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Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions.

Popova TG, Millis B, Chung MC, Bailey C, Popov SG - FEMS Immunol. Med. Microbiol. (2010)

Bottom Line: Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible.Its effect was found to be synergistic with a metabolic product of B. anthracis, succinic acid.Cell death appears to be caused by an acute primary membrane permeabilization by ALO, followed by a burst of reactive radicals from the mitochondria fuelled by the succinate, which is generated by bacteria in the hypoxic environment.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA, USA.

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Catalysts of ROS and peroxynitrite decomposition protect HSAECs against mitochondrial damage by Sups. (a) HSAECs grown on coverslips were treated for 30 min with Sups supplemented with 1 μM MitoSox (red) and 2.5 μg mL−1 Hoechst 33342 nuclear stain (blue). The cells were washed with HBSS, and the fluorescent images for control medium (left panel) and dSterne Sup-treated cells (middle panel) were recorded. The right panel shows a time course of MitoSox mean fluorescence intensity. Similar results were obtained with Sterne 34F2 Sup (not shown). (b) Cells were washed with HBSS and incubated for 30 min at 37°C, 5% CO2 with control CSFM (left panel), dSterne Sup (middle panel), and Sterne 34F2 Sup (right panel). After washing, the cells were stained with JC-1 (10 μg mL−1) and Hoechst 33342 (2.5 μg mL−1) dyes for 30 min. A dissipation of the mitochondrial potential is evident as a loss of red fluorescence characteristic of aggregated JC-1. (c–f) The Sups generated in CSFM (c–e) and DMEM/F-12 supplemented with 300 μM NaNO3 and 1 mg mL−1 BSA (f) were mixed with the indicated catalysts for 30 min and used to treat cells for 2 h. (g) Cells were preincubated with catalysts for 30 min, treated with dSterne Sup (bottom row) or control media (top row) for 30 min, and the MitoSox images were recorded as in (a). Where indicated, CD-cholesterol (1 μg mL−1) was added to the Sup and control medium for 30 min before incubation with the cells. Additional controls included CSFM titrated with HCl to pH 5.3 (left top panel) and the dSterne Sup titrated to pH 7.5 with NaOH (right bottom panel). Error bars represent 95% confidence interval of mean. A quantitative evaluation of MitoSox fluorescence is presented in Fig. S10.
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fig05: Catalysts of ROS and peroxynitrite decomposition protect HSAECs against mitochondrial damage by Sups. (a) HSAECs grown on coverslips were treated for 30 min with Sups supplemented with 1 μM MitoSox (red) and 2.5 μg mL−1 Hoechst 33342 nuclear stain (blue). The cells were washed with HBSS, and the fluorescent images for control medium (left panel) and dSterne Sup-treated cells (middle panel) were recorded. The right panel shows a time course of MitoSox mean fluorescence intensity. Similar results were obtained with Sterne 34F2 Sup (not shown). (b) Cells were washed with HBSS and incubated for 30 min at 37°C, 5% CO2 with control CSFM (left panel), dSterne Sup (middle panel), and Sterne 34F2 Sup (right panel). After washing, the cells were stained with JC-1 (10 μg mL−1) and Hoechst 33342 (2.5 μg mL−1) dyes for 30 min. A dissipation of the mitochondrial potential is evident as a loss of red fluorescence characteristic of aggregated JC-1. (c–f) The Sups generated in CSFM (c–e) and DMEM/F-12 supplemented with 300 μM NaNO3 and 1 mg mL−1 BSA (f) were mixed with the indicated catalysts for 30 min and used to treat cells for 2 h. (g) Cells were preincubated with catalysts for 30 min, treated with dSterne Sup (bottom row) or control media (top row) for 30 min, and the MitoSox images were recorded as in (a). Where indicated, CD-cholesterol (1 μg mL−1) was added to the Sup and control medium for 30 min before incubation with the cells. Additional controls included CSFM titrated with HCl to pH 5.3 (left top panel) and the dSterne Sup titrated to pH 7.5 with NaOH (right bottom panel). Error bars represent 95% confidence interval of mean. A quantitative evaluation of MitoSox fluorescence is presented in Fig. S10.

Mentions: SA is a substrate of succinate dehydrogenase (or Complex II of the mitochondrial electron transport chain), which converts succinate to fumarate. Under some pathophysiological conditions such as hypoxia, the SA-supported mitochondrial respiration could lead to the formation of damaging ROS (Schonfeld & Wojtczak, 2008). Taking into account that the Alamar Blue dye used in our cell viability test largely reflects the redox activity of the mitochondria, we hypothesized that the mitochondria were the likely targets of the Sup-induced respiratory burst, resulting in the release of ROS and consequent self-intoxication. In agreement with this suggestion, a conventional specific inhibitor of Complex II, thenoyltrifluoroacetone, demonstrated protection of cells from the toxicity of Sups (Fig. 3c). Using MitoSox Red, a peroxide-sensitive probe designed for the detection of mitochondrial ROS production (Fauconnier et al., 2007), we observed a faster accumulation of the dye fluorescence in the presence of Sups, compared with the cells incubated in CSFM at pH 5.3 (Fig. 5a). This finding was further supported in the experiments with dehydrorhodamine 123-loaded HSAECs. The cells treated with Sups showed increased oxidation of this dye to the fluorescent rhodamine in the cytoplasm compared with the pH 5.3-titrated CSFM (not shown). Cell death was accompanied by the quick dissipation of the mitochondrial membrane potential detected with the specific dye, JC-1 (Fig. 5b). This observation is consistent with the mitochondrial damage by ROS (Pacher et al., 2007; Schonfeld & Wojtczak, 2008;) and the reduced ATP content of treated cells (Fig. 1a).


Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions.

