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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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Synergistic effect of ALO and SA on HSAEC viability. (a) Recombinant ALO (50 ng mL−1) was spiked into Sups of the hemolysin mutants strains, Sup of the parental strain Sterne 7702, and the solution of SA in CSFM titrated to pH 5.3 with HCl. The mixtures were applied to HSAECs for 2 h. (b) Different concentrations of SA in CSFM were titrated with HCl to pH 5.3, mixed with ALO, and applied to HSAECs for 2 h. Cell viability after treatment is shown relative to incubation in CSFM (pH 7.5). (c) HSAECs were treated with dSterne Sup (gray bars) or incubated in control CSFM (open bars) in the presence of indicated concentrations of thenoyltrifluoroacetone for 30 min. (a–c) The viability of the cells was tested with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.
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fig03: Synergistic effect of ALO and SA on HSAEC viability. (a) Recombinant ALO (50 ng mL−1) was spiked into Sups of the hemolysin mutants strains, Sup of the parental strain Sterne 7702, and the solution of SA in CSFM titrated to pH 5.3 with HCl. The mixtures were applied to HSAECs for 2 h. (b) Different concentrations of SA in CSFM were titrated with HCl to pH 5.3, mixed with ALO, and applied to HSAECs for 2 h. Cell viability after treatment is shown relative to incubation in CSFM (pH 7.5). (c) HSAECs were treated with dSterne Sup (gray bars) or incubated in control CSFM (open bars) in the presence of indicated concentrations of thenoyltrifluoroacetone for 30 min. (a–c) The viability of the cells was tested with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.

Mentions: In order to determine whether the reduction of pH observed during bacterial growth was required for toxicity, we tested the activity of neutralized Sups. Consistent with the results shown in Fig. 2, no toxicity to HSAECs was found at pH>6.0 when Sups were titrated with NaOH or diluted with a neutral culture medium (not shown). However, a concentration-dependent reduction of toxicity was detected when Sups were diluted using CSFM titrated with HCl to pH 5.3, corresponding to the acidity of Sups (Fig. S3). In the control experiments, the titrated medium did not show a substantial toxicity, indicating that it was associated with the pH-dependent activity of secreted bacterial product(s), but not the pH itself. Bacillus anthracis is known to generate a number of fermentation products, including formic, acetic, lactic, and SA, under anaerobic conditions (Puziss & Rittenberg, 1957). The marked pH dependence around 5.5 suggested that among these acids, the SA in its partially protonated state (pKa's 4.19 and 5.57) most likely contributed to the effect of Sups. Analysis of Sups generated by several B. anthracis strains, including Sterne 34F2 and dSterne, confirmed that SA was present at a concentration of about 1.6 ± 0.2 mM (mean ± SD) (Fig. S4). Supplementation of the culture medium with SA showed that it was toxic to HSAECs in a concentration-dependent manner (shown in Fig. 3b, black bars), but less effective than Sups (Fig. 1a), thus suggesting that Sups contained additional pathogenic factor(s).


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)

Synergistic effect of ALO and SA on HSAEC viability. (a) Recombinant ALO (50 ng mL−1) was spiked into Sups of the hemolysin mutants strains, Sup of the parental strain Sterne 7702, and the solution of SA in CSFM titrated to pH 5.3 with HCl. The mixtures were applied to HSAECs for 2 h. (b) Different concentrations of SA in CSFM were titrated with HCl to pH 5.3, mixed with ALO, and applied to HSAECs for 2 h. Cell viability after treatment is shown relative to incubation in CSFM (pH 7.5). (c) HSAECs were treated with dSterne Sup (gray bars) or incubated in control CSFM (open bars) in the presence of indicated concentrations of thenoyltrifluoroacetone for 30 min. (a–c) The viability of the cells was tested with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: Synergistic effect of ALO and SA on HSAEC viability. (a) Recombinant ALO (50 ng mL−1) was spiked into Sups of the hemolysin mutants strains, Sup of the parental strain Sterne 7702, and the solution of SA in CSFM titrated to pH 5.3 with HCl. The mixtures were applied to HSAECs for 2 h. (b) Different concentrations of SA in CSFM were titrated with HCl to pH 5.3, mixed with ALO, and applied to HSAECs for 2 h. Cell viability after treatment is shown relative to incubation in CSFM (pH 7.5). (c) HSAECs were treated with dSterne Sup (gray bars) or incubated in control CSFM (open bars) in the presence of indicated concentrations of thenoyltrifluoroacetone for 30 min. (a–c) The viability of the cells was tested with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.
Mentions: In order to determine whether the reduction of pH observed during bacterial growth was required for toxicity, we tested the activity of neutralized Sups. Consistent with the results shown in Fig. 2, no toxicity to HSAECs was found at pH>6.0 when Sups were titrated with NaOH or diluted with a neutral culture medium (not shown). However, a concentration-dependent reduction of toxicity was detected when Sups were diluted using CSFM titrated with HCl to pH 5.3, corresponding to the acidity of Sups (Fig. S3). In the control experiments, the titrated medium did not show a substantial toxicity, indicating that it was associated with the pH-dependent activity of secreted bacterial product(s), but not the pH itself. Bacillus anthracis is known to generate a number of fermentation products, including formic, acetic, lactic, and SA, under anaerobic conditions (Puziss & Rittenberg, 1957). The marked pH dependence around 5.5 suggested that among these acids, the SA in its partially protonated state (pKa's 4.19 and 5.57) most likely contributed to the effect of Sups. Analysis of Sups generated by several B. anthracis strains, including Sterne 34F2 and dSterne, confirmed that SA was present at a concentration of about 1.6 ± 0.2 mM (mean ± SD) (Fig. S4). Supplementation of the culture medium with SA showed that it was toxic to HSAECs in a concentration-dependent manner (shown in Fig. 3b, black bars), but less effective than Sups (Fig. 1a), thus suggesting that Sups contained additional pathogenic factor(s).

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