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Phagocytic superoxide specifically damages an extracytoplasmic target to inhibit or kill Salmonella.

Craig M, Slauch JM - PLoS ONE (2009)

Bottom Line: The phagocytic oxidative burst is a primary effector of innate immunity that protects against bacterial infection.We found that SodCI acts independently of cytoplasmic SODs, SodA and SodB.Although sodCI did show genetic interaction with recA, this was apparently independent of recombination and is presumably due to the pleiotropic effects of a recA mutation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, USA.

ABSTRACT

Background: The phagocytic oxidative burst is a primary effector of innate immunity that protects against bacterial infection. However, the mechanism by which reactive oxygen species (ROS) kill or inhibit bacteria is not known. It is often assumed that DNA is a primary target of oxidative damage, consistent with known effects of endogenously produced ROS in the bacterial cytoplasm. But most studies fail to distinguish between effects of host derived ROS versus damage caused by endogenous bacterial sources. We took advantage of both the ability of Salmonella enterica serovar Typhimurium to survive in macrophages and the genetic tractability of the system to test the hypothesis that phagocytic superoxide damages cytoplasmic targets including DNA.

Methodology/principal findings: SodCI is a periplasmic Cu-Zn superoxide dismutase (SOD) that contributes to the survival of Salmonella Typhimurium in macrophages. Through competitive virulence assays, we asked if sodCI has a genetic interaction with various cytoplasmic systems. We found that SodCI acts independently of cytoplasmic SODs, SodA and SodB. In addition, SodCI acts independently of the base excision repair system and RuvAB, involved in DNA repair. Although sodCI did show genetic interaction with recA, this was apparently independent of recombination and is presumably due to the pleiotropic effects of a recA mutation.

Conclusions/significance: Taken together, these results suggest that bacterial inhibition by phagocytic superoxide is primarily the result of damage to an extracytoplasmic target.

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Related in: MedlinePlus

Effect of sod mutations on survival in cells exposed to exogenous reactive oxygen species.A) From triplicate cultures, 0.25 mM hypoxanthine with or without 0.1 u/ml xanthine oxidase was added to ∼106 cfu/ml of cells in PBS. Colony forming units were determined at the indicated time points. The cfu of untreated samples remained essentially constant throughout the experiment. The viable count of the treated sample is compared to untreated sample at each time point. B) Triplicate mid-log cultures in LB 0.2% glucose were split and 2.5 mM H2O2 was added to one half. Colony forming units were determined and compared to untreated sample at each time point as above. The cfu of untreated samples increased only slightly during the experiment. The mean and range are plotted for each experiment. Strains used: 14028, JS456, JS831.
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pone-0004975-g001: Effect of sod mutations on survival in cells exposed to exogenous reactive oxygen species.A) From triplicate cultures, 0.25 mM hypoxanthine with or without 0.1 u/ml xanthine oxidase was added to ∼106 cfu/ml of cells in PBS. Colony forming units were determined at the indicated time points. The cfu of untreated samples remained essentially constant throughout the experiment. The viable count of the treated sample is compared to untreated sample at each time point. B) Triplicate mid-log cultures in LB 0.2% glucose were split and 2.5 mM H2O2 was added to one half. Colony forming units were determined and compared to untreated sample at each time point as above. The cfu of untreated samples increased only slightly during the experiment. The mean and range are plotted for each experiment. Strains used: 14028, JS456, JS831.

Mentions: The data above suggest that the most vulnerable target of phagocytic superoxide is extracytoplasmic. But identifying the target(s) is complicated by the fact that sodC mutants of Salmonella show no significant in vitro phenotypes. As shown in Figure 1A, there was no significant difference in sensitivity to 250 µM hypoxanthine/xanthine oxidase of the wild type and isogenic mutants devoid of periplasmic SODs or cytoplasmic SODs. We have performed similar experiments using this and other superoxide-generating systems under a variety of conditions and have never observed a reproducible difference between the wild type and sodCI sodCII double mutant. This should not be surprising. It is estimated that NADPH oxidase is capable of producing a steady state superoxide concentration of 100 µM in a phagosome [12]. Using published data [44], [45], one can calculate that in vitro systems such as that used above are capable of generating only <1 µM of superoxide for a few minutes. When higher concentrations (2.5 mM) of hydrogen peroxide were used, the sodA sodB double mutant showed increased sensitivity (Figure 1B), as previously reported [46]. The sodCI sodCII mutant behaved identically to wild type, again distinguishing loss of cytoplasmic versus periplasmic SOD activity.


