Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa

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 (K d ∼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.

Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis

“…3b). It is important to note that catalase (katA) has been shown to be involved in P. aeruginosa resistance to nitric oxide (Su et al, 2014); however, we measured katA expression in WT PAO1 and FRD1 and in the nirS mutants, and although the nirS mutants were marginally reduced in katA expression, we found no difference in katA expression when the WT isolates and nirS mutants were co-cultured with S. parasanguinis (data not shown). Altogether, these data suggested that nirS was largely induced by nitrogenous intermediates and not H 2 O 2 produced by S. parasanguinis, and that nirS played a role in alleviating endogenous nitrogenous stress.…”

cbb 3 -Type Cytochrome c Oxidases, Aerobic Respiratory Enzymes, Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

“…Cellular NO levels were measured based on a previously described method (20) with a NO detection reagent, diaminofluorescein-2 diacetate (DAF-2 DA) (Sekisui Medical, Tokyo, Japan). Onemilliliter quantities of planktonic shaking cultures grown under anoxic conditions were incubated with 10 M DAF-2 DA.…”

Nitric oxide-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity

“…This disruption includes inactivation of critical microbial enzymes by reacting with iron-containing proteins (especially Fe-S cluster protein) to produce a range of dinitrosyl iron complexes known as DNICs, which are mainly disruptive. 37 As for MRSA, reactive nitrogen intermediates, which are by-products of the reaction between NO and the free radical superoxide (O 2 * -), 11 have been shown to modify DNA, protein, and lipid as well as act indirectly on bacteria by modifying immune responses or other host cell functions. 38 The viability of the bacteria was also visualized by distinguishing live and dead bacteria using a confocal microscope and the results were quantitatively evaluated with the % survival rates by flow cytometry.…”