The peptidoglycan-associated lipoprotein OprL helps protect a Pseudomonas aeruginosa mutant devoid of the transactivator OxyR from hydrogen peroxide-mediated killing during planktonic and biofilm culture

Warunya Panmanee, Francisco Gomez, David Witte, Vijay Pancholi, Bradley E Britigan, Daniel J. Hassett

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

OxyR controls H2O2-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<107/ml) organisms are easily killed by micromolar H2O2. The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H2O2. We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H2O 2 than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H2O2. Given the sensitivity of oxyR oprL bacteria to planktonic H2O2, we next tested the hypothesis that the biofllm mode of growth might protect such organisms from H2O2-mediated killing. Surprisingly, biofllm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H2O2. Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H2O2 in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofllms.

Original languageEnglish (US)
Pages (from-to)3658-3669
Number of pages12
JournalJournal of bacteriology
Volume190
Issue number10
DOIs
StatePublished - May 1 2008

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Trans-Activators
Peptidoglycan
Biofilms
Pseudomonas aeruginosa
Hydrogen Peroxide
Lipoproteins
Catalase
Peroxiredoxins
Bacteria
Proteins
Growth
Transmission Electron Microscopy
Oxidants
Lasers
Antioxidants
Gels
Cell Membrane
Gene Expression
Enzymes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

The peptidoglycan-associated lipoprotein OprL helps protect a Pseudomonas aeruginosa mutant devoid of the transactivator OxyR from hydrogen peroxide-mediated killing during planktonic and biofilm culture. / Panmanee, Warunya; Gomez, Francisco; Witte, David; Pancholi, Vijay; Britigan, Bradley E; Hassett, Daniel J.

In: Journal of bacteriology, Vol. 190, No. 10, 01.05.2008, p. 3658-3669.

Research output: Contribution to journalArticle

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abstract = "OxyR controls H2O2-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<107/ml) organisms are easily killed by micromolar H2O2. The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H2O2. We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H2O 2 than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H2O2. Given the sensitivity of oxyR oprL bacteria to planktonic H2O2, we next tested the hypothesis that the biofllm mode of growth might protect such organisms from H2O2-mediated killing. Surprisingly, biofllm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H2O2. Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H2O2 in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofllms.",
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