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PubMed1996

X-ray absorption spectroscopy study of native and phenylphosphorodiamidate-inhibited Bacillus pasteurii urease.

X-ray absorption spectroscopy (XAS) has been applied to urease from Bacillus pasteurii, a highly ureolytic soil bacterium, with the aim of elucidating the structural details of the nickel-containing active site. The results indicate the presence of octahedrally coordinated Ni2+, in a sphere of six N/O donors at an average distance of 0.203 nm. An average of two histidine residues are bound to nickel. The experimental evidence suggests direct binding of the urease inhibitor phenylphosphorodiamidate to Ni2+. These spectroscopic results are in agreement with previous findings on both plant and microbial ureases, but differ in some respect from the results obtained by X-ray crystallography analysis of Klebsiella aerogenes urease.

Bacillus↗
PubMed2004

Therapeutic vaccination against Helicobacter pylori infection with attenuated recombinant Salmonella typhimurium urease B subunit and catalase in mice.

OBJECTIVE: To investigate the effects of oral immunization with attenuated recombinant Salmonella typhimurium urease B subunit and catalase vaccines in the treatment of Helicobacter pylori infection in a H. pylori infected mouse model. METHODS: Thirty C57BL/6 mice were randomized into three groups and challenged twice with oral administration of H. pylori within 3 days. Four weeks after the second challenge, the mice were immunized by oral administration of attenuated recombinant S. typhimurium urease B subunit (group A), attenuated recombinant S. typhimurium catalase (group B) or saline (group C), and all mice were killed 4 weeks later. The stomachs were collected for a rapid urease test, modified Giemsa staining and quantitative culture to observe the density of H. pylori, hematoxylin-eosin staining was performed to assess the presence of inflammation and lymphocytes from the spleen were used for the lymphoproliferation assay. RESULTS: The gastric H. pylori density of groups A, B and C was 1.58 x 10(5) c.f.u./g, 4.88 x 10(5) c.f.u./g and 1.92 x 10(6) c.f.u./g, respectively. The H. pylori density was significantly decreased in the therapeutic groups (P < 0.05). No significant inflammation was found in any group of mice. The lymphoproliferation assays of groups A and B were positive. CONCLUSION: Immunization with oral attenuated recombinant S. typhimurium urease B subunit and catalase vaccines is effective in reducing the density of H. pylori colonization.

Animals↗
PubMed1996

Effect of omeprazole therapy on the survival of Helicobacter pylori, urease activity, and antral gastric histology in patients with duodenal ulcer.

BACKGROUND: Helicobacter pylori is associated with chronic active gastritis and peptic ulceration (PU). Omeprazole is a proton pump inhibitor that is effective in healing PU and reducing gastritis. Previously it has been found that omeprazole has some bacteriostatic activity against H. pylori both in vitro and in vivo and in inhibiting urease activity in vitro. Our aim was to evaluate the effect of omeprazole on H. pylori colonization of the gastric mucosa, urease activity in vivo, and the presence of associated gastritis in patients with duodenal ulcer (DU). MATERIALS AND METHODS: We studied 12 patients (7 men and 5 women, ages 22-68 yr) with Du larger than 5 mm in diameter with a positive CLOtest (Delta West Ltd., Australia). Omeprazole, 20 mg bid, was given for 8 weeks to each patient, patients were endoscoped at the end of this period to check for healing of DU, and repeat biopsies were obtained from the gastric antrum for histological analysis, CLOtest, and culture. RESULTS: DU healed completely in all patients. Likewise in all patients there was significant reduction in the urease activity, from 22.1 +/- 4.17 to 1.58 +/- 0.92 units/ml (p < .001; 95% confidence interval of the difference between means, 32.7-14.1), and reduced H. pylori density, from 1,403.46 +/- 128.23 to 422.5 +/- 172.39 colony-forming units (CFU) per milligram of tissue biopsy (p < .001; 95% confidence interval of the difference between means, 1,486.1-590.5). The numbers of H. pylori were reduced on the gastric mucosa after omeprazole therapy and disappeared in six patients, a result that correlated with a negative CLOtest reading after 24 hours. CONCLUSION: Omeprazole, 20 mg bid, is capable of reducing H. pylori numbers and urease activity in vivo. There was no significant reduction in the severity of antral gastritis in DU patients studied.

Adult↗
PubMed1998

Prospective comparison of rapid urease tests (PyloriTek, CLO test) for the diagnosis of Helicobacter pylori infection in symptomatic children: a pediatric multicenter study.

