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Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis

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ABSTRACT

Bacterial endophthalmitis, a vision-threatening complication of ocular surgery or trauma, is characterized by increased intraocular inflammation and retinal tissue damage. Although significant vision loss in endophthalmitis has been linked to retinal cell death, the underlying mechanisms of cell death remain elusive. In this study, using a mouse model of Staphylococcus aureus endophthalmitis and cultured human retinal Müller glia (MIO-M1 cell line), we demonstrate that S. aureus caused significant apoptotic cell death in the mouse retina and Müller glia, as evidenced by increased number of terminal dUTP nick end labeling and Annexin V and propidium iodide-positive cells. Immunohistochemistry and western blot studies revealed the reduction in mitochondrial membrane potential (JC-1 staining), release of cytochrome c into the cytosol, translocation of Bax to the mitochondria and the activation of caspase-9 and -3 in S. aureus-infected retina/retinal cells. In addition, the activation of PARP-1 and the release of apoptosis inducing factor from mitochondria was also observed in S. aureus-infected retinal cells. Inhibition studies using pan-caspase (Q-VD-OPH) and PARP-1 (DPQ) inhibitors showed significant reduction in S. aureus-induced retinal cell death both in vivo and in vitro. Together, our findings demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in the both caspase-dependent and independent manners, and mitochondria have a central role in this process. Hence, targeting the identified signaling pathways may provide the rationale to design therapeutic interventions to prevent bystander retinal tissue damage in bacterial endophthalmitis.

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S. aureus induces mitochondrial translocation of Bax in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for Bax and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis for Bax (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.
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fig4: S. aureus induces mitochondrial translocation of Bax in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for Bax and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis for Bax (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.

Mentions: Having seen the release of cytochrome c in the cytoplasm, we next assessed the distribution of Bax, one of the major determinants of the mitochondrial release of cytochrome c and other apoptotic molecules such as Smac and AIF. Bax is generally sequestered in the cytosol and translocate into the mitochondria resulting into permeabilization of mitochondrial membrane and triggering the release of cytochrome c and the induction of apoptosis. As shown in Figure 4, challenge of Müller glia with S. aureus resulted into cellular redistribution of Bax with increased translocation of Bax from cytosol to the mitochondria (Figure 4a). These findings were further confirmed by subcellular fractionation and western blot analysis of Bax, showing increased presence of Bax in the mitochondrial fractions of both 4 and 8 h post S. aureus-infected cells (Figure 4b). Collectively, our data shows the redistribution of cytochrome c and Bax in response to S. aureus challenge.


Mitochondria mediates caspase-dependent and independent retinal cell death in Staphylococcus aureus endophthalmitis
S. aureus induces mitochondrial translocation of Bax in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for Bax and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis for Bax (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979429&req=5

fig4: S. aureus induces mitochondrial translocation of Bax in retinal Müller glia. MIO-M1 cells were left uninfected (control) or challenged with S. aureus RN6390 (multiplicity of infection, 10 : 1) for the indicated time points. Cells were stained with MitoTracker red (Mito Red) followed by immunostaining for Bax and observed under confocal microscope (a). In another experiment, S. aureus-challenged MIO-M1 cells were subjected to subcellular fractionation followed by western blot analysis for Bax (b). Cox 4 and β-actin antibodies were used as protein loading controls for mitochondrial and cytoplasmic fractions, respectively. Results are representative of two independent experiments.
Mentions: Having seen the release of cytochrome c in the cytoplasm, we next assessed the distribution of Bax, one of the major determinants of the mitochondrial release of cytochrome c and other apoptotic molecules such as Smac and AIF. Bax is generally sequestered in the cytosol and translocate into the mitochondria resulting into permeabilization of mitochondrial membrane and triggering the release of cytochrome c and the induction of apoptosis. As shown in Figure 4, challenge of Müller glia with S. aureus resulted into cellular redistribution of Bax with increased translocation of Bax from cytosol to the mitochondria (Figure 4a). These findings were further confirmed by subcellular fractionation and western blot analysis of Bax, showing increased presence of Bax in the mitochondrial fractions of both 4 and 8 h post S. aureus-infected cells (Figure 4b). Collectively, our data shows the redistribution of cytochrome c and Bax in response to S. aureus challenge.

View Article: PubMed Central - PubMed

ABSTRACT

Bacterial endophthalmitis, a vision-threatening complication of ocular surgery or trauma, is characterized by increased intraocular inflammation and retinal tissue damage. Although significant vision loss in endophthalmitis has been linked to retinal cell death, the underlying mechanisms of cell death remain elusive. In this study, using a mouse model of Staphylococcus aureus endophthalmitis and cultured human retinal Müller glia (MIO-M1 cell line), we demonstrate that S. aureus caused significant apoptotic cell death in the mouse retina and Müller glia, as evidenced by increased number of terminal dUTP nick end labeling and Annexin V and propidium iodide-positive cells. Immunohistochemistry and western blot studies revealed the reduction in mitochondrial membrane potential (JC-1 staining), release of cytochrome c into the cytosol, translocation of Bax to the mitochondria and the activation of caspase-9 and -3 in S. aureus-infected retina/retinal cells. In addition, the activation of PARP-1 and the release of apoptosis inducing factor from mitochondria was also observed in S. aureus-infected retinal cells. Inhibition studies using pan-caspase (Q-VD-OPH) and PARP-1 (DPQ) inhibitors showed significant reduction in S. aureus-induced retinal cell death both in vivo and in vitro. Together, our findings demonstrate that in bacterial endophthalmitis, retinal cells undergo apoptosis in the both caspase-dependent and independent manners, and mitochondria have a central role in this process. Hence, targeting the identified signaling pathways may provide the rationale to design therapeutic interventions to prevent bystander retinal tissue damage in bacterial endophthalmitis.

No MeSH data available.


Related in: MedlinePlus