Reactive oxygen and nitrogen species are produced by the human immune system in response to infection. Methods to detoxify these reactive species are vital to the survival of human pathogens, such as Neisseria meningitidis, which is the major aetiological agent of bacterial meningitis. Following activation, macrophages produce superoxide (O2−), hydrogen peroxide (H2O2) and nitric oxide (NO). The toxicity of O2−, generated using X/Xo (xanthine/xanthine oxidase), and H2O2 was investigated in the presence and absence of the NO donor DEA-NONOate [2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt]. Most of the toxicity from X/Xo was due to H2O2. In N. meningitidis, NO decreased the toxicity of the H2O2. In contrast, in the enteric bacterium Escherichia coli, NO increased the toxicity of the H2O2.
- hydrogen peroxide
- Neisseria meningitidis
- nitric oxide
- nitrosative stress
- oxidative stress
Nitrosative stress occurs when micro-organisms inhabit environments where nitric oxide (NO) and related molecules are found. Oxidative stress is mediated by partially reduced states of oxygen, particularly superoxide (O2−), peroxide/hydrogen peroxide (O22−/H2O2), and the hydroxyl radical (OH•). These stresses can occur simultaneously, such as in a microaerobic environment where denitrification is occurring, although there is no consensus on the impact of the combined effect of oxidative and nitrosative stresses on microbes.
Neisseria meningitidis is an exclusively human pathogen that is likely to encounter both nitrosative and oxidative stresses during its life cycle. The nasopharyngeal mucosa it colonizes is rich in macrophages, which produce O2− and NO following activation. Additionally, NO is likely to be encountered as N. meningitidis is a microaerophilic denitrifier . Oxidative stress defences in N. meningitidis include two superoxide dismutases (sodB and sodC), glutathione peroxidase (gpxA) and catalase (kat) , whereas NO reductase (norB) and to a lesser extent cytochrome c′ (cycP) detoxify NO .
Survival of N. gonorrohoeae within activated macrophages is independent of the known oxidative stress defences, as mutants in genes encoding these detoxification systems were not killed more readily than wild-type . In contrast, sodC enhanced the ability of N. meningitidis to avoid internalization by macrophages . Furthermore, norB and cycP increased survival of N. meningitidis in activated macrophages and in a nasopharyngeal mucosa culture system . In the present study, we further analyse the effects of combined oxidative and nitrosative stress on N. meningitidis, in comparison with the model enteric bacterium Escherichia coli.
N. meningitidis strain MC58 was used as wild-type. Mutants were made by standard methods. N. meningitidis strains were cultured on Columbia agar (Oxoid) supplemented with 5% horse blood (Oxoid) at 37°C in an atmosphere of 5% CO2. E. coli strain W3110 was grown on Luria–Bertani agar at 37°C.
Chemicals were obtained from Sigma, with the exception of DEA-NONOate [2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt; A.G. Scientific, San Diego, CA, U.S.A.].
For survival assays, bacteria were grown to mid-exponential phase, centrifuged to pellet cells and resuspended in PBS (pH 7.4) at approx. 1×108 c.f.u. (colony-forming units)/ml. This cell suspension (100 μl) was added to 100 μl of PBS (pH 7.4), in a 96-well plate, containing agents of oxidative and nitrosative stress. Xanthine was used at 2 mM, xanthine oxidase at 400 m-units/ml, DEA-NONOate at 2 mM and catalase at 160 units/ml. H2O2 was used at 1 mM in N. meningitidis and 20 mM in E. coli. At 0, 30 and 60 min, 20 μl was taken and cell viability was assessed by serial dilutions and plating.
X/Xo (xanthine/xanthine oxidase) and DEA-NONOate are donors of extracellular O2− and NO respectively. A survival assay was optimized for X/Xo such that the concentrations used resulted in two to three orders of magnitude reduction in N. meningitidis strain MC58 over 1 h (Figure 1). The concentration of DEA-NONOate used was that used by others .
Surprisingly, when N. meningitidis strain MC58 was simultaneously exposed to X/Xo and DEA-NONOate, less killing was observed than with X/Xo alone (Figure 1A). A decrease in sensitivity to X/Xo and DEA-NONOate, compared with X/Xo alone, was also found in sodC, norB and cycP mutants (results not shown). The presence of both O2− and NO donors suggests that peroxynitrite (ONOO−) might be formed. An efficient ONOO− detoxification system might explain the reduced sensitivity to both X/Xo and DEA-NONOate. However, N. meningitidis strains with mutations in potential ONOO− detoxification systems (gpxA or bcp) also exhibited a decreased sensitivity to X/Xo and DEA-NONOate, compared with X/Xo alone (results not shown).
