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Nitric oxide as a regulator of B. anthracis pathogenicity.

Popova TG, Teunis A, Vaseghi H, Zhou W, Espina V, Liotta LA, Popov SG - Front Microbiol (2015)

Bottom Line: The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH.The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne.This is the first in vivo observation of the bacterial NO effect on the lymphatic system.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Disease, College of Science, George Mason University, Manassas, VA USA ; Center for Applied Proteomics and Molecular Medicine, College of Science, George Mason University, Manassas, VA USA.

ABSTRACT
Nitric oxide (NO) is a key physiological regulator in eukaryotic and prokaryotic organisms. It can cause a variety of biological effects by reacting with its targets or/and indirectly inducing oxidative stress. NO can also be produced by bacteria including the pathogenic Bacillus anthracis; however, its role in the infectious process only begins to emerge. NO incapacitates macrophages by S-nitrosylating the intracellular proteins and protects B. anthracis from oxidative stress. It is also implicated in the formation of toxic peroxynitrite. In this study we further assessed the effects of B. anthracis NO produced by the NO synthase (bNOS) on bacterial metabolism and host cells in experiments with the bNOS knockout Sterne strain. The mutation abrogated accumulation of nitrite and nitrate as tracer products of NO in the culture medium and markedly attenuated growth in both aerobic and microaerobic conditions. The regulatory role of NO was also suggested by the abnormally high rate of nitrate denitrification by the mutant in the presence of oxygen. Anaerobic regulation mediated by NO was reflected in reduced fermentation of glucose by the mutant correlating with the reduced toxicity of bacteria toward host cells in culture. The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH. The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne. Our global proteomic analysis of lymph from the lymph nodes of infected mice harboring bacteria revealed numerous changes in the pattern and levels of proteins associated with the activity of bNOS influencing key cell physiological processes relevant to energy metabolism, growth, signal transduction, stress response, septic shock, and homeostasis. This is the first in vivo observation of the bacterial NO effect on the lymphatic system.

No MeSH data available.


Related in: MedlinePlus

Cytotoxic effect of Sterne and ΔNOS static culture sups. Bacteria were grown in DMEM (10 ml, 24 h, 37°C, 5% CO2) in static conditions and the culture sups were prepared. The monolayers of human small-airway lung epithelial cells (HSAECs) were exposed to the bacterial sups for 2 h and the viability of the cells was determined using Resazurin dye as described in Section “Materials and Methods”. The final pH values of the sups and control medium are shown. Asterisks indicate the sups titrated to the pH 5.2 of Sterne sup after cultivation. The error bars indicate 95% confidence intervals of triplicate measurements.
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Figure 2: Cytotoxic effect of Sterne and ΔNOS static culture sups. Bacteria were grown in DMEM (10 ml, 24 h, 37°C, 5% CO2) in static conditions and the culture sups were prepared. The monolayers of human small-airway lung epithelial cells (HSAECs) were exposed to the bacterial sups for 2 h and the viability of the cells was determined using Resazurin dye as described in Section “Materials and Methods”. The final pH values of the sups and control medium are shown. Asterisks indicate the sups titrated to the pH 5.2 of Sterne sup after cultivation. The error bars indicate 95% confidence intervals of triplicate measurements.

Mentions: Decreased acidification by the ΔNOS mutant in comparison with Sterne was associated with the reduction in the toxicity of culture supernatants (sups) toward cultured host cells. Figure 2 shows the results of the lung epithelial cell viability assay carried out using the Alamar Blue dye reflecting the reducing capacity of viable cells. The mutant cultures were less toxic and less acidic than the Sterne ones. In order to take into account the effect of pH the ΔNOS sups were titrated using HCl to the pH values of corresponding Sterne sups. The acidified ΔNOS sups increased their toxicity, but in control incubations the acidification of the unconditioned medium did not have a toxic effect (Figure 2). The cells were also tested for the extent of membrane permeability using the CellTox assay (Promega) after exposure to sups (Figure 3A). The amount of green fluorescence due to the interaction of the cyanine dye with DNA of dead or permeabilized cells was measured. In correlation with the viability assay, the ΔNOS sups were found to be substantially less active than the Sterne ones but showed increased permeabilization after acidification to the pH of Sterne sups. Cholesterol was able to partially reduce the permeabilization (Figure 3B) in accordance with the presence of the pore-forming cholesterol-sensitive cytolysin ALO in the sups (Popova et al., 2011). These results demonstrated that the membrane-damaging bacterial factor(s) was (were) present in the sups of both ΔNOS and Sterne cultures; however, its full activity required a reduced pH.