OBJECTIVE: Rapid urease tests are reliable methods to diagnose Helicobacter pylori (HP) infection in the endoscopy suite. The PyloriTek test kit is a new rapid urease test that has the advantage of a 1-h final reading. The aim of this study was to compare the accuracy of PyloriTek and the test in the diagnosis of H. pylori infection in children. METHODS: Children from four different pediatric gastroenterology centers were recruited prospectively into the study. These children were >5 yr old and had an upper endoscopy procedure. Antral biopsies were examined for both rapid urease tests in the endoscopy suite, and others were sent for routine histological examination. RESULTS: A total of 242 children were recruited into the study over approximately 1 yr. The concordance between PyloriTek and CLO test was 98% (238 of 242). Twenty-five children were positive for HP organisms by PyloriTek and CLO test, whereas four children were positive by PyloriTek but negative by CLO test. PyloriTek was comparable to CLO test for the diagnosis of HP organisms and HP-associated gastritis. Moreover, in 48% of the positive results, PyloriTek gave significantly faster results than CLO test. CONCLUSIONS: We conclude that PyloriTek is an appropriate rapid urease test to use in children and may have an advantage over the CLO test because of its shorter reading time.

Biopsy↗
PubMed1998

Streptococcus salivarius urease expression: involvement of the phosphoenolpyruvate:sugar phosphotransferase system.

Urease expression in Streptococcus salivarius 57.1 is induced by acidic pH, and further enhanced at high growth rate and with excess carbohydrate. Notably, the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) activity is repressed in oral streptococci under the same conditions. To test the hypothesis that the PTS may be involved in urease regulation, spontaneous mutants (PTS-1 and PTS-4) that were resistant to 2-deoxyglucose were isolated. When compared to wild-type, PTS-1 was devoid of enzyme IIAManH (EIIAManII) and synthesized low amounts of EIIAManL, and PTS-4 was devoid of EIIAManL, but produced wild-type levels of EIIAManH. Urease expression was examined in continuous chemostat cultures at steady state. Induction by acidic pH was still observed in both mutants, but at lower levels compared to wild-type, under carbohydrate limiting conditions. Conversely, the lower level of expression in PTS-4 could be overcome in excess carbohydrate. The data indicated evidence of a molecular link between the PTS, sugar metabolism, and regulation of urease expression.

Blotting, Western↗
PubMed1977

Glial cell reactions in rats with hyperammoniemia induced by urease or porto-caval anastomosis.

The density and size of astrocyte, oligodendrocyte and neurone nuclei were determined in the corpus striatum of rats with urease-induced hyperammoniemia (plasma NH4+-concentration about 800 micronmol/1). No changes in the number of neurone nuclei were found. After 4 days the density of astrocyte nuclei increased from 13 to 23 per cent of the glial nuclei. However, the number of oligodendroxyte nuclei decreased correspondingly and the total glial nuclear number remained constant. The number of astrocyte nuclei was normalized 1 week after the 4-day period of hyperammoniemia. Moreover, intracerebral injections of colchicine at different times of the experimental period revealed no mitoses, indicating that no astrocyte divisions took place in pure hyperammoniemia. The astrocyte nuclei were of normal size in the urease animals in contrast to the animals with porto-caval anastomosis (PCA) which showed enlarged astrocyte nuclei. Thus, hyperammoniemia caused a reversible transformation of glial nuclei, but no real proliferation. A comparison of the glial reactions 4 days after a brain lesion showed the same frequency of astrocyte mitoses in control and urease animlas but a higher incidence of mitoses in the PCA animals. The number of Alzheimer type I astrocytes was the same in control and PCA animals, whereas no such cells were found in the urease animals, indicating that this form of hyperammoniemia did not lead to arrest of astrocyte metaphases with subsequent formation of Alzheimer type 1 cells.

Ammonia↗
PubMed1993

[Antibacterial activity of cefdinir and omeprazole against Helicobacter pylori and their inhibition on H. pylori-producing urease].

