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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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Related in: MedlinePlus

Bacillus anthracis generates toxic secreted products in static cultures. (a, b) Bacteria were grown in the CSFM. The viability of HSAECs relative to untreated controls was tested after incubation with Sups for 2 h at 37°C, 5% CO2 using the Alamar Blue test (a, left panel), LDH test (a, middle panel), intracellular ATP test (a, right panel), and the Trypan Blue permeability test (b). (c) Formation of membrane blebs in HSAECs incubated with Sterne 34F2 Sup. HSAECs were loaded with Calcein AM (5 μM, green), Hoechst 33342 (2.5 μg mL−1, blue), and wheat germ agglutinin conjugated with Alexa Fluor 555 (5 μg mL−1, red), washed with HBSS, and treated with the Sterne Sup for 30 min. The cells demonstrate a transient (up to 30-min postexposure) appearance of numerous membrane blebs (left panel, arrows). The addition of CD-cholesterol (1 μg mL−1) to the Sup for 30 min completely prevents blebbing (middle panel). A typical bleb (right panel) contains a part of the cell cytoplasm (green) surrounded by the cytoplasmic membrane (red). Similar results were obtained with dSterne Sup (not shown). (d) HSAECs grown on slides were incubated with Sups for 2 h at 37°C, 5% CO2, stained with Annexin V-conjugated Cyt3.18 dye (red) and nuclear DAPI dye (blue) for 10 min, fixed, and mounted for imaging. Numbers show the mean intensity of fluorescence (± 95% confidence interval) inred channel relative to the control cells incubated in CSFM. The intensity was evaluated with nis elements software (Nikon) using 10 independent fields of view in three different culture wells for each experimental condition. (e) Viability of Caco-2, HEP-G2, and HUVEC cells in the Alamar Blue test relative to the untreated control cell after the indicated times of exposure to Sups at 37°C, 5% CO2. Error bars represent 95% confidence interval of mean.
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fig01: Bacillus anthracis generates toxic secreted products in static cultures. (a, b) Bacteria were grown in the CSFM. The viability of HSAECs relative to untreated controls was tested after incubation with Sups for 2 h at 37°C, 5% CO2 using the Alamar Blue test (a, left panel), LDH test (a, middle panel), intracellular ATP test (a, right panel), and the Trypan Blue permeability test (b). (c) Formation of membrane blebs in HSAECs incubated with Sterne 34F2 Sup. HSAECs were loaded with Calcein AM (5 μM, green), Hoechst 33342 (2.5 μg mL−1, blue), and wheat germ agglutinin conjugated with Alexa Fluor 555 (5 μg mL−1, red), washed with HBSS, and treated with the Sterne Sup for 30 min. The cells demonstrate a transient (up to 30-min postexposure) appearance of numerous membrane blebs (left panel, arrows). The addition of CD-cholesterol (1 μg mL−1) to the Sup for 30 min completely prevents blebbing (middle panel). A typical bleb (right panel) contains a part of the cell cytoplasm (green) surrounded by the cytoplasmic membrane (red). Similar results were obtained with dSterne Sup (not shown). (d) HSAECs grown on slides were incubated with Sups for 2 h at 37°C, 5% CO2, stained with Annexin V-conjugated Cyt3.18 dye (red) and nuclear DAPI dye (blue) for 10 min, fixed, and mounted for imaging. Numbers show the mean intensity of fluorescence (± 95% confidence interval) inred channel relative to the control cells incubated in CSFM. The intensity was evaluated with nis elements software (Nikon) using 10 independent fields of view in three different culture wells for each experimental condition. (e) Viability of Caco-2, HEP-G2, and HUVEC cells in the Alamar Blue test relative to the untreated control cell after the indicated times of exposure to Sups at 37°C, 5% CO2. Error bars represent 95% confidence interval of mean.

Mentions: In our previous report, we used an experimental system in which cultured HSAECs were infected with B. anthracis spores and the responses of the cells to the germinating spores and vegetative bacteria were studied (Popova et al., 2009). The results indicated that during the first 8 h of incubation, bacteria of the toxigenic Sterne strain 34F2 growing in the culture medium on top of the confluent HSAECs under static conditions at 37 °C and 5% CO2 did not influence HSAEC viability, although during this period of time, bacteria secreted more than 3 μg mL−1 of LeTx and EdTx. In the current study, we found that longer incubation times resulted in a progressive loss of HSAEC viability and wanted to elucidate the nature of this toxic effect. Analysis of the bacterial cultures grown in the absence of HSAECs revealed that the toxicity resided in the supernatant fraction (Sup). The results in Fig. 1a show that Sups of the 24-h cultures grown under the conditions of the experiments with HSAECs demonstrated a high level of toxicity when HSAECs were exposed to Sups for 2 h. The pelleted and washed bacteria resuspended in the original volume of fresh culture medium had no toxicity (not shown), indicating that toxicity was due to the activity of secreted bacterial factors.