Popova TG, Millis B, Chung MC, Bailey C, Popov SG - FEMS Immunol. Med. Microbiol. (2010)

Catalysts of ROS and peroxynitrite decomposition protect HSAECs against mitochondrial damage by Sups. (a) HSAECs grown on coverslips were treated for 30 min with Sups supplemented with 1 μM MitoSox (red) and 2.5 μg mL−1 Hoechst 33342 nuclear stain (blue). The cells were washed with HBSS, and the fluorescent images for control medium (left panel) and dSterne Sup-treated cells (middle panel) were recorded. The right panel shows a time course of MitoSox mean fluorescence intensity. Similar results were obtained with Sterne 34F2 Sup (not shown). (b) Cells were washed with HBSS and incubated for 30 min at 37°C, 5% CO2 with control CSFM (left panel), dSterne Sup (middle panel), and Sterne 34F2 Sup (right panel). After washing, the cells were stained with JC-1 (10 μg mL−1) and Hoechst 33342 (2.5 μg mL−1) dyes for 30 min. A dissipation of the mitochondrial potential is evident as a loss of red fluorescence characteristic of aggregated JC-1. (c–f) The Sups generated in CSFM (c–e) and DMEM/F-12 supplemented with 300 μM NaNO3 and 1 mg mL−1 BSA (f) were mixed with the indicated catalysts for 30 min and used to treat cells for 2 h. (g) Cells were preincubated with catalysts for 30 min, treated with dSterne Sup (bottom row) or control media (top row) for 30 min, and the MitoSox images were recorded as in (a). Where indicated, CD-cholesterol (1 μg mL−1) was added to the Sup and control medium for 30 min before incubation with the cells. Additional controls included CSFM titrated with HCl to pH 5.3 (left top panel) and the dSterne Sup titrated to pH 7.5 with NaOH (right bottom panel). Error bars represent 95% confidence interval of mean. A quantitative evaluation of MitoSox fluorescence is presented in Fig. S10.
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Related In: Results  -  Collection

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fig05: Catalysts of ROS and peroxynitrite decomposition protect HSAECs against mitochondrial damage by Sups. (a) HSAECs grown on coverslips were treated for 30 min with Sups supplemented with 1 μM MitoSox (red) and 2.5 μg mL−1 Hoechst 33342 nuclear stain (blue). The cells were washed with HBSS, and the fluorescent images for control medium (left panel) and dSterne Sup-treated cells (middle panel) were recorded. The right panel shows a time course of MitoSox mean fluorescence intensity. Similar results were obtained with Sterne 34F2 Sup (not shown). (b) Cells were washed with HBSS and incubated for 30 min at 37°C, 5% CO2 with control CSFM (left panel), dSterne Sup (middle panel), and Sterne 34F2 Sup (right panel). After washing, the cells were stained with JC-1 (10 μg mL−1) and Hoechst 33342 (2.5 μg mL−1) dyes for 30 min. A dissipation of the mitochondrial potential is evident as a loss of red fluorescence characteristic of aggregated JC-1. (c–f) The Sups generated in CSFM (c–e) and DMEM/F-12 supplemented with 300 μM NaNO3 and 1 mg mL−1 BSA (f) were mixed with the indicated catalysts for 30 min and used to treat cells for 2 h. (g) Cells were preincubated with catalysts for 30 min, treated with dSterne Sup (bottom row) or control media (top row) for 30 min, and the MitoSox images were recorded as in (a). Where indicated, CD-cholesterol (1 μg mL−1) was added to the Sup and control medium for 30 min before incubation with the cells. Additional controls included CSFM titrated with HCl to pH 5.3 (left top panel) and the dSterne Sup titrated to pH 7.5 with NaOH (right bottom panel). Error bars represent 95% confidence interval of mean. A quantitative evaluation of MitoSox fluorescence is presented in Fig. S10.
Mentions: SA is a substrate of succinate dehydrogenase (or Complex II of the mitochondrial electron transport chain), which converts succinate to fumarate. Under some pathophysiological conditions such as hypoxia, the SA-supported mitochondrial respiration could lead to the formation of damaging ROS (Schonfeld & Wojtczak, 2008). Taking into account that the Alamar Blue dye used in our cell viability test largely reflects the redox activity of the mitochondria, we hypothesized that the mitochondria were the likely targets of the Sup-induced respiratory burst, resulting in the release of ROS and consequent self-intoxication. In agreement with this suggestion, a conventional specific inhibitor of Complex II, thenoyltrifluoroacetone, demonstrated protection of cells from the toxicity of Sups (Fig. 3c). Using MitoSox Red, a peroxide-sensitive probe designed for the detection of mitochondrial ROS production (Fauconnier et al., 2007), we observed a faster accumulation of the dye fluorescence in the presence of Sups, compared with the cells incubated in CSFM at pH 5.3 (Fig. 5a). This finding was further supported in the experiments with dehydrorhodamine 123-loaded HSAECs. The cells treated with Sups showed increased oxidation of this dye to the fluorescent rhodamine in the cytoplasm compared with the pH 5.3-titrated CSFM (not shown). Cell death was accompanied by the quick dissipation of the mitochondrial membrane potential detected with the specific dye, JC-1 (Fig. 5b). This observation is consistent with the mitochondrial damage by ROS (Pacher et al., 2007; Schonfeld & Wojtczak, 2008;) and the reduced ATP content of treated cells (Fig. 1a).

Bottom Line: Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible.Its effect was found to be synergistic with a metabolic product of B. anthracis, succinic acid.Cell death appears to be caused by an acute primary membrane permeabilization by ALO, followed by a burst of reactive radicals from the mitochondria fuelled by the succinate, which is generated by bacteria in the hypoxic environment.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA, USA.

Show MeSH
Related in: MedlinePlus