KatA Activity Research Articles

Antibacterial and Antibiofilm Activities of β-Lapachone by Modulating the Catalase Enzyme

Bacterial infections constantly have a large impact on public health, because of increased rates of resistance and reduced frequency of development of novel antibiotics. The utility of conventional antibiotics for treating bacterial infections has become increasingly challenging. The aim of the study was to assess the antibacterial effect of β-Lapachone (β-Lap), a novel synthetic compound. The antibacterial activity of the β-Lap compound was examined against laboratory strains by agar well diffusion method and broth dilution assay. Growth kinetics in presence of β-Lap on Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa (ATCC 27853) were assessed by microplate alamarBlue assay. Crystal violet blue assay was used for biofilm inhibition and biofilm eradication. P. aeruginosa catalase (KatA) complexed with β-Lap was modeled using molecular docking approach. β-Lap exhibited potent antimicrobial activity against laboratory strains of bacteria with MIC of 0.2 mM for S. saprophyticus and Staphylococcus aureus, and 0.04 mM for Staphylococcus epidermidis and Pseudomonas aeruginosa ATCC 27853. The inhibition of catalase enzyme was found to be the cause for its antibacterial activity. Bioinformatics analysis suggests that β-Lap can inhibit KatA activity by interacting with catalase proximal active site and heme binding site. The activity of some commercial antibiotics was enhanced in association with β-Lap. In addition, β-Lap inhibited the biofilm formation and eradicated the already formed and ultra-mature biofilms of aforesaid bacterial strains. These observations indicated that β-Lap could be a promising antibacterial agent for the treatment and prevention of infectious diseases.

The Catalase KatA Contributes to Microaerophilic H2O2 Priming to Acquire an Improved Oxidative Stress Resistance in Staphylococcus aureus.

Staphylococcus aureus has to cope with oxidative stress during infections. In this study, S. aureus was found to be resistant to 100 mM H2O2 during aerobic growth. While KatA was essential for this high aerobic H2O2 resistance, the peroxiredoxin AhpC contributed to detoxification of 0.4 mM H2O2 in the absence of KatA. In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to be required for detoxification of cumene hydroperoxide (CHP). The high H2O2 tolerance of aerobic S. aureus cells was associated with priming by endogenous H2O2 levels, which was supported by an oxidative shift of the bacillithiol redox potential to −291 mV compared to −310 mV in microaerophilic cells. In contrast, S. aureus could be primed by sub-lethal doses of 100 µM H2O2 during microaerophilic growth to acquire an improved resistance towards the otherwise lethal triggering stimulus of 10 mM H2O2. This microaerophilic priming was dependent on increased KatA activity, whereas aerobic cells showed constitutive high KatA activity. Thus, KatA contributes to the high H2O2 resistance of aerobic cells and to microaerophilic H2O2 priming in order to survive the subsequent lethal triggering doses of H2O2, allowing the adaptation of S. aureus under infections to different oxygen environments.

Helicobacter pylori Outer Membrane Vesicles Protect the Pathogen From Reactive Oxygen Species of the Respiratory Burst.

Outer membrane vesicles (OMVs) play an important role in the persistence of Helicobacter pylori infection. Helicobacter OMVs carry a plethora of virulence factors, including catalase (KatA), an antioxidant enzyme that counteracts the host respiratory burst. We found KatA to be enriched and surface-associated in OMVs compared to bacterial cells. This conferred OMV-dependent KatA activity resulting in neutralization of H2O2 and NaClO, and rescue of surrounding bacteria from oxidative damage. The antioxidant activity of OMVs was abolished by deletion of KatA. In conclusion, enrichment of antioxidative KatA in OMVs is highly important for efficient immune evasion.

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

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.

Protective role of extracellular catalase (KatA) against UVA radiation in Pseudomonas aeruginosa biofilms

One of the more stressful factors that Pseudomonas aeruginosa must face in nature is solar UVA radiation. In this study, the protective role of KatA catalase in both planktonic cells and biofilms of P. aeruginosa against UVA radiation was determined by using the wild-type (PAO1) and an isogenic catalase deficient strain (katA). The katA strain was more sensitive than the wild-type, especially in the case of biofilms. Moreover, the wild-type biofilm was more resistant than its planktonic counterpart, but this was not observed in the katA strain. Striking KatA activity was detected in the matrix of katA+ strains, and to our knowledge, this is the first report of this activity in the matrix of P. aeruginosa biofilms. Provision of bovine catalase or KatA to the matrix of a katA biofilm significantly increased its UVA tolerance, demonstrating that extracellular KatA is essential to optimal defense against UVA in P. aeruginosa biofilms. Efficiency of photocatalytic treatments using TiO2 and UVA was lower in biofilms than in planktonic cells, but KatA and KatB catalases seem not to be responsible for the higher resistance of the sessile cells to this treatment.

IscR Modulates Catalase A (KatA) Activity, Peroxide Resistance, and Full Virulence of Pseudomonas aeruginosa PA14

We have identified the iscR (PA3815) gene encoding an iron-sulfur cluster assembly regulator homologue as one of the genes required for peroxide resistance in Pseudomonas aeruginosa PA14. Here, we present the phenotypic characterization of an iscR deletion mutant in terms of KatA expression, stress responses, and virulence. The iscR null mutant exhibited reduced KatA activity at the posttranslational level, hypersensitivity to hydrogen peroxide, and virulence-attenuation in Drosophila melanogaster and mouse peritonitis models. These phenotypes were fully restored by multi-copy-based expression of katA. These results suggest that the requirement of IscR in P. aeruginosa is related to the proper activity of KatA, which is crucial for peroxide resistance and full virulence of this bacterium.

