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Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase.

Managò A, Becker KA, Carpinteiro A, Wilker B, Soddemann M, Seitz AP, Edwards MJ, Grassmé H, Szabò I, Gulbins E - Antioxid. Redox Signal. (2015)

Bottom Line: Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited.This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Biology, University of Padova , Padova, Italy .

ABSTRACT

Aims: Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.

Results: Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.

Innovation: These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.

Conclusion: These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

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Related in: MedlinePlus

Short-term bioenergetic effects of pyocyanin in intact cells. (A) Mitochondrial membrane potential was measured in Jurkat lymphocytes using TMRM under control conditions and on treatment with pyocyanin or uncoupler FCCP. Mean values±SD are shown (n=3). (B) As in (A), but after preincubation of the cells for 30 min with CSA, an inhibitor of the PTP. The arrows in panels (A) and (B) indicate addition of drugs as outlined. (C, D) OCR measured in intact cells on addition of the indicated compounds. Results shown are representative of three independent experiments. CSA, cyclosporine A; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone; OCR, oxygen consumption rate; PTP, permeability transition pore; TMRM, tetramethylrhodamine, methyl ester. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
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f3: Short-term bioenergetic effects of pyocyanin in intact cells. (A) Mitochondrial membrane potential was measured in Jurkat lymphocytes using TMRM under control conditions and on treatment with pyocyanin or uncoupler FCCP. Mean values±SD are shown (n=3). (B) As in (A), but after preincubation of the cells for 30 min with CSA, an inhibitor of the PTP. The arrows in panels (A) and (B) indicate addition of drugs as outlined. (C, D) OCR measured in intact cells on addition of the indicated compounds. Results shown are representative of three independent experiments. CSA, cyclosporine A; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone; OCR, oxygen consumption rate; PTP, permeability transition pore; TMRM, tetramethylrhodamine, methyl ester. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Mentions: The addition of pyocyanin to intact cells not only led to ROS production but also resulted in a very rapid but incomplete dissipation of the MMP, Δψm (Fig. 3A). This change in Δψm was independent of the opening of the permeability transition pore (PTP) (34), because it also occurred in the presence of cyclosporin A, a potent PTP inhibitor (Fig. 3B). Next, we used an extracellular flux analyzer to determine the effects of pyocyanin on respiration in intact adherent mouse embryonic fibroblasts. The addition of pyocyanin to these cells immediately increased oxygen consumption (Fig. 3C). This apparent increase in respiration was only partially reversible by oligomycin, which blocks ATP synthase (Fig. 3C). The subsequent addition of the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) did not restore respiration, whereas the addition of antimycin A, an inhibitor of complex III, further reduced but did not completely abolish oxygen consumption (Fig. 3C). Antimycin A, by binding to the Qi site of cytochrome c reductase, inhibits the oxidation of ubiquinol in the electron transport chain of oxidative phosphorylation. The inhibition of this reaction disrupts respiration and prevents the formation of the proton gradient across the inner membrane. Thus, pyocyanin drastically reduced the respiratory response to the subsequent addition of oligomycin and FCCP. A massive loss of cells because of death and detachment was excluded by direct microscopic observation of the cells at the end of the experiments. In contrast, if cellular respiration was depressed by oligomycin, the addition of pyocyanin induced a recovery of the respiratory rate (Fig. 3D). This recovery may be simply a reflection of the loss of Δψm induced by pyocyanin: According to the chemiosmotic model, depolarization with associated respiratory stimulation is likely to reflect the appearance of a Δψm-dissipating proton leak. Again, the subsequent addition of FCCP had no effect, whereas antimycin A reduced respiration (Fig. 3D). Similar effects were observed in both settings with 25 μM pyocyanin (not shown). These results suggest that the pyocyanin-induced increase in oxygen consumption is only partially caused by an increase in respiration, given the lack of full reversal by antimycin A. Alternatively, respiration may be increased bypassing complex III. Oxygen consumption is probably also enhanced by the involvement of molecular oxygen as an acceptor of electrons directly from pyocyanin, independent of the respiratory chain.


Pseudomonas aeruginosa pyocyanin induces neutrophil death via mitochondrial reactive oxygen species and mitochondrial acid sphingomyelinase.

