Ozone production by amino acids contributes to killing of bacteria

Abstract

Reactive oxygen species produced by phagocytosing neutrophils are essential for innate host defense against invading microbes. Previous observations revealed that antibody-catalyzed ozone formation by human neutrophils contributed to the killing of bacteria. In this study, we discovered that 4 amino acids themselves were able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway, at comparable level to antibodies. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in our distinct cell-free system and in human neutrophils. The results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defense system, when the host is challenged by high doses of infectious agents. Our findings provide biological insights into the killing of bacteria by neutrophils.

Fig. 1.

Ozone production by immunoglobulins and amino acids in the cell-free system. Indigo carmine was irradiated with UVA in the presence of 6FP. An oxidant with the chemical signature of ozone produced by the addition of immunoglobulins or amino acids converted indigo carmine to isatin sulfonic acids. Loss of indigo carmine was monitored by measuring its absorbance at 610 nm. (A) Effect of Ig, the portion of F(ab)′₂ of antibodies, albumin, or FMLP in the presence of 6FP on ozone production. The data represent mean values ± SD (n = 3; *, P < 0.05, paired t test). (B) Effect of zVAD-fmk in the presence of 6FP on ozone production. The experiments were performed at least 3 times, and representative data are shown. (C) Effect of catalase in the presence of 6FP on IgG-mediated ozone production. The experiments were performed at least 3 times, and representative data are shown. (D) Effect of water-soluble amino acids in the presence of 6FP on ozone production. Representative data are shown. (E) Dose–response curves. Increasing concentrations of Trp, Met, Cys, or His (1 μM to 2 mM) were added to the reaction in the presence of 6FP. (F) Effect of scavengers of singlet oxygen, sodium azide, and edaravone on amino acid (Trp, Met, Cys, or His)-mediated ozone production. The data represent mean values ± SD (n = 3; *, P < 0.05; **, P < 0.01; paired t test). (G) Effect of catalase on amino acid (Trp, Met, Cys, or His)-mediated ozone production. The data represent mean values ± SD (n = 3).

Fig. 2.

HPLC and mass spectral analysis of ozone production in the cell-free system. (A and B) HPLC analysis. Indigo carmine was added to 6FP and Met with (B) or without (A) UVA irradiation. Arrows indicate a peak of indigo carmine (A) and isatin sulfonic acid (B). (C and D) HPLC analysis. Vinylbenzoic acid was added to 6FP and Met with (D) or without (C) UVA irradiation. Arrows indicate a peak of vinylbenzoic acid (C) and 4-carboxybenzaldehyde (D). (E–G) Mass spectral analysis. Indigo carmine was added to 6FP in a reaction mixture containing H₂¹⁶O in the presence of Met (E), H₂¹⁸O in the absence (F) or presence (G) of Met and irradiated with UVA. Note the presence of the mass peak 230 in G.

Fig. 3.

Ozone produced by amino acids kills bacteria in the cell-free system. E. coli were incubated with or without 6FP-tBu-DMF and amino acids under UVA irradiation for 2 h. (A) Effect of Trp or Met on the survival of E. coli. The addition of both 6FP-tBu-DMF and amino acids exhibited strong bactericidal activity after a 2-h irradiation. (B) Effect of Arg or Phe on the survival of E. coli. Note that the amino acids showed no effects on bactericidal activity.

Fig. 4.

H₂O₂ levels in the cell-free system. (A) H₂O₂ levels generated by 6FP-tBu-DMF and Trp after 2-h irradiation. Note that H₂O₂ production was completely repressed by catalase treatment. The data represent mean values ± SD (n = 3). (B) Concentration-dependent toxicity of H₂O₂ on the viability of E. coli. Increasing concentrations of H₂O₂ (0–10³ mM) were added to the E. coli. The experiments were performed at least 3 times, and representative data are shown.

Fig. 5.

Production of singlet oxygen with very little superoxide in a variant type of gp91-phox-deficient CGD neutrophils. (A) SOD-inhibitable reduction of ferricytochrome c in control and CGD neutrophils. Superoxide release was analyzed in unstimulated, OZ-stimulated, or PMA-stimulated neutrophils. (B) DHR assay in neutrophils from a healthy control (Top and Middle) and a CGD patient (Bottom). In Middle the pretreatment of control neutrophils with DPI, an inhibitor of NADPH-oxidase, is revealed. Fluorescence intensity is shown on the logarithmic x axis, and the cell count is shown on the y axis. (C) Superoxide (Upper) and singlet oxygen (Lower) release from control and CDG neutrophils. Neutrophils were incubated with CLA for superoxide detection or MVP for singlet oxygen detection, and luminescence was monitored every 30 s for 30 min. Some control neutrophils were pretreated with DPI for the CLA (Upper) or with ABAH, an inhibitor of MPO, for the MVA (Lower).

Fig. 6.

Ozone production in a variant type of gp91-phox-deficient CGD neutrophils. (A) Effect of Trp on ozone production in activated CGD neutrophils. Indigo carmine was incubated with unstimulated or PMA-stimulated CGD neutrophils. Trp was added to PMA-stimulated CGD neutrophils to analyze ozone production. Loss of indigo carmine was monitored by measuring its absorbance at 610 nm. Note that a scavenger of singlet oxygen, edaravone, partially suppressed the reaction. The experiments were performed at least 3 times, and representative data are shown. (B) HPLC analysis of 4-carboxybenzaldehyde in CGD neutrophils. PMA-stimulated CGD neutrophils with Trp administration produced 4-carboxybenzaldehyde from vinylbenzoic acid.

Fig. 7.

Ozone produced by amino acids augments the bactericidal activity of neutrophils. (A) Bactericidal activity of CGD and healthy control neutrophils (PMN). E. coli were incubated with CGD or control neutrophils for 2 h. (B and C) Effect of Trp on the bactericidal activity of CGD (B) and healthy control (C) neutrophils. CGD or control neutrophils were challenged with increasing amounts of E. coli at a ratio of 1:1, 1:5, or 1:10 in the presence or absence of Trp. The data represent mean values ± SD (n = 3; *, P < 0.05; **, P < 0.01; paired t test). (D) H₂O₂ levels produced by CGD and healthy control neutrophils. The experiments were performed at least 3 times, and representative data are shown.