Pseudomonas aeruginosa OxyR Is Required for Full Virulence in Rodent and Insect Models of Infection and for Resistance to Human Neutrophils

The role of the H2O2-responsive transactivator OxyR was evaluated in mouse and Drosophila models of Pseudomonas aeruginosa infection and by assays of neutrophil killing. Relative to that of wild-type bacteria, oxyR mutant viability was reduced by 99% or greater in mouse models of acute pneumonia and burn sepsis, and the oxyR mutant was more sensitive to killing by human neutrophils and was delayed in the kinetics of Drosophila melanogaster killing. During human infection by the opportunistic bacterium Pseudomonas aeruginosa, the organism faces exposure to toxic reactive oxygen intermediates generated by phagocytic cells. During the phagocytic oxidative burst, hydrogen peroxide (H2O2) is generated at high millimolar levels within the phagosomal vacuole (4). Enzymatic defenses against H2O2 in P. aeruginosa are provided by at least three catalases (KatA, KatB, and KatC) (2, 8), several probable peroxidases (refer to www.pseudomonas.com), and four established alkyl hydroperoxide reductases (AhpA, AhpB, AhpCF, and Ohr); members of the last class can also degrade H2O2 and various alkylhydroperoxides (9). The major gene product involved in endogenous H2O2 detoxification in P. aeruginosa is the 170-kDa heterotrimeric catalase KatA (8). Accordingly, katA gene expression is consistently high during vigorous aerobic growth. In fact, KatA activity is maintained at such high levels that even significant H2O2 stress triggers only a twofold increase in expression, suggesting that a high rate of KatA activity is critical for the detoxification of endogenous H2O2 produced during normal aerobic metabolism. In contrast to that of the katA gene, expression of several other oxidative stress defense genes, including katB-ankB (5), ahpB, and ahpCF, is dramatically increased upon exposure to H2O2, organic peroxides, or the redox cycling agent paraquat, suggesting that a highly specific and tightly regulated stress response exists in this organism (5, 9). We have found that such a response is governed by the 34-kDa H2O2-responsive transactivator known as OxyR in P. aeruginosa (9). P. aeruginosa lacking OxyR is exquisitely susceptible to H2O2, even though it possesses wild-type catalase activity (9). In fact, isolated colonies of oxyR mutant bacteria do not even appear on aerobic Luria-Bertani (LB) agar, because autoxidizable components in the medium itself generate ∼1.2 μM H2O2/min (3). This concentration of H2O2 has been detected in peripheral blood from human donors and is sufficient to kill these organisms (3). In this study, we tested the hypothesis that OxyR is required for the full virulence of P. aeruginosa by use of (i) a mouse intranasal model of acute pneumonia, (ii) a mouse burn sepsis model, (iii) an in vitro model of neutrophil killing, and (iv) a Drosophila melanogaster alternative model for animal infection.

Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-L-homoserine lactone) and PAI-2 (N-butyryl-L-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. beta-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.

We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.

Oxygen-dependent regulation of the expression of the catalase gene katA of Lactobacillus sakei LTH677.

The catalase gene katA of Lactobacillus sakei LTH677 was cloned and expressed in Escherichia coli UM2, Lactobacillus casei LK1, and Lactobacillus curvatus LTH1432. The last host is a catalase-deficient plasmid-cured derivative of a starter organism used in meat fermentation. The regulation of katA expression was found to be the same in L. sakei LTH677 and the recombinant strains. The addition of H2O2 to anaerobic cultures, as well as a switch to aerobic conditions, resulted in a strong increase in KatA activity. The expression was investigated in more detail with L. sakei LTH677 and L. curvatus LTH4002. The recombinant strain LTH4002 did not accumulate H2O2 under glucose-limited aerobic conditions and remained viable in the stationary phase. Under inductive conditions, the katA-specific mRNA and the apoenzyme were synthesized de novo. Deletion derivatives of the katA promoter were produced, and the regulatory response was investigated by fusion to the beta-glucuronidase reporter gene gusA and expression in L. sakei LTH677. The fact that gene expression was subject to induction was confirmed at the level of transcription and protein synthesis. A small putative regulatory sequence of at least 25 bp was identified located upstream of the -35 site. Competition experiments performed with L. sakei LTH677 harboring the fusion constructs consisting of the katA promoter and gusA revealed that an activator protein is involved in the transcriptional induction of katA.

Cloning and characterization of the katA gene of Rhizobium meliloti encoding a hydrogen peroxide-inducible catalase.

To investigate the involvement of bacterial catalases of the symbiotic gram-negative bacterium Rhizobium meliloti in the development of Medicago-Rhizobium functional nodules, we cloned a putative kat gene by screening a cosmid library with a catalase-specific DNA probe amplified by PCR from the R. meliloti genome. Nucleotide sequence analysis of a 1.8-kb DNA fragment revealed an open reading frame, called katA, encoding a peptide of 562 amino acid residues with a calculated molecular mass of 62.9 kDa. The predicted amino acid sequence showed a high homology with the primary structure of monofunctional catalases from eucaryotes and procaryotes. The katA gene was localized on the chromosome, and the katA gene product was essentially found in the periplasmic space. A katA::Tn5 mutant was obtained and showed a drastic sensitivity to hydrogen peroxide, indicating an essential protective role of KatA. However, neither Nod nor Fix phenotypes were impaired in the mutant, suggesting that KatA is not essential for nodulation and establishment of nitrogen fixation. Exposure to a sublethal concentration of H2O2 enhanced KatA activity (100-fold) and also increased survival to subsequent H2O2 exposure at higher concentrations. No protection is observed in katA::Tn5, indicating that KatA is the major component of an adaptive response.