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Hydrogen peroxide produced by oral Streptococci induces macrophage cell death.

Okahashi N, Nakata M, Sumitomo T, Terao Y, Kawabata S - PLoS ONE (2013)

Bottom Line: Although the cytotoxicity of H2O2 has been widely recognized, the effects of H2O2 produced by oral streptococci on host defense systems remain unknown.Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited the cytotoxic effect of S. oralis.Furthermore, H2O2 alone was capable of inducing cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan. okahashi@dent.osaka-u.ac.jp

ABSTRACT
Hydrogen peroxide (H2O2) produced by members of the mitis group of oral streptococci plays important roles in microbial communities such as oral biofilms. Although the cytotoxicity of H2O2 has been widely recognized, the effects of H2O2 produced by oral streptococci on host defense systems remain unknown. In the present study, we investigated the effect of H2O2 produced by Streptococcus oralis on human macrophage cell death. Infection by S. oralis was found to stimulate cell death of a THP-1 human macrophage cell line at multiplicities of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited the cytotoxic effect of S. oralis. S. oralis deletion mutants lacking the spxB gene, which encodes pyruvate oxidase, and are therefore deficient in H2O2 production, showed reduced cytotoxicity toward THP-1 macrophages. Furthermore, H2O2 alone was capable of inducing cell death. The cytotoxic effect seemed to be independent of inflammatory responses, because H2O2 was not a potent stimulator of tumor necrosis factor-α production in macrophages. These results indicate that streptococcal H2O2 plays a role as a cytotoxin, and is implicated in the cell death of infected human macrophages.

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Construction of S. oralis spxB deletion mutant.(A) Black arrow indicates the gene encoding pyruvate oxidase (SMSK23_0092 spxB). A targeted deletion mutant lacking this region was constructed by allelic exchange using the temperature-sensitive shuttle vector pSET4s. (B) S. oralis ATCC35037 wild-type (WT), spxB-deletion mutant (KO), or reverse mutant (Rev) was cultured in BHI broth or 5% RPMI1640 medium at 37°C for 18 h in a 5% CO2 atmosphere. Concentrations of H2O2 in culture supernatants were quantitatively determined using a hydrogen peroxide colorimetric detection kit. Data are shown as the mean ± SD of triplicate samples. *p<0.05 as compared with concentration of wild-type strain.
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pone-0062563-g003: Construction of S. oralis spxB deletion mutant.(A) Black arrow indicates the gene encoding pyruvate oxidase (SMSK23_0092 spxB). A targeted deletion mutant lacking this region was constructed by allelic exchange using the temperature-sensitive shuttle vector pSET4s. (B) S. oralis ATCC35037 wild-type (WT), spxB-deletion mutant (KO), or reverse mutant (Rev) was cultured in BHI broth or 5% RPMI1640 medium at 37°C for 18 h in a 5% CO2 atmosphere. Concentrations of H2O2 in culture supernatants were quantitatively determined using a hydrogen peroxide colorimetric detection kit. Data are shown as the mean ± SD of triplicate samples. *p<0.05 as compared with concentration of wild-type strain.

Mentions: Pyruvate oxidase has been reported as being essential for H2O2 production in the mitis group of streptococci [5], [6], [25]. Therefore, we constructed a deletion mutant of the pyruvate oxidase gene, spxB, via allelic exchange by using a temperature-sensitive shuttle vector (Figure 3A). Deletion of the spxB gene in the mutant was verified by PCR (data not shown). Decreased production of H2O2 by the deletion mutant (spxB KO) was confirmed both in BHI broth and RPMI1640 medium containing 5% FBS at 37°C in a 5% CO2 atmosphere (Figure 3B). The production of H2O2 by the spxB revertant mutant (spxB Rev) was similar to that of a wild type (WT) strain. The mutant strains grew at rates comparable to those of the WT strain (data not shown).


