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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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Sups of attenuated and mutant Bacillus anthracis strains demonstrate different levels of cholesterol-dependent toxicity. (a) Sups of the Sterne 34F2 and dSterne strains were preincubated with the indicated concentrations of cholesterol or CD-cholesterol for 30 min and tested for toxicity. MbCD served as a control to demonstrate the absence of its effect on the activity of CD-cholesterol. The X axis indicates concentrations of added cholesterol, CD-cholesterol, and MbCD. (b) Sups of the indicated B. anthracis strains were preincubated with CD-cholesterol (3 μg mL−1) for 30 min and tested for toxicity with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.
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fig04: Sups of attenuated and mutant Bacillus anthracis strains demonstrate different levels of cholesterol-dependent toxicity. (a) Sups of the Sterne 34F2 and dSterne strains were preincubated with the indicated concentrations of cholesterol or CD-cholesterol for 30 min and tested for toxicity. MbCD served as a control to demonstrate the absence of its effect on the activity of CD-cholesterol. The X axis indicates concentrations of added cholesterol, CD-cholesterol, and MbCD. (b) Sups of the indicated B. anthracis strains were preincubated with CD-cholesterol (3 μg mL−1) for 30 min and tested for toxicity with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.

Mentions: Experiments with a broad-spectrum protease inhibitor cocktail against aspartic, cysteine, serine, metalloproteases, and aminopeptidases did not reveal any influence of inhibitors on Sup toxicity and therefore argued against the proteolytic cells' damage (Fig. S5). On the other hand, quick permeabilization of the Sup-treated cells evident from the LDH and Trypan Blue tests (Fig. 1a and b) strongly suggested that toxicity was associated with the pH-dependent activity of membrane-damaging secreted bacterial factor. Anthrax hemolysins are potent membrane-damaging proteins and their expression is upregulated under anaerobic conditions (Klichko et al., 2003). ALO is a major B. anthracis hemolysin, which belongs to the family of cholesterol-dependent toxins (Tweeten, 2005). We tested the activity of recombinant ALO and found that it caused the formation of membrane blebs similar to the effect of Sups in HSAECs and HUVECs (Movie S1). Damage to membranes of different cell types by PFTs similar to ALO is known to be inhibited by cholesterol (Tweeten, 2005). Recent publications also used a soluble complex of cholesterol with MbCD (CD-cholesterol) (McCormick et al., 2009). In agreement with this, CD-cholesterol protected the cells from membrane blebbing (Fig. 1c). Both cholesterol and CD-cholesterol strongly reduced the toxicity of Sups in the Alamar Blue viability test with HSAECs (Fig. 4a). However, a considerable fraction of toxicity remained cholesterol-independent.


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)

Sups of attenuated and mutant Bacillus anthracis strains demonstrate different levels of cholesterol-dependent toxicity. (a) Sups of the Sterne 34F2 and dSterne strains were preincubated with the indicated concentrations of cholesterol or CD-cholesterol for 30 min and tested for toxicity. MbCD served as a control to demonstrate the absence of its effect on the activity of CD-cholesterol. The X axis indicates concentrations of added cholesterol, CD-cholesterol, and MbCD. (b) Sups of the indicated B. anthracis strains were preincubated with CD-cholesterol (3 μg mL−1) for 30 min and tested for toxicity 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

License
Show All Figures
getmorefigures.php?uid=PMC3040846&req=5

fig04: Sups of attenuated and mutant Bacillus anthracis strains demonstrate different levels of cholesterol-dependent toxicity. (a) Sups of the Sterne 34F2 and dSterne strains were preincubated with the indicated concentrations of cholesterol or CD-cholesterol for 30 min and tested for toxicity. MbCD served as a control to demonstrate the absence of its effect on the activity of CD-cholesterol. The X axis indicates concentrations of added cholesterol, CD-cholesterol, and MbCD. (b) Sups of the indicated B. anthracis strains were preincubated with CD-cholesterol (3 μg mL−1) for 30 min and tested for toxicity with Alamar Blue as described in Materials and methods. Error bars represent 95% confidence interval of mean.
Mentions: Experiments with a broad-spectrum protease inhibitor cocktail against aspartic, cysteine, serine, metalloproteases, and aminopeptidases did not reveal any influence of inhibitors on Sup toxicity and therefore argued against the proteolytic cells' damage (Fig. S5). On the other hand, quick permeabilization of the Sup-treated cells evident from the LDH and Trypan Blue tests (Fig. 1a and b) strongly suggested that toxicity was associated with the pH-dependent activity of membrane-damaging secreted bacterial factor. Anthrax hemolysins are potent membrane-damaging proteins and their expression is upregulated under anaerobic conditions (Klichko et al., 2003). ALO is a major B. anthracis hemolysin, which belongs to the family of cholesterol-dependent toxins (Tweeten, 2005). We tested the activity of recombinant ALO and found that it caused the formation of membrane blebs similar to the effect of Sups in HSAECs and HUVECs (Movie S1). Damage to membranes of different cell types by PFTs similar to ALO is known to be inhibited by cholesterol (Tweeten, 2005). Recent publications also used a soluble complex of cholesterol with MbCD (CD-cholesterol) (McCormick et al., 2009). In agreement with this, CD-cholesterol protected the cells from membrane blebbing (Fig. 1c). Both cholesterol and CD-cholesterol strongly reduced the toxicity of Sups in the Alamar Blue viability test with HSAECs (Fig. 4a). However, a considerable fraction of toxicity remained cholesterol-independent.

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