Managò A, Becker KA, Carpinteiro A, Wilker B, Soddemann M, Seitz AP, Edwards MJ, Grassmé H, Szabò I, Gulbins E - Antioxid. Redox Signal. (2015)

Short-term bioenergetic effects of pyocyanin in intact cells. (A) Mitochondrial membrane potential was measured in Jurkat lymphocytes using TMRM under control conditions and on treatment with pyocyanin or uncoupler FCCP. Mean values±SD are shown (n=3). (B) As in (A), but after preincubation of the cells for 30 min with CSA, an inhibitor of the PTP. The arrows in panels (A) and (B) indicate addition of drugs as outlined. (C, D) OCR measured in intact cells on addition of the indicated compounds. Results shown are representative of three independent experiments. CSA, cyclosporine A; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone; OCR, oxygen consumption rate; PTP, permeability transition pore; TMRM, tetramethylrhodamine, methyl ester. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4403017&req=5

f3: Short-term bioenergetic effects of pyocyanin in intact cells. (A) Mitochondrial membrane potential was measured in Jurkat lymphocytes using TMRM under control conditions and on treatment with pyocyanin or uncoupler FCCP. Mean values±SD are shown (n=3). (B) As in (A), but after preincubation of the cells for 30 min with CSA, an inhibitor of the PTP. The arrows in panels (A) and (B) indicate addition of drugs as outlined. (C, D) OCR measured in intact cells on addition of the indicated compounds. Results shown are representative of three independent experiments. CSA, cyclosporine A; FCCP, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone; OCR, oxygen consumption rate; PTP, permeability transition pore; TMRM, tetramethylrhodamine, methyl ester. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
Mentions: The addition of pyocyanin to intact cells not only led to ROS production but also resulted in a very rapid but incomplete dissipation of the MMP, Δψm (Fig. 3A). This change in Δψm was independent of the opening of the permeability transition pore (PTP) (34), because it also occurred in the presence of cyclosporin A, a potent PTP inhibitor (Fig. 3B). Next, we used an extracellular flux analyzer to determine the effects of pyocyanin on respiration in intact adherent mouse embryonic fibroblasts. The addition of pyocyanin to these cells immediately increased oxygen consumption (Fig. 3C). This apparent increase in respiration was only partially reversible by oligomycin, which blocks ATP synthase (Fig. 3C). The subsequent addition of the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) did not restore respiration, whereas the addition of antimycin A, an inhibitor of complex III, further reduced but did not completely abolish oxygen consumption (Fig. 3C). Antimycin A, by binding to the Qi site of cytochrome c reductase, inhibits the oxidation of ubiquinol in the electron transport chain of oxidative phosphorylation. The inhibition of this reaction disrupts respiration and prevents the formation of the proton gradient across the inner membrane. Thus, pyocyanin drastically reduced the respiratory response to the subsequent addition of oligomycin and FCCP. A massive loss of cells because of death and detachment was excluded by direct microscopic observation of the cells at the end of the experiments. In contrast, if cellular respiration was depressed by oligomycin, the addition of pyocyanin induced a recovery of the respiratory rate (Fig. 3D). This recovery may be simply a reflection of the loss of Δψm induced by pyocyanin: According to the chemiosmotic model, depolarization with associated respiratory stimulation is likely to reflect the appearance of a Δψm-dissipating proton leak. Again, the subsequent addition of FCCP had no effect, whereas antimycin A reduced respiration (Fig. 3D). Similar effects were observed in both settings with 25 μM pyocyanin (not shown). These results suggest that the pyocyanin-induced increase in oxygen consumption is only partially caused by an increase in respiration, given the lack of full reversal by antimycin A. Alternatively, respiration may be increased bypassing complex III. Oxygen consumption is probably also enhanced by the involvement of molecular oxygen as an acceptor of electrons directly from pyocyanin, independent of the respiratory chain.

Bottom Line: Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited.This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Biology, University of Padova , Padova, Italy .

ABSTRACT

Aims: Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.

Results: Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.

Innovation: These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.

Conclusion: These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

Show MeSH
Related in: MedlinePlus