxidase (GPx). Furthermore, the finding that glutathione-deficient yeast strains are hypersensitive to H2O2 points to the importance of glutathione-based peroxide removal in eukaryotes (10, 17).
Haemophilus influenzae is a strictly human commensal organism. As unencapsulated species, the bacterium is found in the upper respiratory tracts of up to 80% of healthy adults and, as encapsulated species, in 3 to 5% of normal individuals (25). Encapsulated strains are capable of invasive infections, including meningitis, pneumonia, and epiglottitis.
An acatalasemic H. influenzae Rd mutant was constructed by transposon-mediated inactivation of one structural gene, hktE. Consequently, hktE accounts for total catalase activity in H. influenzae cells (3). The hktE gene product, HktE, is strongly homologous to E. coli KatE. However, the H. influenzae catalase is downregulated in the stationary growth phase and is upregulated by exposure to H2O2. As such, hktE regulation is similar to that of katG (3). Consistent with findings in other bacteria, disruption of catalase production in H. influenzae causes hypersensitivity to high H2O2 concentrations (2). On the other hand, deletion of the hktE gene produces only a modest reduction in the ability to cause lethal sepsis after parenteral challenge and causes no change in the ability to colonize after intranasal inoculation in the infant rat model of infection (2). Consequently, the H. influenzae hktE gene is not of major importance for the process of colonization and invasive infection.
Because H. influenzae catalase mutants do not show any growth disadvantage either in vitro in rich medium or in vivo after intranasal inoculation in infant rats, it is anticipated that at least one other H2O2-scavenging system supports peroxide removal in H. influenzae. AhpR is not involved because neither an ahpC nor an ahpF homolog is apparent from the H. influenzae Rd genome sequence (7).
Recently, we reported that H. influenzae is naturally deficient in glutathione biosynthesis (42) and showed that H. influenzae instead imports glutathione from the growth medium. In this way, extracellular glutathione (supplemented as GSSG to prevent chemical reactions with the oxidizing test chemicals) protects cultures against methylglyoxal, S-nitrosoglutathione, and t-butylhydroperoxide toxicity. Interestingly, an open reading frame (HI0572) was isolated that encodes glutathione-based t-butylhydroperoxide removal. On the other hand, the presence of GSSG in the growth medium of H. influenzae NCTC 8143 cultures does not influence the sensitivity to exogenous H2O2 stress (42). We show here, however, that glutathione is a crucial component in the metabolism of H2O2 endogenously generated during aerobic growth.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
H. influenzae specific minimal medium (MIc medium) was prepared essentially as described by Herriott et al. (15). L-Cystine (Sigma-Aldrich) was added to a final concentration of 50 µM, and Haemophilus test medium supplement was added according to the manufacturers' instructions. GSSG-supplemented MIc medium [MIc(GSSG)] was prepared by replacing L-cystine with GSSG (Sigma-Aldrich) at the same final concentration. Both L-cystine and GSSG were added from a sterile concentrated stock solution. For anaerobic growth, the recipe for the preparation of MIc minimal medium was adjusted as follows: (i) sodium lactate was replaced by disodium fumarate (final concentration, 5 mM), and (ii) the final glycerol concentration was raised from 0.3 to 0.5%. This medium is referred to as anMIc or anMIc(GSSG) medium throughout the text, depending on whether L-cystine or GSSG was added, respectively; 1.8% agar was added to prepare anMIc and anMIc(GSSG) agar plates.
Bacterial strains and growth conditions. Wild-type strain H. influenzae Rd was purchased from the American Type Culture Collection (Manassas, Va.). The construction of the H. influenzae catalase-negative strain AB2593 (Rd hktE::mini-Tn10Cm) was reported previously (2). Strain AB2593 was kindly provided by William R. Bishai (Department of Medicine, Division of Infectious Diseases, Johns Hopkins University School of Medicine).