Hydrogen peroxide produced by oral Streptococci induces macrophage cell death.

Okahashi N, Nakata M, Sumitomo T, Terao Y, Kawabata S - PLoS ONE (2013)

Construction of S. oralis spxB deletion mutant.(A) Black arrow indicates the gene encoding pyruvate oxidase (SMSK23_0092 spxB). A targeted deletion mutant lacking this region was constructed by allelic exchange using the temperature-sensitive shuttle vector pSET4s. (B) S. oralis ATCC35037 wild-type (WT), spxB-deletion mutant (KO), or reverse mutant (Rev) was cultured in BHI broth or 5% RPMI1640 medium at 37°C for 18 h in a 5% CO2 atmosphere. Concentrations of H2O2 in culture supernatants were quantitatively determined using a hydrogen peroxide colorimetric detection kit. Data are shown as the mean ± SD of triplicate samples. *p<0.05 as compared with concentration of wild-type strain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643943&req=5

pone-0062563-g003: Construction of S. oralis spxB deletion mutant.(A) Black arrow indicates the gene encoding pyruvate oxidase (SMSK23_0092 spxB). A targeted deletion mutant lacking this region was constructed by allelic exchange using the temperature-sensitive shuttle vector pSET4s. (B) S. oralis ATCC35037 wild-type (WT), spxB-deletion mutant (KO), or reverse mutant (Rev) was cultured in BHI broth or 5% RPMI1640 medium at 37°C for 18 h in a 5% CO2 atmosphere. Concentrations of H2O2 in culture supernatants were quantitatively determined using a hydrogen peroxide colorimetric detection kit. Data are shown as the mean ± SD of triplicate samples. *p<0.05 as compared with concentration of wild-type strain.
Mentions: Pyruvate oxidase has been reported as being essential for H2O2 production in the mitis group of streptococci [5], [6], [25]. Therefore, we constructed a deletion mutant of the pyruvate oxidase gene, spxB, via allelic exchange by using a temperature-sensitive shuttle vector (Figure 3A). Deletion of the spxB gene in the mutant was verified by PCR (data not shown). Decreased production of H2O2 by the deletion mutant (spxB KO) was confirmed both in BHI broth and RPMI1640 medium containing 5% FBS at 37°C in a 5% CO2 atmosphere (Figure 3B). The production of H2O2 by the spxB revertant mutant (spxB Rev) was similar to that of a wild type (WT) strain. The mutant strains grew at rates comparable to those of the WT strain (data not shown).

Bottom Line: Although the cytotoxicity of H2O2 has been widely recognized, the effects of H2O2 produced by oral streptococci on host defense systems remain unknown.Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited the cytotoxic effect of S. oralis.Furthermore, H2O2 alone was capable of inducing cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan. okahashi@dent.osaka-u.ac.jp

ABSTRACT
Hydrogen peroxide (H2O2) produced by members of the mitis group of oral streptococci plays important roles in microbial communities such as oral biofilms. Although the cytotoxicity of H2O2 has been widely recognized, the effects of H2O2 produced by oral streptococci on host defense systems remain unknown. In the present study, we investigated the effect of H2O2 produced by Streptococcus oralis on human macrophage cell death. Infection by S. oralis was found to stimulate cell death of a THP-1 human macrophage cell line at multiplicities of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited the cytotoxic effect of S. oralis. S. oralis deletion mutants lacking the spxB gene, which encodes pyruvate oxidase, and are therefore deficient in H2O2 production, showed reduced cytotoxicity toward THP-1 macrophages. Furthermore, H2O2 alone was capable of inducing cell death. The cytotoxic effect seemed to be independent of inflammatory responses, because H2O2 was not a potent stimulator of tumor necrosis factor-α production in macrophages. These results indicate that streptococcal H2O2 plays a role as a cytotoxin, and is implicated in the cell death of infected human macrophages.

Show MeSH
Related in: MedlinePlus