For comparison, the toxicity of X/Xo and/or DEA-NONOate in E. coli strain W3110 was investigated. When cells were exposed to X/Xo and DEA-NONOate simultaneously, the protective effect observed for N. meningitidis was not seen (Figure 1B). Instead, the cells were more sensitive to X/Xo and DEA-NONOate than to X/Xo or DEA-NONOate alone.
Bacteria exposed to X/Xo suffer toxicity from both H2O2 and O2−, so to help explain the observations from X/Xo studies, survival assays with H2O2 were performed (Figure 2). N. meningitidis was much more sensitive to H2O2 than E. coli. Interestingly, N. meningitidis was more sensitive to H2O2 alone than both H2O2 and DEA-NONOate, yet E. coli was more sensitive to H2O2 and DEA-NONOate than H2O2 alone (Figure 2). In N. meningitidis, catalase eliminated the toxicity of H2O2, with and without DEA-NONOate, and reduced the toxicity of X/Xo, with and without DEA-NONOate.
Simultaneous exposure to donors of NO and O2− might be expected to lead to an increased toxicity compared with either of these species individually, since NO and O2− react rapidly to form ONOO−, which is highly oxidative and damaging to proteins, lipids and nucleic acids. However, in the present study, H2O2, and not O2−, was largely responsible for the toxicity of X/Xo. E. coli were very sensitive to simultaneous exposure to H2O2 and DEA-NONOate, which is consistent with previous reports [6,7]. In contrast, N. meningitidis was much more sensitive to H2O2 alone.
The apparent resistance of N. meningitidis to the O2− produced by X/Xo may be due to an effective O2− detoxification system although the sodC mutant was less sensitive to X/Xo and DEA-NONOate compared with X/Xo alone. The resistance of N. meningitidis to X/Xo and DEA-NONOate may suggest that this organism has an effective ONOO− detoxification system, but it is noteworthy that N. meningitidis strains containing mutations in genes predicted to encode peroxynitrite detoxification enzymes were also protected from the toxicity of X/Xo by DEA-NONOate.
The results of the present study indicate a positive role for NO by preventing the damage associated with H2O2. The decrease in H2O2 toxicity in the presence of NO has also been observed for animal and plant cell lines [8,9] although the mechanism explaining this observation is not clear. The protection afforded by NO is likely to be due to NO scavenging species involved in oxidative stress with three possible targets being: (i) a direct interaction between H2O2 and NO, (ii) interaction with metal ions to prevent the formation of reactive species through Fenton chemistry and (iii) interaction with reactive oxygen species formed by the Fenton chemistry . A direct interaction between H2O2 and NO is unlikely to explain the reduced toxicity in N. meningitidis, as we would expect to observe a similar decrease in E. coli, which was not observed.
Hydroxyl radicals generated from H2O2 via the Fenton chemistry are extremely damaging but the reaction of OH• with NO may decrease the impact of Fenton chemistry on cellular viability . The amount of OH• produced will be influenced by the amount of iron present. Differences in the toxicity of H2O2 and NO in N. meningitidis and E. coli may relate to differences in intracellular iron, as well as the detoxification systems that the organisms have available to deal with the consequences of Fenton chemistry. Further work will focus on the role of iron in the toxicity of H2O2, with and without DEA-NONOate.
J.M. acknowledges the Biotechnology and Biological Sciences Research Council for funding this work through grant BBS/B/02835. A plasmid containing the sodC::tet construct was kindly provided by Kate Seib and Michael Jennings (University of Queensland, Brisbane, Australia).
The 11th Nitrogen Cycle Meeting 2005: Independent Meeting held at Estación Experimental del Zaidín, Granada, Spain, 15–17 September 2005. Organized and Edited by E.J. Bedmar (Granada, Spain), M.J. Delgado (Granada, Spain) and C. Moreno-Vivián (Córdoba, Spain).
Abbreviations: c.f.u., colony-forming units; DEA-NONOate, 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt; X/Xo, xanthine/xanthine oxidase
- © 2006 The Biochemical Society