Cloning and characterization of the catalase gene of Neisseria gonorrhoeae: use of the gonococcus as a host organism for recombinant DNA.

The structural gene for the catalase of Neisseria gonorrhoeae was cloned into a Kat- strain of that organism by using a recombinant vector derived from one of the beta-lactamase-specifying plasmids found in that organism. The kat gene was then successfully subcloned into both pUC8 and pGB2, transformed into Escherichia coli, and shown to complement the E. coli katE mutants UM2 and UMRl. The gene was subsequently mutagenized and returned to the gonococcus to generate a Kat- strain that was phenotypically identical to the strain originally used to clone the gene. The sequence of the gene and the derived amino acid sequence showed that the gonococcal kat gene closely resembles the hktE gene of Haemophilus influenzae. The sequence of the promoter region of the gonococcal kat gene is unusual and may explain the extremely high, loosely regulated expression of the gene.     

Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella.

We examined 40 strains of Legionella for reduced-oxygen scavenging enzymes. Using a simple reaction chamber with a Swinney filter for the Beers and Sizer assay, we determined the catalase activity of live cells grown on buffered charcoal-yeast extract agar. For 29 strains of Legionella pneumophila, the apparent first-order rate constants for catalase ranged from 0.000 to 0.005. Similarly, low values ranging from 0.001 to 0.005 were observed for Legionella wadsworthii, Legionella oakridgensis, and Legionella gormanii. High catalase activities were found for Legionella jordanis, Legionella longbeachae, Legionella micdadei, and Legionella bozemanii, with first-order rate constant values of 0.010 to 0.035. Cell-free extracts were analyzed for catalase, peroxidase, and superoxide dismutase. Cell-free extracts of all strains had superoxide dismutase levels ranging from 8.2 to 30.5 U per mg of protein. The species could be characterized by their catalase and peroxidase since L. pneumophila and L. gormanii had only peroxidase (relative molecular weight [Mr], 150,000); L. dumoffii had a peroxidase (Mr, 150,000) plus a catalase (Mr, 174,000); and all remaining species had catalase only (Mr, 300,000, 220,000, or 150,000).     

Age-related changes in Cu,Zn superoxide dismutase, Se-dependent and -independent glutathione peroxidase and catalase activities in specific areas of rat brain.

Oxidative injury of tissues involves both accumulation of damage due to persistent oxidative stress and loss of the proper balance of antioxidative enzymes. These events may produce a faster rate of tissue senescence. In this regard, we have assayed the antioxidative enzyme activities (Cu,Zn superoxide dismutase, glutathione peroxidase and catalase), in various areas of rat brain (prefrontal cortex, parietal cortex, hippocampus, hypothalamus, caudate nucleus, mesencephalon and lower brain stem) for the age groups of 3, 6, 12, 24 months. The results obtained show that the levels of antioxidant enzyme activities differed considerably in the various brain parts studied. Furthermore, changes in the specific activities of superoxide dismutase, catalase, and glutathione peroxidase did not follow the same pattern as a function of aging. In particular, in prefrontal cortex and caudate nucleus, superoxide dismutase and glutathione peroxidase activities did not change, while catalase activity decreased. In parietal cortex and mesencephalon, superoxide dismutase and glutathione peroxidase activities increased, but the catalase activity decreased in parietal cortex and did not change in mesencephalon. In lower brain stem, the activities of glutathione peroxidase and catalase decreased in 3-12-month-old rats. The activity of glutathione peroxidase was increased in the hippocampus and was decreased in hypothalamus during aging. In this area the catalase activity was also significantly diminished.     

Enzymatic degradation of H2O2 by Leptospira.

The enzymes responsible for reducing H2O2 were surveyed in 49 strains of Leptospira by using semiquantitative assays for catalase and peroxidase. The survey revealed a differential distribution of catalase and peroxidase activities between the two leptospiral complexes. The pathogenic Leptospira interrogans strains gave strong catalase and weak or negative peroxidase reactions. Conversely, the nonpathogenic Leptospira biflexa strains gave strong peroxidase and negative or weak catalase reactions. An intermediate group of four L. biflexa strains, which were isolated from mammals, fell into the high peroxidase, low or negative catalase group. One water isolate, H-23, gave strong reactions for both enzymes and was examined for virulence and in vitro growth parameters. Results indicate metabolic differences between pathogens and water forms in their abilities to reduce H2O2.     

