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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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Microscopic images of macrophage cell death.THP-1 macrophages were infected with S. oralis wild-type strain (WT), mutant strain defective in H2O2 production (spxB KO), or reverse mutant strain (spxB Rev) for 2 h, washed with PBS, and cultured in fresh medium containing antibiotics for 18 h. Macrophages were stained with trypan blue and Live/Dead cell staining kit. EthD-III (red fluorescence) stained the nuclear DNA of dead THP-1 cells, while calcein AM (green fluorescence) stained live cells. Bar, 50 μm.
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pone-0062563-g004: Microscopic images of macrophage cell death.THP-1 macrophages were infected with S. oralis wild-type strain (WT), mutant strain defective in H2O2 production (spxB KO), or reverse mutant strain (spxB Rev) for 2 h, washed with PBS, and cultured in fresh medium containing antibiotics for 18 h. Macrophages were stained with trypan blue and Live/Dead cell staining kit. EthD-III (red fluorescence) stained the nuclear DNA of dead THP-1 cells, while calcein AM (green fluorescence) stained live cells. Bar, 50 μm.

Mentions: In order to evaluate the contribution of H2O2 produced by S. oralis to macrophage cell death, differentiated THP-1 cells were exposed to S. oralis WT strain, spxB KO mutant, and spxB Rev mutant. Macrophages were then stained with trypan blue to determine their viability (Figure 4, left). At an MOI of 200, macrophages infected with S. oralis WT and spxB Rev strains were found dead, whereas most of the spxB KO-infected cells were still viable. Live/Dead fluorescence staining also revealed reduced cell death of macrophages infected with spxB KO mutant (Figure 4, right).


Hydrogen peroxide produced by oral Streptococci induces macrophage cell death.

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

Microscopic images of macrophage cell death.THP-1 macrophages were infected with S. oralis wild-type strain (WT), mutant strain defective in H2O2 production (spxB KO), or reverse mutant strain (spxB Rev) for 2 h, washed with PBS, and cultured in fresh medium containing antibiotics for 18 h. Macrophages were stained with trypan blue and Live/Dead cell staining kit. EthD-III (red fluorescence) stained the nuclear DNA of dead THP-1 cells, while calcein AM (green fluorescence) stained live cells. Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062563-g004: Microscopic images of macrophage cell death.THP-1 macrophages were infected with S. oralis wild-type strain (WT), mutant strain defective in H2O2 production (spxB KO), or reverse mutant strain (spxB Rev) for 2 h, washed with PBS, and cultured in fresh medium containing antibiotics for 18 h. Macrophages were stained with trypan blue and Live/Dead cell staining kit. EthD-III (red fluorescence) stained the nuclear DNA of dead THP-1 cells, while calcein AM (green fluorescence) stained live cells. Bar, 50 μm.
Mentions: In order to evaluate the contribution of H2O2 produced by S. oralis to macrophage cell death, differentiated THP-1 cells were exposed to S. oralis WT strain, spxB KO mutant, and spxB Rev mutant. Macrophages were then stained with trypan blue to determine their viability (Figure 4, left). At an MOI of 200, macrophages infected with S. oralis WT and spxB Rev strains were found dead, whereas most of the spxB KO-infected cells were still viable. Live/Dead fluorescence staining also revealed reduced cell death of macrophages infected with spxB KO mutant (Figure 4, right).

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