Since Helicobacter pylori is isolated very frequently from gastric ulcer specimens, the combination therapy of antimicrobial agent and proton pump inhibitor has recently been used. A study was made on whether cefdinir (CFDN), amoxicillin (AMPC), metronidazole (MNZ), omeprazole (OPZ), and omeprazole-M (OPZ-M) have antimicrobial activity against H. pylori and whether they can inhibit H. pylori-producing urease. 1) CFDN, AMPC and MNZ showed a potent antimicrobial activity against H. pylori, and especially, AMPC showed a marked bactericidal activity in a short time. 2) OPZ is reported to be converted to OPZ-M, and active form, in the body. OPZ and OPZ-M showed a moderate antimicrobial activity against H. pylori, and scarcely any bactericidal activity. 3) CFDN and OPZ or AMPC and OPZ in combination did not show any synergistic effect on the antimicrobial activity, but MNZ and OPZ in combination showed additive effect on the antimicrobial activity against H. pylori. 4) OPZ and OPZ-M inhibited H. pylori-producing urease and the inhibitory effect of OPZ-M was more stronger than that of OPZ. CFDN, AMPC and MNZ did not show any inhibitory effect on H. pylori-producing urease at 10 micrograms/ml. From these data, antimicrobial agents and proton pump inhibitors in combination are expected to exert the in vivo synergistic effect since these drugs eradicate H. pylori and inhibit H. pylori-producing urease.

Cefdinir↗
PubMed2000

A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization.

Acidic media trigger cytoplasmic urease activity of the unique human gastric pathogen Helicobacter pylori. Deletion of ureI prevents this activation of cytoplasmic urease that is essential for bacterial acid resistance. UreI is an inner membrane protein with six transmembrane segments as shown by in vitro transcription/translation and membrane separation. Expression of UreI in Xenopus oocytes results in acid-stimulated urea uptake, with a pH profile similar to activation of cytoplasmic urease. Mutation of periplasmic histidine 123 abolishes stimulation. UreI-mediated transport is urea specific, passive, nonsaturable, nonelectrogenic, and temperature independent. UreI functions as a H+-gated urea channel regulating cytoplasmic urease that is essential for gastric survival and colonization.

Amino Acid Sequence↗
PubMed2002

Three-dimensional quantitative structure-activity relationship and comparative molecular field analysis of dipeptide hydroxamic acid Helicobacter pylori urease inhibitors.

A homology model of Helicobacter pylori urease was developed by using the crystal structure of urease from Klebsiella aerogenes (EC 3.5.1.5) as a template. The acetohydroxamic acid moiety was docked into the active pocket of the enzyme model, followed by relaxation of the complex by use of molecular dynamics. The resulting conformation was used as a template to construct 24 potential dipeptide hydroxamic acid inhibitors with which comparative molecular field analysis (CoMFA) was performed. The resulting model provided a cross-validation correlation coefficient (q(2)(L00)) of 0.610, a conventional r(2) value of 0.988, and an F (Fisher indication of statistical significance) value of 294.88. We were able to validate the CoMFA model by using the 50% inhibitory concentrations of six compounds that were not included in the construction of the model. A very good structural correlation was observed between the amino acids in the model urease's active pocket and the contour maps derived from the CoMFA model. This correlation, accompanied by the validation supplied by use of the CoMFA data, illustrates that the model can aid in the prediction and design of novel H. pylori urease inhibitors.

Computational Biology↗
PubMed1974

Isolation of ureolytic Peptostreptococcus productus from feces using defined medium; failure of common urease tests.

Colony counts of fecal samples from three persons, obtained by using a chemically defined anaerobic roll-tube medium (containing glucose, maltose, glycerol, minerals, hemin, B-vitamins, methionine, volatile fatty acids, sulfide, bicarbonate, agar, carbon dioxide (gas phase), and 1 mM NH(4) (+) as main nitrogen source), averaged 60% of the 8.8 x 10(10) bacteria per g obtained when 0.2% Trypticase and 0.05% yeast extract were added to the otherwise identical medium. When 0.2% vitamin-free Casitone replaced Trypticase and yeast extract, counts were 94% those of the more complex medium. When urea-nitrogen was added to the defined medium as the main nitrogen source in place of NH(4) (+), counts of relatively large colonies averaged 1.0 x 10(9) per g of feces from five persons-1.1% of counts on the medium containing Trypticase and yeast extract. All of the organisms from the large colonies in the urea roll tubes were morphologically similar, and all six representative strains isolated were identified as urease-forming Peptostreptococcus productus, a species not previously known to produce urease. Ureolytic strains of Selenomonas ruminantium and P. productus were negative for urease activity in three assay media when inocula were from media containing complex nitrogen sources. The study documents that P. productus is the most numerous ureolytic species so far found in human feces and suggests that NH(4) (+) and more complex organic nitrogen sources strongly repress its production of urease. The study also indicates the efficacy of chemically defined media for direct selective isolation of nutritional groups of bacteria from feces.

