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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

Mortality curves and bacterial load in LNs and the spleen of spore-challenged DBA/2J mice. Animals were challenged with the toxinogenic, non-encapsulated Bacillus anthracis Sterne 34F2 or ΔNOS knockout Sterne spores (4 × 106 spores in 20 μl of PBS), intradermally into both hind footpads. Twenty animals were used for each strain to obtain the mortality curves. Four animals were euthanized at each time point to determine a bacterial load in the LNs and spleen. The organs were surgically removed and homogenized. The homogenates were seeded onto agar plates to determine the number of colony-forming units (CFUs). The CFU values are shown for each animal, and the lines in the panels are drawn through the average CFU values for each time point. A statistically reliable difference (t-test, p < 0.001) between Sterne and ΔNOS in the spleen at 48 h post challenge is indicated by an asterisk.
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Figure 5: Mortality curves and bacterial load in LNs and the spleen of spore-challenged DBA/2J mice. Animals were challenged with the toxinogenic, non-encapsulated Bacillus anthracis Sterne 34F2 or ΔNOS knockout Sterne spores (4 × 106 spores in 20 μl of PBS), intradermally into both hind footpads. Twenty animals were used for each strain to obtain the mortality curves. Four animals were euthanized at each time point to determine a bacterial load in the LNs and spleen. The organs were surgically removed and homogenized. The homogenates were seeded onto agar plates to determine the number of colony-forming units (CFUs). The CFU values are shown for each animal, and the lines in the panels are drawn through the average CFU values for each time point. A statistically reliable difference (t-test, p < 0.001) between Sterne and ΔNOS in the spleen at 48 h post challenge is indicated by an asterisk.

Mentions: The mortality curves (Figure 5) show that it took the ΔNOS strain approximately 40 h longer than Sterne to reach 50% mortality. However, as in the case of Sterne, all mice ultimately died demonstrating that deletion of bNOS did not abrogate the toxicity but rather delayed it. The time course of dissemination of infectious material to draining popliteal LN and spleen was tested by seeding the organ homogenates onto the LB agar plates. At 3 h post challenge a large number of CFUs representing viable spores and vegetative cells were detected in the LNs and spleens of mice challenged with Sterne and ΔNOS strains. The amount of infectious material continued to increase steadily during infection with Sterne spores until all mice succumbed to the disease at 72 h (at this time point no survived animals could be tested). In comparison with Sterne the ΔNOS strain showed a reduced virulence correlating with a slower propagation of bacteria in the LNs and a reduced dissemination to the spleen. Although the observed difference between strains in the LNs did not reach statistical reliability, the spleen counts were reliably different at 48 h post challenge (p < 0.001).


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)

Mortality curves and bacterial load in LNs and the spleen of spore-challenged DBA/2J mice. Animals were challenged with the toxinogenic, non-encapsulated Bacillus anthracis Sterne 34F2 or ΔNOS knockout Sterne spores (4 × 106 spores in 20 μl of PBS), intradermally into both hind footpads. Twenty animals were used for each strain to obtain the mortality curves. Four animals were euthanized at each time point to determine a bacterial load in the LNs and spleen. The organs were surgically removed and homogenized. The homogenates were seeded onto agar plates to determine the number of colony-forming units (CFUs). The CFU values are shown for each animal, and the lines in the panels are drawn through the average CFU values for each time point. A statistically reliable difference (t-test, p < 0.001) between Sterne and ΔNOS in the spleen at 48 h post challenge is indicated by an asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Mortality curves and bacterial load in LNs and the spleen of spore-challenged DBA/2J mice. Animals were challenged with the toxinogenic, non-encapsulated Bacillus anthracis Sterne 34F2 or ΔNOS knockout Sterne spores (4 × 106 spores in 20 μl of PBS), intradermally into both hind footpads. Twenty animals were used for each strain to obtain the mortality curves. Four animals were euthanized at each time point to determine a bacterial load in the LNs and spleen. The organs were surgically removed and homogenized. The homogenates were seeded onto agar plates to determine the number of colony-forming units (CFUs). The CFU values are shown for each animal, and the lines in the panels are drawn through the average CFU values for each time point. A statistically reliable difference (t-test, p < 0.001) between Sterne and ΔNOS in the spleen at 48 h post challenge is indicated by an asterisk.
Mentions: The mortality curves (Figure 5) show that it took the ΔNOS strain approximately 40 h longer than Sterne to reach 50% mortality. However, as in the case of Sterne, all mice ultimately died demonstrating that deletion of bNOS did not abrogate the toxicity but rather delayed it. The time course of dissemination of infectious material to draining popliteal LN and spleen was tested by seeding the organ homogenates onto the LB agar plates. At 3 h post challenge a large number of CFUs representing viable spores and vegetative cells were detected in the LNs and spleens of mice challenged with Sterne and ΔNOS strains. The amount of infectious material continued to increase steadily during infection with Sterne spores until all mice succumbed to the disease at 72 h (at this time point no survived animals could be tested). In comparison with Sterne the ΔNOS strain showed a reduced virulence correlating with a slower propagation of bacteria in the LNs and a reduced dissemination to the spleen. Although the observed difference between strains in the LNs did not reach statistical reliability, the spleen counts were reliably different at 48 h post challenge (p < 0.001).

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