Role of superoxide dismutase and catalase as determinants of pathogenicity of Nocardia asteroides: importance in resistance to microbicidal activities of human polymorphonuclear neutrophils.

The roles of nocardial superoxide dismutase (SOD) and catalase in the resistance of Nocardia asteroides to the microbicidal properties of human polymorphonuclear leukocytes were determined in vitro. The neutrophils killed ca. 80% of the cells of the less virulent N. asteroides 10905 and ca. 50% of the log phase of the more virulent N. asteroides GUH-2 after 180 min of incubation. These phagocytes were not able to kill early-stationary-phase cells of strain GUH-2 that contained 10 times more intracytoplasmic catalase than log-phase cells of the same culture. However, the polymorphonuclear leukocytes were able to kill more than 50% of the cells of early-stationary-phase strain GUH-2 after treatment with purified antibody specific for surface-associated SOD. No killing was observed when the bacteria were treated with normal rabbit immunoglobulin G or with serum obtained from rabbits immunized against whole nocardial cells (containing little or no activity against SOD). These phagocytes killed more than 99% of Listeria monocytogenes used as a control. Chlorpromazine-treated polymorphonuclear leukocytes killed L. monocytogenes (70%) but they were not able to kill antibody-treated cells of N. asteroides GUH-2. Exogenously added SOD partially protected strain 10905, which lacked surface-associated enzyme, but it had no effect on the killing of strain GUH-2, which already possessed significant amounts of surface-bound SOD. In contrast, catalase added to the nocardiae provided almost complete protection to the log-phase cells of strain GUH-2, but strain 10905 was only partially protected. SOD combined with catalase had additive activity which completely protected the cells of strain 10905. A mutant of N. asteroides GUH-2 (SCII-C) is more virulent during the log phase than is the parental strain. This mutant contained at least 7 times more catalase at this stage of growth than did the parent. No other differences between these two strains were observed during the log phase. In sharp contrast to those of the parent, log-phase cells of this high-catalase mutant were not killed by polymorphonuclear phagocytes. These data indicate a role for both SOD and catalase in the resistance of Nocardia spp. to human neutrophils, and they represent at least two factors associated with virulence.     

OxyR acts as a repressor of catalase expression in Neisseria gonorrhoeae

It has been reported that Neisseria gonorrhoeae possesses a very high level of catalase activity, but the regulation of catalase expression has not been investigated extensively. In Escherichia coli and Salmonella enterica serovar Typhimurium, it has been demonstrated that OxyR is a positive regulator of hydrogen peroxide-inducible genes, including the gene encoding catalase. The oxyR gene from N. gonorrhoeae was cloned and used to complement an E. coli oxyR mutant, confirming its identity and function. The gene was inactivated by inserting a kanamycin resistance cassette and used to make a knockout allele on the chromosome of N. gonorrhoeae strain 1291. In contrast to E. coli, the N. gonorrhoeae oxyR::kan mutant expressed ninefold-more catalase activity and was more resistant to hydrogen peroxide killing than the wild type. These data are consistent with OxyR in N. gonorrhoeae acting as a repressor of catalase expression.     

A Neisseria gonorrhoeae catalase mutant is more sensitive to hydrogen peroxide and paraquat, an inducer of toxic oxygen radicals

Catalase is hypothesized to be critical in the protection of Neisseria gonorrhoeae from H2O2 produced during aerobic respiration and by phagocytes during infection. Here we cloned the catalase (kat) gene of gonococcal strain FA1090 and constructed a genetically defined N. gonorrhoeae kat mutant to assess the role of catalase in defense against oxidative stress. The gonococcal kat gene conferred increased H2O2 resistance to a catalase-deficient Escherichia coli strain. Mutation of the kat gene in strain FA1090 via an in-frame deletion resulted in increased sensitivity to H2O2 and paraquat, an inducer of toxic oxygen radicals. Expression of catalase in trans from a shuttle vector restored catalase activity and paraquat resistance to the kat mutant, but not resistance to H2O2. The inability to fully complement the mutant was perhaps due to a modification in the catalase, as evidenced by altered mobility of the recombinant catalase on activity gels when expressed from the shuttle vector in N. gonorrhoeae. Additionally, we showed a 262 base pair region upstream of the kat gene is required for expression in E. coli and a putative fumarate-nitrate regulator (FNR) binding site is located in this region.     