Cultures were routinely grown at 37°C under anaerobic conditions in anMIc medium. Anaerobic cultures were prepared in a Coy chamber (Coy Laboratory Products, Inc.) under an atmosphere of 85% N2-10% H2-5% CO2 in culture tubes that fit tightly into the cuvette holder of a Shimadzu 1240 Mini Single-Beam UV-VIS spectrophotometer (Shimadzu, Duisburg, Germany). The system enables cell density measurements without the need to transfer culture samples into regular cuvettes. The tubes were closed with a silicone stopper and were shaken outside the Coy chamber. As such, anaerobiosis was preserved, a finding confirmed in preliminary experiments by adding 0.002% resazurine to the growth medium (resazurine is a redox-sensitive dye added to media as a simple, qualitative indicator of the redox conditions of the media).
Tn10-linked mutations in hktE were confirmed by assaying catalase activity in whole cells as described earlier (12).
For aerobic shift experiments, precultures grown anaerobically overnight were diluted 1:100 in anMIc or anMIc(GSSG) medium to an optical density at 600 nm (OD600) of 
0.005 and then grown anaerobically to an OD600 of 0.15. The silicone stopper was then removed in order to shift the cells into air. In the case of aerobic-shift experiments to monitor growth in the presence of nonenzyme H2O2 scavenging, pyruvate was added to the anMIc or anMIc(GSSG) cultures from a buffered sterile stock solution to a final concentration of 0.75%. In the case of aerobic-shift experiments to evaluate catalase activity in response to aerobiosis, cell extracts were prepared, and catalase activity was measured, as described previously (42), 20 min after the removal of the silicone stopper. These catalase activities were then compared to those derived from continuously anaerobically grown counterparts.
Heat-killed bacteria were prepared by incubating suspensions at 60°C for 1 h.
Disk diffusion.
Overnight anaerobically grown precultures were diluted 1:100 times in anMIc or anMIc(GSSG) medium to an OD600 of 0.005 and then grown anaerobically to an OD600 of 0.5 (late exponential phase). The following manipulations were performed inside the Coy chamber. Using a sterile cotton swab, cells were inoculated onto the entire surface of anMIc or anMIc(GSSG) plates. Round sterile filters (5.2-mm diameter) were placed in the center of the plates and spotted with either 5 µl of 3% H2O2 or 5 µl of 500 mM t-butyl hydroperoxide. The plates were placed in an anaerobic jar and incubated for 2 days at 37°C. The diameter of the zone of complete inhibition was recorded in millimeters. The experiment was performed in triplicate; mean values are reported.
H2O2 detection. The Amplex red hydrogen peroxide/peroxidase assay kit (Molecular Probes, Eugene, Oreg.) was used to detect H2O2 according to the manufacturer's instructions. This assay can be performed either fluorometrically or spectrophotometrically. We chose the latter approach because the extinction coefficient of the Amplex red reagent oxidation product (resorufin) is high (54,000 cm-1 M-1), resulting in a detection limit of ca. 0.2 µM. The latter value is low enough to complete the experiments described here. Absorbance was measured at 563 nm by using a Shimadzu 1240 Mini Single-Beam UV-VIS spectrophotometer. The assay procedure was performed in a total volume of 400 µl. For each assay, no-H2O2 controls were monitored.
H2O2 scavenging by whole cells.
Overnight anaerobically grown precultures were diluted 1:100 times in anMIc or anMIc(GSSG) medium to an OD600 of 0.005 and grown anaerobically to an OD600 of 0.15. Cells were pelleted in a microcentrifuge, washed twice, and resuspended in room temperature phosphate-buffered saline (PBS) at an OD600 of 0.15. H2O2 was added to the appropriate final concentration (see the figure legends). At intervals, 200-µl samples were removed, and the reactions were terminated (i.e., the cells were removed) by filtering the reaction mixtures with sterile Millex-GV13 0.22-µm-pore-size filter units (Millipore Products Division, Bedford, Mass.). H2O2 was then assayed by the Amplex red method.
