Catalase (KatA) Plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa

Shengchang Su, Warunya Panmanee, Jeffrey J. Wilson, Harry K. Mahtani, Qian Li, Bradley D. VanderWielen, Thomas M. Makris, Melanie Rogers, Cameron McDaniel, John D Lipscomb, Randall T. Irvin, Michael J. Schurr, Jack R. Lancaster, Rhett A. Kovall, Daniel J. Hassett

Research output: Contribution to journalArticle

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Abstract

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ,50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.

Original languageEnglish (US)
Article numbere91813
JournalPLoS One
Volume9
Issue number3
DOIs
StatePublished - Mar 24 2014

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Pseudomonas aeruginosa
Catalase
nitric oxide
catalase
Nitric Oxide
Transcription
Heme
mutants
Paramagnetic resonance
Bacteria
Nitrite Reductases
Poisons
X ray crystallography
anaerobiosis
Biofilms
Pathogens
iron
Nitrites
Respiration
Hydrogen Peroxide

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Su, S., Panmanee, W., Wilson, J. J., Mahtani, H. K., Li, Q., VanderWielen, B. D., ... Hassett, D. J. (2014). Catalase (KatA) Plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa. PLoS One, 9(3), [e91813]. https://doi.org/10.1371/journal.pone.0091813

Catalase (KatA) Plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa. / Su, Shengchang; Panmanee, Warunya; Wilson, Jeffrey J.; Mahtani, Harry K.; Li, Qian; VanderWielen, Bradley D.; Makris, Thomas M.; Rogers, Melanie; McDaniel, Cameron; Lipscomb, John D; Irvin, Randall T.; Schurr, Michael J.; Lancaster, Jack R.; Kovall, Rhett A.; Hassett, Daniel J.

In: PLoS One, Vol. 9, No. 3, e91813, 24.03.2014.

Research output: Contribution to journalArticle

Su, S, Panmanee, W, Wilson, JJ, Mahtani, HK, Li, Q, VanderWielen, BD, Makris, TM, Rogers, M, McDaniel, C, Lipscomb, JD, Irvin, RT, Schurr, MJ, Lancaster, JR, Kovall, RA & Hassett, DJ 2014, 'Catalase (KatA) Plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa' PLoS One, vol. 9, no. 3, e91813. https://doi.org/10.1371/journal.pone.0091813
Su, Shengchang ; Panmanee, Warunya ; Wilson, Jeffrey J. ; Mahtani, Harry K. ; Li, Qian ; VanderWielen, Bradley D. ; Makris, Thomas M. ; Rogers, Melanie ; McDaniel, Cameron ; Lipscomb, John D ; Irvin, Randall T. ; Schurr, Michael J. ; Lancaster, Jack R. ; Kovall, Rhett A. ; Hassett, Daniel J. / Catalase (KatA) Plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa. In: PLoS One. 2014 ; Vol. 9, No. 3.
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abstract = "Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ,50{\%} the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.",
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AU - Su, Shengchang

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AU - Wilson, Jeffrey J.

AU - Mahtani, Harry K.

AU - Li, Qian

AU - VanderWielen, Bradley D.

AU - Makris, Thomas M.

AU - Rogers, Melanie

AU - McDaniel, Cameron

AU - Lipscomb, John D

AU - Irvin, Randall T.

AU - Schurr, Michael J.

AU - Lancaster, Jack R.

AU - Kovall, Rhett A.

AU - Hassett, Daniel J.

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