Age-dependent basal level and induction capacity of copper-zinc and manganese superoxide dismutase and other scavenging enzyme activities in leukocytes from young and elderly adults.

Several enzymes, including superoxide dismutase (SOD), catalase, glutathione peroxidase, and D-glucose-6-phosphate dehydrogenase are capable of scavenging reactive oxygen species in in vivo. We assessed both basal levels and the capacity of these enzyme activities to be induced in human leukocytes in response to a variety of agents. Basal activity of copper-zinc SOD, and manganese SOD showed little variation with age. In contrast, the basal activity of the three H2O2 scavenging enzymes, catalase, glutathione peroxidase, and D-glucose-6-phosphate dehydrogenase, was significantly higher in younger adults than in elderly individuals. Both manganese SOD and copper, zinc SOD activities were significantly induced by paraquat, interleukin-1, tumor necrosis factor, adriamycin, and bleomycin in lymphocytes and neutrophils from asymptomatic non-aged adults, whereas neither activity was induced in aged individuals. In contrast, glutathione peroxidase activity was significantly induced in both groups of subjects, whereas catalase and D-glucose-6-phosphate dehydrogenase were only slightly induced in either. Enzyme induction with paraquat, adriamycin, or bleomycin was inhibitable by neutralizing antibody to interleukin-1 and tumor necrosis factor, suggesting that the inductions observed with these three drugs are due to the distal mediators, interleukin-1 or tumor necrosis factor released from the cells. Finally, as observed in the regulation of genes in eukaryotes (Storz et al: Bacterial defenses against oxidative stress. Trends Genetics 1990, 6:363-368, ref. 1) O2- and H2O2 seem to differ in the rate of change with age in both basal levels and inducibility under oxygen stress.     

Analysis of Aspergillus fumigatus catalases possessing antigenic activity.

Analysis of Aspergillus fumigatus water soluble fractions by electrophoresis on non-denaturing polyacrylamide gels (PAGE) showed the presence of at least three catalase bands. They were designated F, S1 and S2 in order of descending electrophoretic mobility with respect to the anode. The multiple enzyme forms appear to be distinct in their physicochemical properties. Enzyme bands S1 and S2 were simple catalases; the F band had an additional peroxidase function. All of the components were antigenic and differed in their binding to specific antibodies raised in rabbits with separate fractions of A. fumigatus mycelium. When serum from patients with aspergilloma, allergic bronchopulmonary aspergillosis, cystic fibrosis and chronic asthma were pre-incubated with A. fumigatus antigens and analysed by PAGE, 17 of 26 samples either abolished or reduced catalase activity. Enzyme F was a non-Concanavalin A (ConA)-binding antigen; the S1 and S2 enzymes were ConA-binding glycoprotein antigens. The major catalase band present in A. niger preparations represented only a minor component in A. fumigatus.     

Presence of hydrogen peroxide in media used for cultivation of Neisseria gonorrhoeae.

Defined complex media used for cultivation of Neisseria gonorrhoeae were tested for the presence of H2O2 by both a spectrophotometric and a polarographic assay. H2O2 (35 to 165 microM) was present in all media tested. In the defined media, H2O2 was generated by the interaction of cysteine with other amino acids. The addition of the chelator 8-hydroxyquinoline prevented formation of detectable H2O2, suggesting that metal ions were necessary. The persistence of H2O2 varied greatly among different media. Medium components which affected the presence of H2O2 were pyruvate, oxalacetate, and sodium sulfite. Sodium sulfite also generated superoxide radical. In liquid medium containing H2O2, the endogenous gonococcal catalase present in an inoculum of about 2 X 10(7) colony-forming units/ml destroyed detectable H2O2. The long lag phase which resulted from a 10-fold lower inoculum could not be shortened by the addition of exogenous catalase. Small amounts of residual H2O2 in agar plates of complex medium affected the viability of gonococci which had been suspended in buffer and incubated for 60 min at 37 degrees C. Addition of pyruvate or catalase increased viable counts in medium containing H2O2.     