Measurement of H2O2 accumulation in cell cultures. Cultures were grown as described for the aerobic-shift experiments. Immediately before and at intervals after the shift to air, the OD600s were measured, and 200-µl samples were removed. Cells were immediately removed from the samples by filtration with sterile Millex-GV13 0.22-µm-pore-size filter units. The cell-free culture media were then assayed for H2O2. H2O2 levels were also determined in sterile medium that was incubated at 37°C for an equivalent time period. These background levels, however, did not exceed the detection limit of ca. 0.2 µM. The rate of H2O2 production was normalized to the cytoplasmic volume of the suspended cells by using a standard ratio of 0.94-µl internal volume per 1 ml of H. influenzae at 1 OD600. The latter value is derived from the assumption that H. influenzae cells can be modeled as approximate cylinders with a length of 1.2 µm and a radius of 0.25 µm; as such, the volume per cell is 0.236 x 10-15 liters. The number of cells present in 1 ml of a 1-OD600 anMIc culture was determined by dilution plating to be 4.0 x 109.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
Exogenous glutathione conceals the aerobic growth defect in minimal medium of H. influenzae AB2593. The fact that even fairly dense cultures of AB2593 died as a result of an aerobic shift (Fig. 2) rules out the possibility that a dilution effect causes growth inhibition in minimal medium. More likely, the aerobic growth defect is the result of the different composition of the growth media. One particular component that is not present in (an)MIc medium compared to rich medium is glutathione. When supplied to the growth medium in oxidized form, this thiol tripeptide was recently shown to strengthen the oxidative defense of naturally glutathione-deficient H. influenzae (42). We therefore repeated the aerobic-shift experiments in the presence of GSSG (Fig. 2) and observed that AB2593 cultures grown in glutathione-supplemented minimal medium are completely relieved from defective growth under aerobic conditions.
Effects of intracellular glutathione-based peroxide reduction on the metabolism of H2O2 produced during aerobic respiration. In order to elucidate whether glutathione-deficient AB2593 cells indeed exhibit problems in detoxifying the H2O2 that is produced during aerobic respiration, we repeated our aerobic-shift experiment and monitored the H2O2 concentrations in the medium (Fig. 3). For all conditions tested, we measured higher H2O2 concentrations immediately after the aerobic shift. This most likely represents the adaptation time required to build up adequate antioxidant activity. Table 1 illustrates that the shift to oxidative metabolism caused a substantial 6.6-fold increase in catalase activity in glutathione-deficient H. influenzae Rd cells, indeed suggesting that, in response to the shift to air, H2O2 is generated at a rate greater than what could be metabolized to a steady-state level (i.e., <0.2 µM) by the antioxidant machinery present in anaerobically growing cells. This hypothesis, however, appears not to be valid for glutathione-supplemented cells. Note that the experiment illustrated in Table 1 only focuses on catalase activity, which, as described below, appears to be less efficient compared to glutathione-based peroxide removal. Thus, in response to the aerobic shift, induction of the latter system may be sufficient to alleviate the toxicity of respiratory-generated H2O2 to levels too low to induce the synthesis of catalase.
|
|
Once we had determined that both glutathione-based H2O2 removal and catalase activity are important during aerobic growth, we wondered whether these two systems represent the entire H2O2-scavenging activity of H. influenzae. Therefore, we set up a time-response experiment exemplifying the in vivo H2O2-scavenging activities of whole metabolically active cells (Fig. 4). We observed that, whereas glutathione-supplemented AB2593 scavenged 1.5 µM H2O2 as well as did its wild-type parent, the glutathione-deficient hktE mutant exhibited virtually no H2O2-scavenging activity. Also, heat-killed glutathione-supplemented AB2593 cells failed to remove H2O2 from solution. Thus, H. influenzae relies on two systems for the destruction of H2O2 generated through respiration: a glutathione-based system that requires metabolically active cells on the one hand and catalase on the other.