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)

Bacillus anthracis generates toxic secreted products in static cultures. (a, b) Bacteria were grown in the CSFM. The viability of HSAECs relative to untreated controls was tested after incubation with Sups for 2 h at 37°C, 5% CO2 using the Alamar Blue test (a, left panel), LDH test (a, middle panel), intracellular ATP test (a, right panel), and the Trypan Blue permeability test (b). (c) Formation of membrane blebs in HSAECs incubated with Sterne 34F2 Sup. HSAECs were loaded with Calcein AM (5 μM, green), Hoechst 33342 (2.5 μg mL−1, blue), and wheat germ agglutinin conjugated with Alexa Fluor 555 (5 μg mL−1, red), washed with HBSS, and treated with the Sterne Sup for 30 min. The cells demonstrate a transient (up to 30-min postexposure) appearance of numerous membrane blebs (left panel, arrows). The addition of CD-cholesterol (1 μg mL−1) to the Sup for 30 min completely prevents blebbing (middle panel). A typical bleb (right panel) contains a part of the cell cytoplasm (green) surrounded by the cytoplasmic membrane (red). Similar results were obtained with dSterne Sup (not shown). (d) HSAECs grown on slides were incubated with Sups for 2 h at 37°C, 5% CO2, stained with Annexin V-conjugated Cyt3.18 dye (red) and nuclear DAPI dye (blue) for 10 min, fixed, and mounted for imaging. Numbers show the mean intensity of fluorescence (± 95% confidence interval) inred channel relative to the control cells incubated in CSFM. The intensity was evaluated with nis elements software (Nikon) using 10 independent fields of view in three different culture wells for each experimental condition. (e) Viability of Caco-2, HEP-G2, and HUVEC cells in the Alamar Blue test relative to the untreated control cell after the indicated times of exposure to Sups at 37°C, 5% CO2. Error bars represent 95% confidence interval of mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig01: Bacillus anthracis generates toxic secreted products in static cultures. (a, b) Bacteria were grown in the CSFM. The viability of HSAECs relative to untreated controls was tested after incubation with Sups for 2 h at 37°C, 5% CO2 using the Alamar Blue test (a, left panel), LDH test (a, middle panel), intracellular ATP test (a, right panel), and the Trypan Blue permeability test (b). (c) Formation of membrane blebs in HSAECs incubated with Sterne 34F2 Sup. HSAECs were loaded with Calcein AM (5 μM, green), Hoechst 33342 (2.5 μg mL−1, blue), and wheat germ agglutinin conjugated with Alexa Fluor 555 (5 μg mL−1, red), washed with HBSS, and treated with the Sterne Sup for 30 min. The cells demonstrate a transient (up to 30-min postexposure) appearance of numerous membrane blebs (left panel, arrows). The addition of CD-cholesterol (1 μg mL−1) to the Sup for 30 min completely prevents blebbing (middle panel). A typical bleb (right panel) contains a part of the cell cytoplasm (green) surrounded by the cytoplasmic membrane (red). Similar results were obtained with dSterne Sup (not shown). (d) HSAECs grown on slides were incubated with Sups for 2 h at 37°C, 5% CO2, stained with Annexin V-conjugated Cyt3.18 dye (red) and nuclear DAPI dye (blue) for 10 min, fixed, and mounted for imaging. Numbers show the mean intensity of fluorescence (± 95% confidence interval) inred channel relative to the control cells incubated in CSFM. The intensity was evaluated with nis elements software (Nikon) using 10 independent fields of view in three different culture wells for each experimental condition. (e) Viability of Caco-2, HEP-G2, and HUVEC cells in the Alamar Blue test relative to the untreated control cell after the indicated times of exposure to Sups at 37°C, 5% CO2. Error bars represent 95% confidence interval of mean.
Mentions: In our previous report, we used an experimental system in which cultured HSAECs were infected with B. anthracis spores and the responses of the cells to the germinating spores and vegetative bacteria were studied (Popova et al., 2009). The results indicated that during the first 8 h of incubation, bacteria of the toxigenic Sterne strain 34F2 growing in the culture medium on top of the confluent HSAECs under static conditions at 37 °C and 5% CO2 did not influence HSAEC viability, although during this period of time, bacteria secreted more than 3 μg mL−1 of LeTx and EdTx. In the current study, we found that longer incubation times resulted in a progressive loss of HSAEC viability and wanted to elucidate the nature of this toxic effect. Analysis of the bacterial cultures grown in the absence of HSAECs revealed that the toxicity resided in the supernatant fraction (Sup). The results in Fig. 1a show that Sups of the 24-h cultures grown under the conditions of the experiments with HSAECs demonstrated a high level of toxicity when HSAECs were exposed to Sups for 2 h. The pelleted and washed bacteria resuspended in the original volume of fresh culture medium had no toxicity (not shown), indicating that toxicity was due to the activity of secreted bacterial factors.

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