The activity of liver oxidative enzymes after single and multiple grapefruit juice ingestion.

In the recent time, several in vitro and in vivo studies have shown the inhibitory effect of grapefruit juice on metabolism of xenobiotics catalyzed by liver oxidative enzymes including cytochrome P450 izoenzymes. However, all these experiments were done with a single dose of grapefruit juice. The primary aim of this study was to evaluate if the chronical ingestion of grapefruit juice can cause enzyme activity alteration as well as a single dose. Three groups of male mice were used: the control group, the group which was administered 0.2 mL of grapefruit juice per os 10 days and the group which was administered single dose of 0.5 mL grapefruit juice per os 90 min. before the sacrificing. After the sacrificing of animals, liver was homogenized with appropriate buffer, and the activity of oxidative liver enzymes: xanthine oxidase (XOD), peroxidase (Px), catalase (CAT), lipid peroxidase (LPx), glutathion peroxidase (GSH-Px) and liver glutathion contents (GSH) were detected by standard methods. The results show that the enzyme activity of liver MFO was changed according to a single or multiple grapefruit juice ingestion. The grapefruit juice in a single oral dose significantly decreases the activity of xanthine oxidase, glutathion peroxidase, lipid peroxidase and liver glutathion contents, and has no effect on activity of catalase and peroxidase. The multiple grapefruit ingestion increases the activity of XOD, GSH-Px, LPx, Px and GSH, while the activity of CAT enzyme is unchanged. The chronical and single grapefruit ingestion has no effect on relative liver weight, but the liver protein content is significantly decreased after the multiple oral grapefruit juice ingestion.     

Thyroid peroxidase deficiency in Batten-Spielmeyer-Vogt disease.

Thyroid tissue from two patients with Batten-Spielmeyer-Vogt disease (BSV) was studied for peroxidase enzyme activity and for morphological abnormalities by light and electron microscopy. Diagnosis was confirmed by the demonstration of intracytoplasmic material identical to that described in other reported cases of BSV. There was a substantial decrease in peroxidase activity in the thyroid tissue from both patients. An abnormally low level of peroxidase activity had previously been demonstrated in the white blood cells of these patients. Thyroid biopsy offers obvious advantages over brain biopsy, provides adequate tissue for enzyme analysis, and allows the demonstration of the intracytoplasmic structures characteristic of BSV.     

Distribution of superoxide dismutase, catalase, and peroxidase activities among Treponema pallidum and other spirochetes.

Representative members of Spirochaetales were surveyed for their content of superoxide dismutase (SOD), catalase, and peroxidase activities. Only Leptospira exhibited peroxidase activity. Obligately anaerobic cultivable Treponema and Spirochaeta possessed no SOD or peroxidative capabilities. Upon polyacrylamide gel electrophoresis, Spirochaeta aurantia, Borrelia hermsi, and five Leptospira biflexa serovars showed SOD activity associated with one electrophoretic band which was inhibited by H2O2, suggesting that they were iron-containing dismutases. These spirochetes could be distinguished by differences in relative mobilities of their SODs. SOD activity, but not catalase activity, was induced aerobically in S. aurantia. All Leptospira interrogans serovars and two L. biflexa serovars lacked significant SOD activity. These SOD-deficient strains of Leptospira, with one exception, possessed high levels of catalase activity. The Nichols strain of virulent Treponema pallidum possessed SOD and catalase activities, but lacked peroxidase activity. The SOD in T. pallidum exhibited two electrophoretic bands containing copper and zinc, and its relative mobility was identical to that of purified rabbit SOD. Immunization of sheep with purified rabbit SOD resulted in antiserum which inhibited both rabbit SOD and T. pallidum SOD assayed by spectrophotometric analysis or activity staining following polyacrylamide gel electrophoresis. In agarose gel diffusion, precipitin lines of identity were observed between purified rabbit SOD and cell extracts of T. pallidum. These data indicated that the SOD activity detected in T. pallidum was host derived.     

Effect of cell substrate on antioxidant enzyme activities in cultured renal glomerular epithelium.

