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Involvement of a 1-Cys peroxiredoxin in bacterial virulence.

Kaihami GH, Almeida JR, Santos SS, Netto LE, Almeida SR, Baldini RL - PLoS Pathog. (2014)

Bottom Line: This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase.In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria.LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H(2)O(2) and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

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LsfA confers PA14 resistance to hydrogen peroxide and reduces it in vitro.(A) The recombinant proteins His-LsfA and His-C45A were incubated with 200 µM H2O2. The amount of H2O2 remaining in the reaction mixtures was determined by ferric-thiocyanate assays at the indicated time points. In a control assay, H2O2 was incubated with buffer without recombinant proteins. The data are the means ± SD from at least three independent experiments performed in triplicate. (B) Bacteria were grown to an OD600 nm of 1 and spread on LB plates. A 6 mm filter disk containing 2.5% H2O2 was placed on top of the bacterial lawn, and the plates were incubated at 37°C for 16 hours. The data represent 1 of 5 independent experiments and the diameters of the inhibition haloes are shown as the means ± SD (mm).
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ppat-1004442-g001: LsfA confers PA14 resistance to hydrogen peroxide and reduces it in vitro.(A) The recombinant proteins His-LsfA and His-C45A were incubated with 200 µM H2O2. The amount of H2O2 remaining in the reaction mixtures was determined by ferric-thiocyanate assays at the indicated time points. In a control assay, H2O2 was incubated with buffer without recombinant proteins. The data are the means ± SD from at least three independent experiments performed in triplicate. (B) Bacteria were grown to an OD600 nm of 1 and spread on LB plates. A 6 mm filter disk containing 2.5% H2O2 was placed on top of the bacterial lawn, and the plates were incubated at 37°C for 16 hours. The data represent 1 of 5 independent experiments and the diameters of the inhibition haloes are shown as the means ± SD (mm).

Mentions: A previous sequence alignment revealed that LsfA belongs to the Prx6 subfamily [16], with the Cys45 of LsfA as the putative peroxidasic cysteine (Fig. S1). To determine whether P. aeruginosa LsfA is indeed endowed with thiol-dependent peroxidase activity, the recombinant wild-type (His-LsfA) protein and a mutant protein without the putative catalytic cysteine (His-C45A) were expressed in Escherichia coli and purified by affinity chromatography. As predicted, H2O2 was reduced in the presence of wild-type His-LsfA but not when His-C45A was employed (Fig. 1A), showing that Cys45 is essential for catalysis and confirming that LsfA is an active 1-Cys Prx.


Involvement of a 1-Cys peroxiredoxin in bacterial virulence.

Kaihami GH, Almeida JR, Santos SS, Netto LE, Almeida SR, Baldini RL - PLoS Pathog. (2014)

LsfA confers PA14 resistance to hydrogen peroxide and reduces it in vitro.(A) The recombinant proteins His-LsfA and His-C45A were incubated with 200 µM H2O2. The amount of H2O2 remaining in the reaction mixtures was determined by ferric-thiocyanate assays at the indicated time points. In a control assay, H2O2 was incubated with buffer without recombinant proteins. The data are the means ± SD from at least three independent experiments performed in triplicate. (B) Bacteria were grown to an OD600 nm of 1 and spread on LB plates. A 6 mm filter disk containing 2.5% H2O2 was placed on top of the bacterial lawn, and the plates were incubated at 37°C for 16 hours. The data represent 1 of 5 independent experiments and the diameters of the inhibition haloes are shown as the means ± SD (mm).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199769&req=5

ppat-1004442-g001: LsfA confers PA14 resistance to hydrogen peroxide and reduces it in vitro.(A) The recombinant proteins His-LsfA and His-C45A were incubated with 200 µM H2O2. The amount of H2O2 remaining in the reaction mixtures was determined by ferric-thiocyanate assays at the indicated time points. In a control assay, H2O2 was incubated with buffer without recombinant proteins. The data are the means ± SD from at least three independent experiments performed in triplicate. (B) Bacteria were grown to an OD600 nm of 1 and spread on LB plates. A 6 mm filter disk containing 2.5% H2O2 was placed on top of the bacterial lawn, and the plates were incubated at 37°C for 16 hours. The data represent 1 of 5 independent experiments and the diameters of the inhibition haloes are shown as the means ± SD (mm).
Mentions: A previous sequence alignment revealed that LsfA belongs to the Prx6 subfamily [16], with the Cys45 of LsfA as the putative peroxidasic cysteine (Fig. S1). To determine whether P. aeruginosa LsfA is indeed endowed with thiol-dependent peroxidase activity, the recombinant wild-type (His-LsfA) protein and a mutant protein without the putative catalytic cysteine (His-C45A) were expressed in Escherichia coli and purified by affinity chromatography. As predicted, H2O2 was reduced in the presence of wild-type His-LsfA but not when His-C45A was employed (Fig. 1A), showing that Cys45 is essential for catalysis and confirming that LsfA is an active 1-Cys Prx.

Bottom Line: This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase.In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria.LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H(2)O(2) and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

Show MeSH
Related in: MedlinePlus