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Infection and Immunity, September 2006, p. 5058-5066, Vol. 74, No. 9
0019-9567/06/$08.00+0     doi:10.1128/IAI.01932-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Interplay of Pneumococcal Hydrogen Peroxide and Host-Derived Nitric Oxide

Olaf Hoffmann,^1, Janine Zweigner,^2,3, Shannon H. Smith,³ Dorette Freyer,¹ Cordula Mahrhofer,¹ Emilie Dagand,¹ Elaine I. Tuomanen,^3, and Joerg R. Weber^1,*

Department of Neurology,¹ Department of Microbiology and Hygiene, Charité—Universitaetsmedizin Berlin, Berlin, Germany,² Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee³

Received 25 November 2005/ Returned for modification 1 February 2006/ Accepted 7 June 2006

	ABSTRACT

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Reactive oxygen and nitrogen species are released by immune-competent cells and contribute to cellular damage. On the other hand, certain pathogens, including Streptococcus pneumoniae, are known to produce hydrogen peroxide (H₂O₂), while production of nitrogen radicals by bacteria presumably occurs but has been poorly studied. We determined the relative contributions of bacterial versus host-derived oxygen and nitrogen radicals to cellular damage in pneumococcal infection. A special focus was placed on peroxynitrite as a hypothetical common product formed by the reaction of H₂O₂ and NO. In microglial cultures, reduction of the formation of 3-nitrotyrosine and cellular damage required H₂O₂-deficient (spxB or carB) pneumococci and inhibition of host NO synthesis with aminoguanidine. In infected C57BL/6 mice, neuronal loss and immunopositivity for nitrotyrosine in the dentate gyrus were markedly reduced with spxB or carB bacterial mutants and in inducible nitric oxide synthase knockout mice. We conclude that although host and bacteria both produce oxygen and nitrogen radicals, the interplay of prokaryotic H₂O₂ and eukaryotic NO is a major contributor to cellular damage in pneumococcal meningitis.

	INTRODUCTION

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The host response to invading bacteria involves not only immune responses but also release of nonspecific and chemically highly reactive molecules. Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) can damage membrane structures and DNA of prokaryotic as well as eukaryotic cells. Considerable quantities of ROI are produced by macrophages and polymorphonuclear leukocytes in response to different bacterial stimuli. Additionally, macrophages are significant sources of RNI such as nitric oxide (NO). As a joint product of ROI and RNI, peroxynitrite is a particularly destructive molecule that exerts antimicrobial effects but also initiates host cell damage (6, 7).

Reactive oxygen species (ROS) are generated by oxidative metabolism of all aerobic cells, but particularly efficient production is achieved by the NADPH (phagocyte) oxidase of neutrophilic and eosinophilic granulocytes and mononuclear phagocytes (3). This enzyme complex produces superoxide (O₂^–) from oxygen, most of which is then converted by superoxide dismutase (SOD) to hydrogen peroxide (H₂O₂) (22). Further reduction yields hydroxyl radicals (OH) and ultimately H₂O. In the host, NO is generated by a family of NO synthases (1). Of these enzymes, the calcium-dependent neuronal and endothelial isoforms are constitutively active and produce nanomolar amounts of NO as a strictly local neurotransmitter and modulator of vascular tone. Conversely, a calcium-independent, inducible isoform (inducible nitric oxide synthase [iNOS]) is inactive in most resting cells and is induced in cells with phagocytic capacities under pathological conditions, e.g., in infection, trauma, or ischemia (11).

While the production of ROS and RNI in the host has been extensively studied, it is less well appreciated that bacteria also produce these compounds. In particular, Streptococcus pneumoniae releases large amounts of H₂O₂ due to the absence of catalase to neutralize H₂O₂ produced by pyruvate oxidase (SpxB) (pyruvate plus O₂ plus P_i yields acetyl phosphate plus H₂O₂ plus CO₂) (2). The antimicrobial effect of H₂O₂ provides pneumococci with a significant advantage over other, non-H₂O₂-producing bacteria. Streptococci also produce nitrogen radicals, for instance, through the metabolism of arginine by carbamoyl-phosphate synthase (CarB) (13). Pneumococci are the leading cause of invasive infections such as community-acquired pneumonia and meningitis (15, 32). Pneumococcal meningitis is associated with 34% mortality (14) and with persistent neurological sequelae in 30 to 50% of survivors (8, 35). Apoptotic loss of neurons during meningitis may contribute to this particularly poor outcome (10, 25). While the mechanisms of host toxicity are not resolved in detail, pneumococcal H₂O₂ has been identified as one important apoptosis-inducing pneumococcal toxin (5, 9).