The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, were monitored in isolated guinea pig glomeruli and primary or subcultured glomerular epithelial cells. Cell injury was assessed by morphologic studies and by measurement of cellular lipid peroxidation (levels of malondialdehyde). Antioxidant enzyme activities were very different in cultured cells than in parent glomeruli. The possible effect of culture substrates (tissue culture plastic, bovine corneal endothelial [BCE] cell basement membrane, and PF-HR-9 endodermal cell basement membrane) on antioxidant enzyme status, cell morphology, and lipid peroxidation was also assessed. Glomerular epithelial cells cultured on the BCE cell basement membrane substrate survived longer and showed less lipid peroxidation than cells cultured on plastic or the HR-9 substrate. Cells cultured on a plastic substrate had substantially less glutathione peroxidase activity than cells cultured on either BCE or HR-9 basement membranes.     

Characterization of Mycobacterium paratuberculosis and "wood-pigeon" mycobacteria by isoenzyme profile and selective staining of immunoprecipitates

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.     

Guinea-pig alveolar macrophage killing of Mycobacterium tuberculosis, in vitro, does not require hydrogen peroxide or hydroxyl radical.

Alveolar macrophages from the lungs of guinea-pigs that had been vaccinated, boosted and then intravenously challenged with Mycobacterium bovis BCG, killed both a hydrogen-peroxide-resistant and a hydrogen-peroxide-sensitive strain of M. tuberculosis, in vitro. Pretreatment of the alveolar macrophages, in vitro, with catalase or mannitol, agents which remove hydrogen peroxide and the hydroxyl radical, respectively, did not decrease the extent of killing of either strain. In contrast, pretreatment of alveolar macrophages with catalase, reversed the inhibition of growth of Listeria monocytogenes.     

Catalase, a novel antigen for Helicobacter pylori vaccination.

The efficacy of an orogastric vaccine comprised of purified Helicobacter pylori catalase plus the mucosal adjuvant cholera toxin (CT) was examined with both the Helicobacter felis and H. pylori mouse models with BALB/c mice. Native H. pylori catalase (200 microg) plus CT was initially used as a vaccine antigen in the H. felis mouse model and protected 80% (8 of 10) of the challenged animals, while all control animals were infected (20 of 20). In a follow-up experiment, recombinant H. pylori catalase plus CT was used for immunization, and groups of mice were challenged with the Sydney strain of H. pylori. Immunization with recombinant catalase protected a significant proportion (9 of 10) of the mice from H. pylori challenge, indicating that this enzyme should be considered as a candidate for a future vaccine. This study provides the first available data on the efficacy of protective immunization with the new Sydney strain of H. pylori in a mouse model. These data also provide indirect evidence that proteins which are normally intracellular, such as catalase, may be present on the surface of H. pylori and thus may provide targets for immunization.     

Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus.

Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1+ strain in a murine model of aspergillosis.     

Isoniazid resistance and the point mutation of codon 463 of katG gene of Mycobacterium tuberculosis.

It has long been known that almost all isoniazid (INH) resistant mycobacteria lose the catalase and peroxidase activities along with reduced or no virulence for guinea pigs. Recently resistance to INH has become known to be associated with mutations of katG gene encoding the HPI (Hydroperoxidase I) type catalase and peroxidase. Among these mutations, the point mutation of codon 463 of katG gene is found frequently, and is suggested as being associated with INH resistance. Therefore we performed this study in order to confirm the correlation between the point mutation of codon 463 of the katG gene and INH resistance of M. tuberculosis in Korea. Fifty isolates, 32 of which were resistant to INH, and 18 of which were sensitive to INH, were selected for this study. We used PCR-SSCP and RFLP analysis to detect the point mutation of the codon 463 of katG gene and confirmed the CGG (arginine) to CTG (leucine) mutation by direct sequencing analysis. Among 32 resistant isolates, 7 isolates (22%) had the same restriction pattern compared with that of the reference strain (H37Rv), and 25 isolates (78%) showed a different restriction pattern. Among 18 sensitive isolates, 7 isolates (39%) had the same restriction pattern compared with that of H37Rv, and 11 isolates (61%) showed a different restriction pattern. These results suggest that the CGG to CTG change of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance.