Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide

Abstract

Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced by S. oralis is sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2 is a cytotoxin. In the present study, we investigated whether streptococcal H2O2 impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death. S. oralis infection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2 also induced cell death. Infection with either a mutant lacking spxB, which encodes pyruvate oxidase responsible for H2O2 production, or Streptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection with S. oralis or exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection with S. oralis or exposure to H2O2 The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2 induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells.

FIG 1

Death of RAW 264 macrophages. RAW 264 cells were exposed to the S. oralis WT or spxB KO mutant, S. mutans MT8148, or H₂O₂ for 3 h. Following washing steps, the cells were cultured in fresh medium containing antibiotics for 21 h (see also Fig. S1 in the supplemental material). Viable cells were counted after trypan blue staining. Data are shown as the mean ± SD for triplicate samples. *, P < 0.05 compared with the untreated control (None). The results of live/dead fluorescent staining are shown in Fig. S3 in the supplemental material.

FIG 2

H₂O₂ concentration in culture medium of RAW 264 cells infected with S. oralis. (A) RAW 264 cells were infected with the S. oralis WT for 3 h. The cells were washed with PBS and cultured for an additional 21 h (total, 24 h) in fresh medium containing antibiotics. The H₂O₂ concentration of the culture supernatants was determined using a hydrogen peroxide colorimetric detection kit. (B) The H₂O₂ concentration of the culture supernatants of S. oralis cultured in the same medium was also determined.

FIG 3

S. oralis and H₂O₂ mediate lysosomal damage. RAW 264 cells were exposed to the S. oralis WT or H₂O₂ for 3 h. Then, the cells were cultured for an additional 3 h (total, 6 h) in fresh medium containing antibiotics. At 1, 3, and 6 h after exposure, the cells were stained with LysoTracker red and SYBR green II. Lysosomal deacidification was monitored using LysoTracker red (red), which accumulates in acidic organelles. Bar = 20 μm.

FIG 4

The H₂O₂ produced by S. oralis induces lysosomal deacidification. (A) RAW 264 cells were exposed to the S. oralis WT or spxB KO mutant, S. mutans MT8148, or H₂O₂ for 3 h. (Top) The cells were stained with LysoTracker red and SYBR green II or acridine orange. LysoTracker red accumulates in acidic organelles. (Bottom) The acidic environment in lysosomes was also monitored by acridine orange staining. Acridine orange emits red fluorescence in acidic environments, and thus, the disappearance of red fluorescence reflects lysosome deacidification. Bar = 20 μm. (B) Measurements of the LysoTracker-stained fluorescent areas were conducted using ImageJ software. The average fluorescence area of control cells (None) was set to 100%. The results are shown as the mean ± SD for four samples. *, P < 0.05 compared with the untreated control (None).

FIG 5

S. oralis and H₂O₂ mediate lysosomal destruction. RAW 264 cells were exposed to the S. oralis WT or H₂O₂ for 3 h and cultured for an additional 3 and 21 h (total, 6 and 24 h, respectively) in fresh medium containing antibiotics. At the indicated times after exposure, nuclei and LAMP-1 were fluorescently labeled with DAPI and Alexa Fluor 488-conjugated anti-LAMP-1 monoclonal antibody, respectively. Bar = 10 μm.

FIG 6

The H₂O₂ produced by S. oralis induces lysosomal destruction. (A) RAW 264 cells were exposed to the S. oralis WT or spxB KO mutant, S. mutans MT8148, or H₂O₂ for 3 h. The cells were then washed and cultured for an additional 3 h in fresh medium containing antibiotics. (Top) LAMP-1 and DNA were labeled with Alexa Fluor 488-conjugated anti-LAMP-1 monoclonal antibody and DAPI, respectively. (Bottom) Cathepsin B was also labeled with Alexa Fluor 594-conjugated anti-goat IgG. Bar = 10 μm. (B) Measurements of the LAMP-1-positive areas were conducted using ImageJ software. The average fluorescence area for untreated control cells (None) was set to 100%. The results are shown as the mean ± SD for four samples. *, P < 0.05 compared with the control (None).

FIG 7

Effect of catalase on lysosomal damage in S. oralis-infected macrophages. RAW 264 cells pretreated with 50 or 200 U ml⁻¹ of catalase were infected with viable S. oralis WT for 3 h. The cells were washed with PBS and cultured in fresh medium containing catalase and antibiotics for 21 h. (A) Viability was determined by trypan blue staining. *, P < 0.05 compared with the untreated control (None). (B) The cells were stained with LysoTracker after 3 h of infection (top) and were also immunostained using LAMP-1 antibodies after 6 h of infection (bottom). Bar = 10 μm.

FIG 8

Effect of deferoxamine on lysosomal damage in S. oralis-infected macrophages. RAW 264 cells were treated with 0.5 or 2 mM deferoxamine and then infected with viable S. oralis strains for 3 h. The cells were washed with PBS and cultured in fresh medium containing antibiotics for 21 h. (A) Viability was determined by trypan blue staining. *, P < 0.05 compared with the untreated control (None). (B) The cells were stained with LysoTracker after 3 h of infection (top) and were also immunostained using LAMP-1 antibodies after 6 h of infection (bottom). Bar = 10 μm.

FIG 9

Effect of a cathepsin B inhibitor on cell death and detachment of dead cells. RAW 264 cells pretreated with the cathepsin B inhibitor CA-074 Me (20 μM) were exposed to the S. oralis WT (MOI = 100 or 200) or H₂O₂ (1 mM) for 3 h. The cells were washed and cultured for an additional 21 h (total, 24 h) in fresh medium containing CA-074 Me (10 μM) and antibiotics (see also Fig. S2 in the supplemental material). (A) The viable cells were counted after trypan blue staining. (B) The adherent cells (both viable and dead) were also counted. Data are shown as the mean ± SD for triplicate samples. *, P < 0.05 compared with the untreated control (None, No inhibitor). (C) The cells were stained with SYBR green II (green; nuclei) together with Alexa Fluor 594-conjugated phalloidin (red; actin filaments). Bar = 10 μm.

FIG 10

Proposed model illustrating the role of H₂O₂ in S. oralis-induced macrophage death. H₂O₂ causes lysosomal deacidification and the destruction of lysosomal integrity via Fenton's reaction, followed by leakage of cathepsins and other lysosomal hydrolytic enzymes. These cytotoxic enzymes degrade cellular components and induce self-damage in S. oralis-infected cells. Cathepsins are implicated in the detachment of the dead cells from the culture plates.