Reactive oxygen and reactive nitrogen compounds converge to form peroxynitrite (ONOO^–), an extremely toxic oxidant. Reaction of O₂^– with NO is regarded as the classical path for ONOO^– formation, occurring at near-diffusion-limited rates in aqueous solution. At elevated concentrations, NO may compete with superoxide dismutase for O₂^–, leading to increased production of ONOO^– (4). In addition to O₂^–, H₂O₂ may also be used for the generation of ONOO^–. In contrast to its normal O₂^–-detoxifying function, Cu²⁺-containing SOD-1 may become a peroxidase in the presence of elevated H₂O₂ concentrations, catalyzing the formation of O₂^– (18, 20). Moreover, SOD-1 will catalyze the formation of ONOO^– when both H₂O₂ and NO are present (23). Notably, bacterial manganese-containing SOD (MnSOD) has been identified as a virulence factor of pneumococci in experimental pneumonia (37). ONOO^– causes damage to cells in various ways, including lipid peroxidation (30), DNA breakage (31), and modification of proteins through nitration or oxidation of aromatic or thiol residues. Ultimately, ONOO^–-induced toxicity results in cell death (34).

The present study is based on the hypothesis that in pneumococcal infections, an interplay between eukaryotically and prokaryotically derived oxidants contributes to detrimental neurotoxicity.

	MATERIALS AND METHODS

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Bacterial strains and growth. D39, an encapsulated strain of S. pneumoniae serotype 2, was used as the wild type in all experiments. For liquid cultures, the strains were grown in standard casein plus yeast (C+Y) medium (21) or in microglial culture medium (9, 28). Mutant bacteria were grown in the presence of 1 µg/ml erythromycin to maintain the chromosomally integrated plasmid pJDC9 (see below). After centrifugation and resuspension in pyrogen-free 0.1 M phosphate-buffered saline (PBS), CFU per milliliter was determined photometrically (by absorption at 620 nm) using a standard curve. The correctness of CFU calculations was verified by plating of serial dilutions.

Recombinant DNA methods. Pneumococcal mutants were made by insertion-duplication mutagenesis (26). Table 1 shows a synopsis of targeted genes, primers used to amplify a 300- to 400-bp internal region of the gene of interest, and amplified fragments. After amplification, the resulting fragment was digested with EcoRI and BamHI and then ligated into pJDC9 (12), and the resulting plasmid was then transformed into Escherichia coli. Positive clones were selected on agar plates containing 1 µg/ml erythromycin. Plasmid DNA from these colonies was purified using a QIAGEN (Valencia, CA) Miniprep kit according to the manufacturer's recommendations and verified by sequencing. Insertion-duplication was accomplished by natural transformation, and transformants were selected on blood agar plates containing 1 µg/ml erythromycin. Mutations were confirmed by PCR.

Cytochemistry for fluorescent microscopy. Loss of mitochondrial membrane integrity, an early marker of apoptosis, was studied by adding MitoTracker CMX-Ros (200 nM for 30 min; Invitrogen, Karlsruhe, Germany). Other wells were incubated for 5 min with 1 µg/ml propidium iodide, a nuclear stain that is excluded by healthy cells. Cells were then fixed with 4% paraformaldehyde (PFA) in PBS for 3 min. Following repeated changes of PBS, cells were blocked with serum, incubated with a polyclonal nitrotyrosine antibody (Upstate, Waltham, MA; 1:100) at 4°C overnight, and visualized using an Alexa 488-labeled secondary antibody (Invitrogen). For a positive control, microglia were chemically nitrosylated by incubation with 1 mM H₂O₂ and 1 mM NaNO₂ (Sigma) in pH 5 acetate buffer for 30 min. The specificity of this protocol was ensured by a further experiment where preincubation of the primary antibody with 10 mM nitrotyrosine completely abolished the nitrotyrosine signal in chemically nitrosylated microglia.