Nitric oxide as a regulator of B. anthracis pathogenicity.

Popova TG, Teunis A, Vaseghi H, Zhou W, Espina V, Liotta LA, Popov SG - Front Microbiol (2015)

Cytotoxic effect of Sterne and ΔNOS static culture sups. Bacteria were grown in DMEM (10 ml, 24 h, 37°C, 5% CO2) in static conditions and the culture sups were prepared. The monolayers of human small-airway lung epithelial cells (HSAECs) were exposed to the bacterial sups for 2 h and the viability of the cells was determined using Resazurin dye as described in Section “Materials and Methods”. The final pH values of the sups and control medium are shown. Asterisks indicate the sups titrated to the pH 5.2 of Sterne sup after cultivation. The error bars indicate 95% confidence intervals of triplicate measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557104&req=5

Figure 2: Cytotoxic effect of Sterne and ΔNOS static culture sups. Bacteria were grown in DMEM (10 ml, 24 h, 37°C, 5% CO2) in static conditions and the culture sups were prepared. The monolayers of human small-airway lung epithelial cells (HSAECs) were exposed to the bacterial sups for 2 h and the viability of the cells was determined using Resazurin dye as described in Section “Materials and Methods”. The final pH values of the sups and control medium are shown. Asterisks indicate the sups titrated to the pH 5.2 of Sterne sup after cultivation. The error bars indicate 95% confidence intervals of triplicate measurements.
Mentions: Decreased acidification by the ΔNOS mutant in comparison with Sterne was associated with the reduction in the toxicity of culture supernatants (sups) toward cultured host cells. Figure 2 shows the results of the lung epithelial cell viability assay carried out using the Alamar Blue dye reflecting the reducing capacity of viable cells. The mutant cultures were less toxic and less acidic than the Sterne ones. In order to take into account the effect of pH the ΔNOS sups were titrated using HCl to the pH values of corresponding Sterne sups. The acidified ΔNOS sups increased their toxicity, but in control incubations the acidification of the unconditioned medium did not have a toxic effect (Figure 2). The cells were also tested for the extent of membrane permeability using the CellTox assay (Promega) after exposure to sups (Figure 3A). The amount of green fluorescence due to the interaction of the cyanine dye with DNA of dead or permeabilized cells was measured. In correlation with the viability assay, the ΔNOS sups were found to be substantially less active than the Sterne ones but showed increased permeabilization after acidification to the pH of Sterne sups. Cholesterol was able to partially reduce the permeabilization (Figure 3B) in accordance with the presence of the pore-forming cholesterol-sensitive cytolysin ALO in the sups (Popova et al., 2011). These results demonstrated that the membrane-damaging bacterial factor(s) was (were) present in the sups of both ΔNOS and Sterne cultures; however, its full activity required a reduced pH.

Bottom Line: The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH.The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne.This is the first in vivo observation of the bacterial NO effect on the lymphatic system.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Disease, College of Science, George Mason University, Manassas, VA USA ; Center for Applied Proteomics and Molecular Medicine, College of Science, George Mason University, Manassas, VA USA.

ABSTRACT
Nitric oxide (NO) is a key physiological regulator in eukaryotic and prokaryotic organisms. It can cause a variety of biological effects by reacting with its targets or/and indirectly inducing oxidative stress. NO can also be produced by bacteria including the pathogenic Bacillus anthracis; however, its role in the infectious process only begins to emerge. NO incapacitates macrophages by S-nitrosylating the intracellular proteins and protects B. anthracis from oxidative stress. It is also implicated in the formation of toxic peroxynitrite. In this study we further assessed the effects of B. anthracis NO produced by the NO synthase (bNOS) on bacterial metabolism and host cells in experiments with the bNOS knockout Sterne strain. The mutation abrogated accumulation of nitrite and nitrate as tracer products of NO in the culture medium and markedly attenuated growth in both aerobic and microaerobic conditions. The regulatory role of NO was also suggested by the abnormally high rate of nitrate denitrification by the mutant in the presence of oxygen. Anaerobic regulation mediated by NO was reflected in reduced fermentation of glucose by the mutant correlating with the reduced toxicity of bacteria toward host cells in culture. The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH. The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne. Our global proteomic analysis of lymph from the lymph nodes of infected mice harboring bacteria revealed numerous changes in the pattern and levels of proteins associated with the activity of bNOS influencing key cell physiological processes relevant to energy metabolism, growth, signal transduction, stress response, septic shock, and homeostasis. This is the first in vivo observation of the bacterial NO effect on the lymphatic system.

No MeSH data available.


Related in: MedlinePlus