|
|
|
12.4 µM per s. |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We demonstrated here that the acatalasaemic H. influenzae mutant strain AB2593 requires glutathione in its growth medium in order to survive endogenous H2O2 stress associated with aerobic growth. In a previous study (42), we showed that H. influenzae, although it possesses glutathione metabolizing enzymes, is naturally glutathione deficient and that glutathione is acquired by importing the thiol tripeptide (either as GSH or as reducible symmetrical and asymmetrical [mixed] disulfide forms of GSH) from the growth medium. Therefore, since H. influenzae AB2593 is deficient in catalase, the restoration of wild-type growth rates in glutathione-supplemented (an)MIc medium could be the result of pure glutathione-based nonenzymatic H2O2 scavenging inside the cell (28). However, the fact that glutathione-supplemented AB2593 scavenges H2O2 at wild-type rates (Fig. 4) and the fact that the glutathione-based H2O2 removal attains a maximum initial velocity with increasing H2O2 concentrations (Fig. 5) indicate that intracellular GSH donates electrons to a powerful peroxidase.
Glutathione-dependent peroxidatic H2O2 removal requires a continuous flow of GSH. Glutathione reductase is the central enzyme in glutathione redox cycling, and this reductase exclusively exploits NADPH as a reductant. Heat-killed glutathione-supplemented AB2593 is not able to generate reducing power. This probably explains our observation that these cells were totally ineffective in H2O2 scavenging (Fig. 4). Consequently, the rate of glutathione-based H2O2 scavenging most likely depends on the rate at which metabolism generates NADPH. This may well explain why exogenous H2O2 stress results in a twofold increase in glucose-6-phosphate dehydrogenase (G6PD) activity (42), whereas G6PD levels remain unaltered in response to peroxides in E. coli. Indeed, G6PD is the first and rate-limiting enzyme of the pentose phosphate pathway, and for both lower and higher eukaryotes G6PD expression is regulated in order to maintain NADPH levels for the glutathione-based enzymatic pathway of peroxide removal (40). In E. coli, NADH is the preferred reductant for AhpR-dependent peroxidatic activity (26) and, it therefore appears that no G6PD induction is required in response to H2O2 stress.
Recently, we reported that the H. influenzae genome encloses a gene (ORF HI0572) encoding a novel GSH-dependent t-butylhydroperoxide reductase (Prx/Grx) (42). Naturally glutathione-peroxidase-deficient E. coli expresses a vast amount of GSH-dependent t-butylhydroperoxide reductase activity when transformed with a multicopy plasmid carrying the HI0572 locus. The Prx/Grx protein shares 63% sequence identity with the garA gene product of Chromatium gracile (30). Because the C. gracile peroxidase reduces both t-butylhydroperoxide and H2O2 at high rates (43), we anticipated that Prx/Grx might be the major peroxidase involved in H2O2 metabolism of H. influenzae. To examine this premise, we are currently generating both an H. influenzae HI0572 mutant and an H. influenzae HI0572 hktE double mutant. In the meantime, we recently obtained supportive biochemical evidence through the characterization of the H. influenzae Rd Prx/Grx peroxidase. We observed that H2O2 binds very efficiently to the GSH-dependent peroxidase, as can be demonstrated by deducing the Km value from an empirical fit to the data given by the concentration-response curve shown in Fig. 5; note that the abscissa displays extracellular H2O2 concentrations and, therefore, the deduced Km value cannot be interpreted as real Michaelis constant. The extracellular concentration at half-maximal rate was ca. 5.8 µM, which is very similar to the Michaelis constant (2.29 µM) obtained via in vitro kinetics of the Prx/Grx peroxidase (30).