Flow cytometry. For quantification using a fluorescence-activated cell sorter (FACS), 2.5 x10⁵ to 5 x10⁵ cells/well were challenged with bacteria. Following incubation with MitoTracker (500 nM, 30 min), cells were fixed with 4% PFA for 15 min and permeabilized with 70% ethanol at –20°C overnight. In other experiments, we assessed apoptotic cells by exposure of phosphatidylserine on the plasma membrane. For this purpose, 0.5 x10⁶ to 1 x10⁶ cells were resuspended in 100 µl binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) containing fluorescein-conjugated annexin V (1.0 µg; Becton Dickinson, Franklin Lakes, NJ) and incubated for 15 min at 20°C. Propidium iodide was added prior to analysis in 250 µl binding buffer at a final concentration of 1.0 µg. A total of 10,000 cells were analyzed by FACS using the excitation of a 488-nm line of an argon ion laser. Nitrotyrosine labeling was performed as described above. Fluorescein isothiocyanate-labeled annexin V fluorescence was measured at 530 ± 20 nm, while MitoTracker red 580 (final concentration, 200 nM; Molecular Probes) and propidium iodide were measured at >580 nm.

H₂O₂ production and nitrite measurements. The H₂O₂ production assay was based on a published method (29) and its adaptation (16). Nitrite was assayed by the Griess reaction (17). Cell culture supernatants or bacterial supernatants were harvested at 4 h or at late-logarithmic phase.

Murine meningitis model. All experimental designs fully complied with federal and institutional guidelines and were reviewed and authorized by the hospital research boards or state authorities as applicable. Experiments were conducted on 4- to 5-week-old mice weighing about 20 g. B6 129P2-Nos2^tm1Lau and B6 129PF2-J mice (The Jackson Laboratory, Bar Harbor, ME) were used in experiments with live D39 bacteria. Mutants were first confirmed to exhibit wild-type rates of growth in blood prior to comparisons in the meningitis model. Meningitis was induced with 5 x 10⁵ CFU pneumococci by using a modification of a previously described method (19, 24). Cerebrospinal fluid (CSF) leukocytes were counted and bacterial concentrations determined as described elsewhere (24). The animals were perfused transcardially with PBS followed by 4% PFA in PBS, pH 7.4; after removal, brains were postfixed in 4% PFA for 4 h and then transferred to PBS until paraffin embedding.

Histological techniques. For the evaluation of neuronal damage in the dentate gyrus, 5-µm-thick paraffin sections were stained with standard hematoxylin and eosin as well as terminal deoxynucleotidyltransferase-mediated UTP nick end labeling (TUNEL). For quantification, the number of damaged neurons in the dentate gyrus was counted and divided by the area of the dentate gyrus on multiple sections. Planimetry for this purpose was performed using Stereo Investigator software (MicroBrightfield Europe, Magdeburg, Germany). For TUNEL labeling, sections were deparaffinized and digested with 10 µg/ml proteinase K. The TUNEL reaction was performed for 60 min at 37°C using a commercially available kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. Following incorporation of digoxigenin-labeled dUTP, sites of DNA single-strand breaks were visualized using a fluorescein-conjugated anti-digoxigenin Fab fragment (1:250). Additional slides were doubly stained by the TUNEL reaction as described above, followed by incubation with antibodies directed against the neuronal antigen NeuN or by incubation integrin alpha M (CD11b) as a marker of activated microglia. In these experiments, slides were deparaffinized and subjected to microwave antigen retrieval. Following TUNEL staining and blocking in 10% normal goat serum with 0.3% Triton X-100 in PBS for 1 h, slides were incubated overnight at 4°C with an anti-NeuN antibody (Chemicon; mouse monoclonal antibody; 1:500 in blocking solution) or anti-CD11b (Chemicon; rat monoclonal antibody; 1:100 in blocking solution). Hoechst 33258 (Invitrogen; 1:10,000) was used as a nuclear counterstain. For the detection of nitrotyrosine, deparaffinized sections were blocked with serum and incubated overnight at 4°C with a polyclonal anti-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY; 1:100). The signal was visualized using an Alexa 488-conjugated secondary antibody (1:200 in blocking solution; Molecular Probes, Eugene, OR). To ensure the specificity of the signal, additional sections were incubated in blocking solution without primary antibody or with primary antibody solution competed with 10 mM 3-nitrotyrosine (Sigma).

iNOS in situ hybridization. Murine iNOS-specific antisense and sense RNA probes were synthesized from commercially available templates (Cayman Chemical, Ann Arbor, MI) using T7 and T3 RNA polymerases as appropriate (Stratagene, La Jolla, CA). The probes were labeled with digoxigenin using a commercially available kit according to the manufacturer's instructions (Roche, Mannheim, Germany). For in situ hybridization, 4-µm-thick paraffin sections were deparaffinized, rehydrated, and rinsed with Tris-buffered saline. Following incubation with 0.2 M HCl for 10 min and digestion with proteinase K (100 µg/ml in Tris-buffered saline with 2 mM CaCl₂) for 20 min at 37°C, the sections were acetylated with 0.5% acetic anhydride in 0.1 M Tris (pH 8.0) for 10 min. After dehydration through graded ethanol, sections were immersed in chloroform for 5 min and then rehydrated with 100% and 95% ethanol. After preincubation in a humid chamber at 60°C for 30 min, the sections were incubated with hybridization mix under glass coverslips [50% deionized formamide, 4x standard saline citrate (SSC; 1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10% dextran sulfate, 5x Denhardt's solution, 200 µg/ml salmon sperm DNA, 100 µg/ml poly(A), 25 mM sodium phosphate, 1 mM sodium pyrophosphate, and 5% dithiothreitol plus 3 ng of the RNA probe]. The sections were heated to 94°C for 4 min on a hot plate and then incubated overnight at 68°C in a humid chamber. Following repeated washing steps, sections were blocked with 10% fetal calf serum and a proprietary blocking agent (Boehringer, Ingelheim, Germany). Bound probe was then detected using an alkaline phosphatase-labeled anti-digoxigenin antibody (1:500 in blocking solution for 60 min; Boehringer) with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (BCIP) as the chromogenic substrate as stated by the manufacturer (Roche).

Statistical analysis. Data are presented as means ± standard deviations. Statistical analysis was performed using SigmaStat (SPSS Inc., Chicago, IL). After normal distribution and equal variance were ensured, differences between groups were evaluated using Student's t tests. The correlation between H₂O₂ production and viable cells was detected by Spearman's rank test.

	RESULTS

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Metabolic characterization of bacterial mutant strains. To study the impact of specific bacterial metabolic pathways on oxidative damage to the host during pneumococcal meningitis, we used isogenic bacterial mutants of D39 bearing functional inactivation of genes encoding key proteins of oxidative metabolism (Fig. 1; Table 1). All mutant strains exhibited growth rates similar to that of wild-type D39 in C+Y medium and in microglial tissue culture medium. Release of H₂O₂ and NO was quantified for each strain and was similar in tissue culture medium or bacterial culture medium. Whereas D39 accumulated 111 ± 61 µM H₂O₂ in the microglial tissue culture medium, reduced release of H₂O₂ was observed for the pyruvate oxidase-negative (spxB) and carbamoyl phosphate synthase-negative (carB) mutants (Fig. 2A), while no significant reduction was observed for bacteria lacking the formate-nitrite transporter family member (nex mutant) or NADH oxidase (nox mutant) (Fig. 2A). Cultivating D39 in C+Y medium revealed comparable H₂O₂ concentrations (data nor shown). The specificity of the spxB effect was affirmed by a similar decrease in H₂O₂ accumulation in D39 in the presence of catalase. As an indication of NO release, 8.3 ± 4.4 µM nitrite was detected in the supernatant of wild-type D39 in microglial tissue culture medium (Fig. 2B). Nitrite production was significantly reduced for the carB mutant (3.6 ± 0.7 µM), while no relevant change was found with the remaining mutants (Fig. 2B). The reduction in nitrite production was not due to H₂O₂ production, since addition of 130 µM H₂O₂ to D39 did not appreciably change nitrite accumulation (data not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 1. Overview of the production of reactive oxygen and nitrogen species by pneumococci. Reactive oxygen species, such as H2O2 and O2–, can cause oxidative stress on their own or interact with NO to generate the potent peroxynitrite radical. NO alone can cause nitrosative stress. ROI and RNI are by-products of metabolic reactions involving the enzymes indicated (boxed). Proteins encoded by genes targeted by insertion duplication mutagenesis are boxed. For protein designations, see Table 1.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Metabolic characterization and cytotoxicity of the pneumococcal mutants. (A and B) Measurement of H2O2 production (A) and nitrite formation (B) in the supernatant of 1 x 107/ml bacteria following incubation in microglial culture medium for 4 h at 37°C (medium alone or medium plus dead pneumococci produced no detectable H2O2 or nitrite). Asterisk indicates a statistically significant difference between wild-type and mutant bacteria (P < 0.01; n = 4). (C) Correlation between H2O2 production and viability of microglia 4 h after infection with 1 x 107 CFU/ml of D39 or the spxB or carB mutant (Spearman correlation coefficient [r], –0.74; P < 0.05). Each symbol represents an independent experiment. (D) H2O2 production in tissue culture medium by bacteria alone or in the presence of microglia (n = 3). (E) Production of nitrite by D39 bacteria in tissue culture medium in the presence and absence of microglia (n = 3).

Radical production in eukaryotic cell and bacterial cocultures. To address the relative contributions of prokaryotic and eukaryotic metabolism to the production of H₂O₂ and NO, both were measured in the supernatants of microglial cultures 4 h after infection with pneumococci. In comparison to bacteria alone in tissue culture medium (132 ± 56 µM), total H₂O₂ accumulation decreased when bacteria were exposed to microglia (73 ± 46 µM) (P < 0.05) (Fig. 2D). Elimination of the bacterial H₂O₂ source (spxB mutant) nearly abolished H₂O₂ accumulation in the presence or absence of microglia (Fig. 2D), indicating bacteria, and specifically SpxB, as the primary source of this oxidant. Addition of D39 bacteria to microglia resulted in an increase in the nitrite level above that of microglia alone, an effect that was reduced for the carB mutant (Fig. 2E). These data suggest that both the pneumococcus and microglia contribute to nitrite production and that loss of CarB function eliminates most of the NO produced by bacteria. Thus, spxB and carB were identified as valid tools to probe the bacterial contributions to radical-induced damage.

Interplay of bacterial H₂O₂ and microglial NO. Infection of microglial cultures with D39 led to increased nitrotyrosine immunostaining as a footprint of peroxynitrite formation (Fig. 3A and K) and to a severe loss of mitochondrial membrane and cellular integrity (Fig. 3B, L, and C). Double labeling with anti-nitrotyrosine was observed in a subset of propidium iodide-positive cells (Fig. 3C, inset). Treatment of D39-infected cells with aminoguanidine, an inhibitor of iNOS, decreased the nitrotyrosine signal (Fig. 3K) and preserved an intense MitoTracker signal (Fig. 3L). However, spxB mutant-infected cells were able to maintain mitochondrial integrity (Fig. 3L) despite modest nitrotyrosine staining (Fig. 3K). The absence of bacterial H₂O₂ in conjunction with inhibition of host NO (Fig. 3E) had a similar effect as the absence of bacterial H₂O₂ alone. These results suggest that bacterial H₂O₂ is an important source of cellular damage.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Interplay of bacterial H2O2 and microglial NO affects nitrotyrosine formation, mitochrondrial membrane potential, and cell death. (A through J) Healthy microglia stain negative for nitrotyrosine (G), positive for mitochondrial integrity with MitoTracker (I), and negative for cell death with propidium iodide (J). Treatment of microglia with H2O2 and NaNO2 induced positive nitrotyrosine staining (H). Infection of microglia with D39 induced the formation of nitrotyrosine (A), decreased the uptake of MitoTracker (B), and caused cell death as shown by the uptake of propidium iodide (C). The inset in panel C shows the colocalization (yellow) of propidium iodide (red) and nitrotyrosine (green). The absence of bacterial H2O2 (spxB) resulted in a decrease in nitrotyrosine staining (D) and cell death (F) and restored mitochondrial function (E). (K and L) Staining was quantitated by FACS analysis comparing a control (CON) to D39 bacteria with or without the iNOS inhibitor aminoguanidine (AG). All experiments were performed at least in triplicate. Symbols indicate statistically significant (P < 0.05) differences between D39 and the control (*), D39 alone versus D39 plus AG (# in panel K), D39 versus the spxB mutant (# in panel L), and D39 versus the spxB mutant plus AG (##).

Compared to controls, infected mice displayed strongly increased immunoreactivity for nitrotyrosine in inflammatory cells in the subarachnoid space, in the adjacent superficial cerebral cortex, and also within hippocampal structures, predominantly in the dentate gyrus and hilus region (Fig. 4A versus C). Less nitrotyrosine signal was observed in meningitis due to spxB (Fig. 4E) or carB (data not shown) mutants. iNOS knockout mice in all instances displayed markedly less signal than wild-type mice infected with the same bacterial strain (Fig. 4B, D, and F). In situ hybridization for iNOS mRNA was positive in the cortex, ependyma, and dentate gyrus of wild-type mice treated with D39 (Fig. 4G), but not in iNOS-deficient mice (Fig. 4H) or in saline-treated controls. These data suggest that bacterial H₂O₂ and host iNOS-derived NO contribute to the formation of peroxynitrite in vivo but that host-derived NO production has a stronger influence.

View larger version (71K): [in this window] [in a new window]   FIG. 4. Interplay of bacterial H2O2 and host-derived NO effects on nitrotyrosine formation in experimental meningitis. (A through F) Immunohistochemistry for nitrotyrosine in the dentate gyri of wild-type (A, C, and E) or iNOS–/– (B, D, and F) mice under control conditions (A and B) or 24 h after meningitis induction with D39 (C and D) or the H2O2-deficient (spxB) mutant (E and F). (G and H) In situ labeling for iNOS mRNA in the dentate gyrus after induction of meningitis with D39 in wild-type (G) and iNOS–/– (H) mice. Data are representative of three to six animals per condition.

View larger version (55K): [in this window] [in a new window]   FIG. 5. Contribution of host inducible nitric oxide and bacterial H2O2 to neuronal loss in vivo. (A through F) Detection of apoptotic cells via the TUNEL reaction in the dentate gyrus under control conditions (A and B) or 24 h after meningitis induction with D39 (C and D) or the spxB mutant (E and F). (G) Double labeling with the TUNEL signal (green) and an anti-NeuN antibody (red) identified most apoptotic cells as neurons. (H) No colocalization of the TUNEL signal (green) with the microglial marker CD11b (red) was observed. Hoechst 33258 was used as a nuclear counterstain (blue). (I) Quantitation of TUNEL-positive cells per square millimeter of dentate gyrus in wild-type (filled bars) and iNOS–/– (open bars) mice infected with D39, spxB, or carB bacteria. Data represent three to six animals per condition. *, P = 0.037 for comparison with D39 in wild-type mice; #, P = 0.021 for comparison with D39 in wild-type mice.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Genes for bacterial enzymes with potential influence on the generation of H₂O₂ and NO were disrupted by insertion-duplication mutagenesis. As previously reported, the disruption of pyruvate oxidase in spxB pneumococci led to a significant reduction of H₂O₂ release (27, 33). Conversely, disruption of NADH oxidase (nox, an enzyme required for the complete reduction of O₂ to H₂O) had no effect on H₂O₂, suggesting that, at least under the conditions in culture, these pathways do not contribute significantly to the release of H₂O₂ by S. pneumoniae. NO release by S. pneumoniae was also detected and is a novel observation in the context of potential bacterially induced cytotoxicity. Presumably because carbamoyl phosphate is required for the synthesis of citrulline as a precursor of arginine (13), lack of carbamoyl phosphate synthase activity resulted in reduced ability of S. pneumoniae to release NO. Surprisingly, we also observed markedly reduced H₂O₂ release by a carB mutant. The mechanism of this effect is unclear but does not arise from polar effects, since the gene is not in an operon.

