Protective role of D-amino acid oxidase against Staphylococcus aureus infection

Abstract

D-Amino acid oxidase (DAO) is a hydrogen peroxide-generating enzyme that uses a D-amino acid as a substrate. We hypothesized that DAO may protect against bacterial infection, because hydrogen peroxide is one of the most important molecules in the antibacterial defense systems in mammals. We show here that DAO suppressed the growth of Staphylococcus aureus in a manner that depended on the concentration of DAO and D-amino acid in vitro. Addition of catalase abolished the bacteriostatic activity of DAO. Although DAO plus D-Ala showed less bactericidal activity, addition of myeloperoxidase (MPO) greatly enhanced the bactericidal activity of DAO. Furthermore, DAO was able to utilize bacterial lysate, which contains D-Ala derived from peptidoglycan; this could produce hydrogen peroxide with, in the presence of myeloperoxidase, formation of hypochlorous acid. This concerted reaction of DAO and MPO led to the bactericidal action. In vivo experiments showed that DAO(-/-) (mutant) mice were more susceptible to S. aureus infection than were DAO(+/+) (wild-type) mice. These results suggest that DAO, together with myeloperoxidase, may play an important role in antibacterial systems in mammals.

Fig 1

Bacteriostatic activity of DAO against S. aureus. (a and b) S. aureus (1 × 10⁶ CFU/ml), which was grown until the mid-log phase in SCD broth, was incubated with increasing concentrations of DAO in the presence of 10 mM d-Ala (a) or increasing concentrations of d-Ala in the presence of 10 μg/ml DAO (b) in SCD broth at 37°C for 5 h, and then the optical density at 570 nm was measured. (c) S. aureus (1 × 10⁶ CFU/ml) was incubated with DAO and d-Ala, with or without 1 mg/ml (5,000 to 10,000 U/ml) of bovine catalase, and then the optical density at 570 nm was measured. (d and e) S. aureus (1 × 10⁶ CFU/ml) was incubated with DAO in the presence of d-amino acids (d-Pro [0.3 to 10 mM], d-Glu [10 mM], or d-Asp [10 mM]) in SCD broth at 37°C for 5 h, and then the optical density at 570 nm was measured. The values are means and standard deviations (SD). **, statistically significant differences (P < 0.01) by Student's t test.

Fig 2

Generation of HOCl and bacterial killing by the DAO–MPO–d-amino acid system. (a and d) S. aureus (1 × 10⁶ CFU/ml) was incubated with DAO (10 to 50 μg/ml) and d-amino acid (d-Ala [0.3 to 5 mM], d-Pro [10 mM], d-Glu [10 mM], or d-Asp [10 mM]) in 10 mM phosphate-buffered saline (pH 7.4) at 37°C for 30 min, and then each reaction mixture was diluted in physiological saline, mixed with warm SCD agar, and plated on petri dishes. The bacterial colonies were counted after overnight culture at 37°C. The values are expressed relative to the control. (b) DAO (200 μg/ml) was incubated with or without MPO in 50 mM phosphate citrate buffer (pH 5.5) for the indicated time in the system containing 10 mM d-Ala and 0.1 mg/ml SBT, an indicator of HOCl generation, and absorbance at 675 nm was then measured. (c, e, and f) S. aureus (1 × 10⁶ CFU/ml) was incubated with a combination of DAO (10 to 200 μg/ml), d-amino acid (d-Ala [0.6 to 10 mM], d-Pro [0.6 to 10 mM], d-Glu [10 mM], or d-Asp [10 mM]), and MPO (3.2 U/ml) for 30 min in 50 mM phosphate citrate buffer (pH 5.5). The viable bacteria were counted by colony-forming assay as described above. The values are means and SD. N.D., not detected. ** and *, statistically significant differences (P < 0.01 and P < 0.05, respectively) by Student's t test.

Fig 3

Utilization of bacterial cell wall-derived d-amino acid by DAO and generation of the bactericidal molecule HOCl. (a) Lysate of S. aureus (1 mg/ml) was incubated with 0.2 mg/ml DAO for 30 min at 37°C, and then pyruvic acid production was quantified as described in Materials and Methods. (b) Lysate of S. aureus (0.5 to 1 mg/ml) was incubated with DAO (5 μg/ml) in 50 mM phosphate buffer at 25°C for the indicated time to generate H₂O₂. H₂O₂ was measured by use of peroxidase-coupled oxidation of o-dianisidine (460 nm) as described in Materials and Methods. (c) Lysate of S. aureus (1 mg/ml) was incubated with DAO (200 μg/ml) plus MPO (1.6 U/ml) in the presence of SBT in acetate buffer (pH 5.5), and then absorbance at 675 nm was measured for HOCl generation. (d) S. aureus (1 × 10⁶ CFU/ml) was incubated with the lysate of S. aureus (0.5 to 1.0 mg/ml), DAO (10 to 25 μg/ml), and MPO (1.6 U/ml) in 50 mM phosphate citrate buffer (pH 5.5) at 37°C for 30 min. Each reaction mixture was diluted with physiological saline, mixed with warm SCD agar, and plated on petri dishes. The bacterial colonies were counted after overnight culture at 37°C. The values are means and SD. **, statistically significant differences (P < 0.01) by Student's t test.

Fig 4

The Dao variant gene in ddY mice and its role in bacterial infection. (a) DNA sequences of the Dao genes in wild-type (DAO^+/+) and mutant (DAO^−/−) mice. Asterisks indicate identical nucleotide sequences. (b) The mouse Dao gene was amplified via a pair of 5′-mDAO and 3′-mDAO primers, followed by digestion with PflMI restriction enzyme. These digested fragments were applied to gel electrophoresis in a 1% agarose gel. (c) DAO activity in kidney homogenates was measured as described in Materials and Methods. (d) Fate of S. aureus after injection of 1 × 10⁷ CFU/mouse. Three days after i.v. bacterial infection, viable bacteria in kidney homogenates were counted as described in Materials and Methods (n = 7 to 8). The values are means and SD. **, statistically significant differences (P < 0.01) by Student's t test. (e) Female ddY mice, 6 weeks old, were injected with S. aureus, 1 × 10⁸ CFU/mouse, via the tail vein. The survival rates of the DAO^+/+ and DAO^−/− groups were checked daily (n = 9). *, statistically significant differences (P < 0.05) by the chi-square test.

Fig 5

DAO activity of mouse peritoneal neutrophils and antibacterial activity of DAO^−/− and DAO^+/+ mouse peritoneal neutrophils. (a) Peritoneal neutrophils were elicited using 6% casein sodium salt. The DAO activities of mouse neutrophils and kidney were measured, as evaluated by pyruvic acid formation. (b) S. aureus (4 × 10⁶ CFU/ml) was incubated with or without equal numbers of mouse peritoneal neutrophils (1:1) at 37°C for 30 min. Serial dilutions were mixed with SCD agar on petri dishes, and the bacterial colonies were counted after overnight culture at 37°C. The values are means and SD. **, statistically significant differences (P < 0.01) by Student's t test.