The role of catalase in gonococcal resistance to peroxynitrite

Abstract

We have reported that Neisseria gonorrhoeae is extremely resistant to reactive nitrogen species (RNS) including peroxynitrite (PN). Recent literature suggests that catalase can provide protection against commercial preparations of PN. Though wild-type gonococci were shown to be highly resistant to 2 mM PN, Neisseria meningitidis and a gonococcal katA mutant were both shown to be extremely sensitive to 2 mM PN. Analysis of translational fusions to lacZ of the catalase promoters from N. gonorrhoeae and N. meningitidis demonstrated that basal katA expression from gonococci is 80-fold higher than in meningococci, though meningococcal katA retains a greater capacity to be activated by OxyR. This activation capacity was shown to be due to a single base pair difference in the -10 transcription element between the two kat promoters. PN resistance was initially shown to be associated with increasing catalase expression; however, commercial preparations of PN were later revealed to contain higher levels of contaminating hydrogen peroxide (H2O2) than expected. Removal of H2O2 from PN preparations with manganese dioxide markedly reduced PN toxicity in a gonococcal katA mutant. Simultaneous treatment with non-lethal concentrations of PN and H2O2 was highly lethal, indicating that these agents act synergistically. When treatment was separated by 5 min, high levels of bacterial killing occurred only when PN was added first. Our results suggest that killing of N. gonorrhoeae ΔkatA by commercial PN preparations is likely due to H2O2, that H2O2 is more toxic in the presence of PN, and that PN, on its own, may not be as toxic as previously believed.

Fig. 1.

Survival following 2 mM PN exposure and catalase activity of N. gonorrhoeae and N. meningitidis strains. All cultures were grown to exponential phase and diluted to OD₆₀₀ 0.2. One millilitre was exposed to 2 mM PN and grown out for 45 min before plating to determine viability (a), and 2 ml was used to determine catalase activity (b). For strains incubated with IPTG, cultures were exposed to 0.2 mM IPTG for 90 min before determination of survival and catalase activity. Strains: F62 (parental), ΔkatA (ΔkatA), katA-/+ (ΔkatA strain containing a chromosomal insertion of a P_lac-inducible katA allele), MC58 (meningococcal strain). Results are representative of at least three independent determinations; error bars, sd.

Fig. 2.

Schematic representation of the gonococcal and meningococcal katA upstream regions. The OxyR binding site, promoter elements and transcription start site are based on those described for meningococcal katA (Ieva et al., 2008).

Fig. 3.

Catalase activity and survival of inducible katA, katE and katG strains of F62 ΔkatA. Exponential phase cultures were diluted to OD₆₀₀ 0.2 and exposed to the indicated concentration of IPTG. After 90 min, the cultures were diluted back to OD₆₀₀ 0.2. (a) An aliquot of the culture was used for measuring catalase activity. Data are representative of three separate experiments. (b) The remainder of the culture was exposed to 2 mM PN and grown out for 45 min before determination of viability. Results are plotted as survival versus catalase activity. Data points are individual values determined from three repetitions of the experiment. Symbols used: gonococcal katA (open and closed diamonds), E. coli katE (open and closed circles), E. coli katG (open and closed triangles).

Fig. 4.

Dose–response of individual and combined PN/H₂O₂. All cultures were grown to exponential phase and diluted to OD₆₀₀ 0.2 in GCK (0.042 % NaHCO₃). (a) Cultures were exposed to a range of PN concentrations (0–1.75 mM), with (squares) or without (diamonds) addition of 0.2 mM H₂O₂. (b) Cultures were exposed to a range of H₂O₂ concentrations (0–0.3 mM), with (squares) or without (diamonds) immediate previous addition of 2.0 mM PN. Results are representative of one of three independent trials, all of which generated a similarly shaped curve.

Fig. 5.

Survival of F62 ΔkatA after exposure to PN in the presence or absence of sodium bicarbonate. (a) Cultures were grown to exponential phase in GCK (0.042 % NaHCO₃), filtered, diluted to OD₆₀₀ 0.2 in fresh medium containing 0.042 % NaHCO₃, and treated with H₂O₂ and/or PN. (b) Cultures were grown to exponential phase in GCK (0.042 % NaHCO₃), filtered, diluted to OD₆₀₀ 0.2 in fresh medium lacking NaHCO₃, and treated with H₂O₂ and/or PN. All cultures were allowed to grow out for 45 min before plating to determine viability. Abbreviations: NA (no addition), PN (2 mM PN), H (0.2 mM H₂O₂), PN+H (simultaneous 0.2 mM H₂O₂/2 mM PN treatment). Results are representative of three independent determinations; error bars, sd.

Fig. 6.

Survival of F62 Δkat following sequential exposure to PN and H₂O₂. All cultures were grown to exponential phase and diluted to OD₆₀₀ 0.2 in GCK (0.042 % NaHCO₃). Abbreviations: NA (no addition), H→PN (0.2 mM H₂O₂ addition followed by 2 mM PN addition 5 min later), PN→H (2 mM PN addition followed by 0.2 mM H₂O₂ addition 5 min later), PN+H (simultaneous 0.2 mM H₂O₂/2 mM PN addition). All cultures were allowed to grow out for 45 min before plating to determine viability. Results are representative of three independent determinations; error bars, sd.