Phagocytic superoxide specifically damages an extracytoplasmic target to inhibit or kill Salmonella.

Craig M, Slauch JM - PLoS ONE (2009)

Effect of sod mutations on survival in cells exposed to exogenous reactive oxygen species.A) From triplicate cultures, 0.25 mM hypoxanthine with or without 0.1 u/ml xanthine oxidase was added to ∼106 cfu/ml of cells in PBS. Colony forming units were determined at the indicated time points. The cfu of untreated samples remained essentially constant throughout the experiment. The viable count of the treated sample is compared to untreated sample at each time point. B) Triplicate mid-log cultures in LB 0.2% glucose were split and 2.5 mM H2O2 was added to one half. Colony forming units were determined and compared to untreated sample at each time point as above. The cfu of untreated samples increased only slightly during the experiment. The mean and range are plotted for each experiment. Strains used: 14028, JS456, JS831.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654757&req=5

pone-0004975-g001: Effect of sod mutations on survival in cells exposed to exogenous reactive oxygen species.A) From triplicate cultures, 0.25 mM hypoxanthine with or without 0.1 u/ml xanthine oxidase was added to ∼106 cfu/ml of cells in PBS. Colony forming units were determined at the indicated time points. The cfu of untreated samples remained essentially constant throughout the experiment. The viable count of the treated sample is compared to untreated sample at each time point. B) Triplicate mid-log cultures in LB 0.2% glucose were split and 2.5 mM H2O2 was added to one half. Colony forming units were determined and compared to untreated sample at each time point as above. The cfu of untreated samples increased only slightly during the experiment. The mean and range are plotted for each experiment. Strains used: 14028, JS456, JS831.
Mentions: The data above suggest that the most vulnerable target of phagocytic superoxide is extracytoplasmic. But identifying the target(s) is complicated by the fact that sodC mutants of Salmonella show no significant in vitro phenotypes. As shown in Figure 1A, there was no significant difference in sensitivity to 250 µM hypoxanthine/xanthine oxidase of the wild type and isogenic mutants devoid of periplasmic SODs or cytoplasmic SODs. We have performed similar experiments using this and other superoxide-generating systems under a variety of conditions and have never observed a reproducible difference between the wild type and sodCI sodCII double mutant. This should not be surprising. It is estimated that NADPH oxidase is capable of producing a steady state superoxide concentration of 100 µM in a phagosome [12]. Using published data [44], [45], one can calculate that in vitro systems such as that used above are capable of generating only <1 µM of superoxide for a few minutes. When higher concentrations (2.5 mM) of hydrogen peroxide were used, the sodA sodB double mutant showed increased sensitivity (Figure 1B), as previously reported [46]. The sodCI sodCII mutant behaved identically to wild type, again distinguishing loss of cytoplasmic versus periplasmic SOD activity.

Bottom Line: The phagocytic oxidative burst is a primary effector of innate immunity that protects against bacterial infection.We found that SodCI acts independently of cytoplasmic SODs, SodA and SodB.Although sodCI did show genetic interaction with recA, this was apparently independent of recombination and is presumably due to the pleiotropic effects of a recA mutation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, USA.

ABSTRACT

Background: The phagocytic oxidative burst is a primary effector of innate immunity that protects against bacterial infection. However, the mechanism by which reactive oxygen species (ROS) kill or inhibit bacteria is not known. It is often assumed that DNA is a primary target of oxidative damage, consistent with known effects of endogenously produced ROS in the bacterial cytoplasm. But most studies fail to distinguish between effects of host derived ROS versus damage caused by endogenous bacterial sources. We took advantage of both the ability of Salmonella enterica serovar Typhimurium to survive in macrophages and the genetic tractability of the system to test the hypothesis that phagocytic superoxide damages cytoplasmic targets including DNA.

Methodology/principal findings: SodCI is a periplasmic Cu-Zn superoxide dismutase (SOD) that contributes to the survival of Salmonella Typhimurium in macrophages. Through competitive virulence assays, we asked if sodCI has a genetic interaction with various cytoplasmic systems. We found that SodCI acts independently of cytoplasmic SODs, SodA and SodB. In addition, SodCI acts independently of the base excision repair system and RuvAB, involved in DNA repair. Although sodCI did show genetic interaction with recA, this was apparently independent of recombination and is presumably due to the pleiotropic effects of a recA mutation.

Conclusions/significance: Taken together, these results suggest that bacterial inhibition by phagocytic superoxide is primarily the result of damage to an extracytoplasmic target.

Show MeSH
Related in: MedlinePlus