By infecting microglial cell cultures with the mutant pneumococcal strains, we were able to demonstrate their relative contributions to radical production. A significant link between the absolute concentration of H₂O₂ and the rate of eukaryotic cell death was identified. The H₂O₂ concentration was lower in supernatants from D39-infected microglia than from bacteria cultured alone, suggesting a cellular capacity to either partially scavenge or chemically degrade bacterially derived H₂O₂. In support of the previously described role of H₂O₂ as a pneumococcal exotoxin (5, 10), microglia were not identified as a relevant source of H₂O₂ in these experiments. In contrast, eukaryotic cells were identified as the primary source of damaging NO.

H₂O₂ toxicity is generally thought to involve the generation of hydroxyl (HO) radicals by interacting with Fe²⁺ ions (Fenton's reaction), ultimately leading to peroxidation and cross-linking of cellular and mitochondrial membrane lipids. However, we hypothesized that in infected microglial cultures, bacterial H₂O₂ could interact with iNOS-derived NO to form ONOO^–. As a footprint of ONOO^– formation, 3-nitrotyrosine was detected in D39-infected microglial cultures. Using spxB bacteria in combination with a pharmacological inhibitor of iNOS, we found a contribution of both bacterial H₂O₂ and host NO to the formation of ONOO^–. In these experiments, inhibition of NO production with aminoguanidine appeared to have as strong an effect on nitrotyrosine formation as lack of bacterial pyruvate oxidase activity.

The significance of peroxynitrite as an agent of microglial toxicity was then determined by comparing the survival of cells exposed to H₂O₂ or NO individually or in combination as ONOO^–. Bacterial H₂O₂ had a greater impact on the loss of mitochondrial and cellular integrity than the formation of NO, and indeed, cellular damage showed no strong correlation with the detection of nitrotyrosine. Therefore under the conditions of the microglial culture system, it appears that pneumococcal H₂O₂ participates in the formation of ONOO^– but that ONOO^– is not a required intermediate for H₂O₂ toxicity.

To determine the influence of host NO and pneumococcal H₂O₂ on neuronal cell death in vivo, we performed experiments in C57BL/6 and iNOS^–/– mice. Neuronal loss in the dentate gyrus has been demonstrated in human autopsy tissue after meningitis and in different experimental animals (24, 25). Our finding of increased local nitrotyrosine formation suggests a role for peroxynitrite in meningitis-induced neuronal damage in the hippocampus. Here we found additional evidence supporting an interplay of bacterial H₂O₂ and host NO, resulting in the formation of highly toxic peroxynitrite. Marked upregulation of iNOS mRNA at 24 h after meningitis induction was previously demonstrated in hippocampus-enriched brain tissue by using reverse transcription-PCR (36). As with the cell culture findings, significant neuroprotection was observed with removal of either host iNOS or pneumococcal H₂O₂. Neuronal damage was paralleled by the formation of nitrotyrosine, but apoptotic nuclei were not uniformly colocalized with the nitrotyrosine signal. From the reduction of the nitrotyrosine signal in the respective knockout experiments, bacterial H₂O₂ and host iNOS-derived NO are likely to contribute to the formation of ONOO^– but do not appear to be exclusive sources. Interestingly, no nitrotyrosine signal was observed in meningitis due to the carB mutant, pointing to a possible additional role of bacterial NO production.

We conclude that in vivo and in vitro, bacterial and eukaryotic oxidants contribute to host toxicity, that part of this toxicity is related to ONOO^– formation, and that part of the nitrotyrosine signal is the consequence of an interplay between host NO and bacterial H₂O₂.

	ACKNOWLEDGMENTS

 
This work was supported by NIAID grants R01 27913 and 39482 (both to E.I.T.), Deutsche Forschungsgemeinschaft grant SFB 507/B6 (to J.R.W.), the Meningitis Research Foundation (to J.R.W.), the American Lebanese Syrian Associated Charities (to E.I.T.), and the Hermann and Lilly Schilling Foundation (to J.R.W.).

The authors do not have a commercial or other association that might pose a conflict of interest.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Neurology, Charité—Universitaetsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Phone: 49 30 450 560192. Fax: 49 30 450 560942. E-mail: joerg.weber{at}charite.de.

Editor: J. N. Weiser

O.H. and J.Z. contributed equally to this paper.

E.I.T. and J.R.W. contributed equally to this paper.

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