As observed for E. coli (36), our results strongly suggest that aerobically grown log-phase H. influenzae scavenges the majority of endogenous H2O2 through one catalase and one peroxidase. Indeed, as reported for catalase and AhpR-deleted E. coli (36), glutathione-deficient H. influenzae lacking catalase is almost completely devoid of H2O2-scavenging activity (Fig. 4). Notably, in contrast to the fact that H2O2 scavengerless E. coli grows, albeit poorly, in rich and minimal media in air (36), we observed that glutathione-deficient AB2593 is completely unable to grow aerobically. So, the continual accumulation of H2O2 inside scavengerless aerobically growing cells appears to be more damaging to H. influenzae than to E. coli. In growing cells, the steady-state concentration of H2O2 depends on the rates of its formation and of its dissipation. Given that H2O2 removal is essentially absent in both strains, the growth difference could be the result of differences in H2O2 formation rates. We observed rates (12.4 µM per s) that are 1 order of magnitude higher than those reported by Gonzales-Flecha and Demple (ca. 1 to 2 µM per s) for E. coli (9). However, Seaver and Imlay (36) reported rates of H2O2 production in exponentially growing E. coli (14 µM) similar to those reported here for H. influenzae. This inconsistency is the result of differences in experimental procedure to measure H2O2 formation rates (36). Since our procedure is very similar to the one used by Seaver and Imlay (i.e., actual H2O2 formation rates are measured by using H2O2 scavengerless log-phase cultures grown at 37°C), we suggest that H. influenzae and E. coli cells generate equal amounts of H2O2 per s. Thus, the growth difference between H2O2 scavengerless H. influenzae and E. coli cannot be explained in terms of differences in H2O2 formation rates. Since GSH is a potent nonenzyme scavenger of reactive oxygen species and since it also functions as a cofactor for enzymes involved in oxidative stress defense, such as glutaredoxins and glutathione S-transferases, deprivation of intracellular GSH not only blocks GSH-dependent H2O2 removal but also prevents all other processes that require GSH in the H. influenzae cytoplasm. As such, glutathione-deficient AB2593 is subjected to additional, e.g., thiol-disulfide, stress (32) compared to H2O2 scavengerless E. coli, which explains why glutathione-deficient AB2593 is not viable under aerobic conditions.
For E. coli, it was proposed that AhpR and HPI have discrete roles in scavenging H2O2. AhpR scavenges low levels of H2O2, and HPI scavenges high levels of H2O2 (36). We obtained analogous results showing that glutathione-based peroxidatic activity is more effective at scavenging very low concentrations of H2O2, whereas HktE is the more effective enzyme at higher concentrations. Omitting GSSG from the media of H. influenzae NCTC 8143 cultures results in a twofold increase in catalase activity (42). Moreover, the catalase activity of glutathione-deficient H. influenzae NCTC 8143 is similar compared to the activity of glutathione-supplemented counterparts stressed with 50 µM H2O2 for 1 h. This implies that glutathione deficiency and micromolar H2O2 stress affect hktE gene expression to a similar extent (42). Moreover, Table 1 illustrates that aerobiosis caused a drastic 6.6-fold increase in catalase activity in glutathione-deficient H. influenzae Rd cells. We and others observed no such regulation of catalase for E. coli deficient in glutathione biosynthesis compared to the isogenic parent (28; data not shown). Thus, in H. influenzae, debilitation of the glutathione-based peroxidatic system causes catalase induction, which may be a compensatory response to provide enough, though less efficient, H2O2-scavenging activity. This compensatory response may well explain our observation illustrated in Fig. 4 that either catalase or glutathione-based peroxidase activities alone give the same rate of H2O2 scavenging by whole cells as when both are present. Compensatory interactions between catalase and peroxidase (AhpR) synthesis have been observed previously in a wide range of bacteria. In E. coli (36), Xanthomonas campestris (24), Bacteroides fragilis (34), and Pseudomonas aeruginosa (27), mutations in the peroxidatic system, AhpR, cause catalase induction.
|
ACKNOWLEDGMENTS |
|---|
J.J.V.B. is indebted to the Fund for Scientific Research-Flanders (grant 3G003601). F.P. is a beneficiary of a doctorate research grant provided by the Institute IWT, Flanders, Belgium.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B: studies with isolated mitochondria and rat hepatocytes. Mol. Pharmacol. 48:825-834.[Abstract]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |
; AB2593 (1), 
], and growth was monitored at 600 nm. After 8 h of growth, these cultures were subcultured (1:100) in MIc broth, and growth was recorded [Rd (2), 
). Again, 8 h later, growth was monitored of the third-generation subcultures in MIc broth [Rd (3), 
].
; AB2593, 
) were prepared by incubating the PBS suspension at 60°C for 1 h. H2O2 was added to a final concentration of 1.5 µM. The H2O2 concentration was measured at various time points after addition of H2O2 as described in Materials and Methods.