Anaerobiosis↗
PubMed1990

Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a urease-producing uropathogen, causes serious urinary tract infections in humans. To specifically evaluate the contribution of urease to virulence, a mutation was introduced into P. mirabilis HI4320 by homologous recombination. Virulence was assessed in the CBA mouse model of ascending urinary tract infection. Twenty mice each were challenged transurethrally with P. mirabilis HI4320 and its urease-negative derivative (1 x 10(9) to 2 x 10(9) CFU). At 48 h animals were sacrificed and the mean log10 CFU per milliliter of urine (parent, 6.23; mutant, 4.19; P = 0.0014) or per gram of bladder (parent, 6.29; mutant, 4.28; P = 0.0002), left kidney (parent, 4.11; mutant, 1.02; P = 0.00009), and right kidney (parent, 4.11; mutant, 2.43; P = 0.036) were all shown to be significantly different. These data demonstrate a role for urease as a critical virulence determinant for uropathogenic P. mirabilis.

Animals↗
PubMed1991

Virulence of ureaplasmal urease for mice.

Ureaplasmas killed mice within 5 min after intravenous injection. The 50% lethal dose of whole ureaplasmal organisms was 32 micrograms per mouse, a value also found for crystalline jackbean urease. The reaction was specific to urease, since protection was afforded by intraperitoneal injection of 200 micrograms of flurofamide, a potent urease inhibitor. The finding that a similar lethal effect was produced by injection of 200 mumol of NH4+ indicates that the toxicity of urease is mediated by ammonium ions or free ammonia.

Ammonium Chloride↗
PubMed1993

The putative arthritogenic cationic 19-kilodalton antigen of Yersinia enterocolitica is a urease beta-subunit.

The gene coding for a putative 19-kDa arthritogenic antigen of Yersinia enterocolitica O:3 (A. K. H. Mertz et al., J. Clin. Invest. 87:632-642, 1991) was cloned and sequenced after amplification of part of the gene by the polymerase chain reaction using degenerate primers, inferred from the amino acid sequence. The deduced amino acid sequence of the antigen showed similarity to small subunits of ureases from several different organisms, including the jack bean urease. Screening of a genomic library of Y. enterocolitica O:3 with a 19-kDa-antigen-specific DNA probe allowed recombinant clones containing the entire urease operon to be obtained. These clones expressed urease activity in Escherichia coli.

Amino Acid Sequence↗
PubMed1993

Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme.

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.

Amino Acid Sequence↗
PubMed1995

Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach.

Eight ferrets specific-pathogen-free for Helicobacter mustelae were given, per dose, approximately 3.0 x 10(7) CFU of either the wild-type parent strain of H. mustelae (NCTC 12032) (two ferrets) the isogenic urease-negative mutant strain of H. mustelae (10::Tn3Km) (four ferrets), or sterile culture broth (two ferrets). Infection status was monitored by endoscopic gastric biopsy for urease activity, histopathology, and culture and by serology at 3, 6, 10, and 21 weeks. All ferrets were necropsied at 25 weeks. Both negative control ferrets remained uninfected, both ferrets receiving the H. mustelae wild-type parent strain became infected after two doses of the organism, and all four ferrets given two doses of the isogenic urease-negative mutant strain of H. mustelae remained uninfected throughout the 6-month study. Histopathology correlated with infection status. H. mustelae-infected ferrets exhibited diffuse mononuclear inflammation in the subglandular region and the lamina propria of the gastric mucosa, while uninfected ferrets showed no or minimal inflammation. These results suggest that urease activity is essential for colonization of the ferret stomach by H. mustelae.

Animals↗
PubMed1996

The urease enzyme of Helicobacter pylori does not function as an adhesin.

Helicobacter pylori urease is essential for colonization of the gastric mucosa irrespective of whether the stomach is acidic or hypochlorhydric. It has therefore been speculated that the enzyme functions as an adhesin. The aim of this study was to compare the adherence of H. pylori N6 with the adherence of an isogenic urease-negative mutant, strain N6(ureB::TnKm), to gastric cells. Strain N6 originated from a patient with gastritis. Strain N6(ureB::TnKm) is specifically modified in the gene which encodes the large subunit of urease, UreB, and hence does not form a UreA-UreB enzyme complex. We have used flow cytometry to assess the adherence of H. pylori to the cells. We have also used phase-contrast microscopy to assess the adherence of the organism to Kato III cells. In the absence of urea both strains bound to Kato III cells and to primary gastric cells. Binding of both strains to the cells occurred rapidly. The presence of urea in the incubation medium decreased the binding of strain N6 to the cells. This was due to a rise in the pH of the incubation medium, which caused loss of viability of the organism. Urea had no effect on the adherence of strain N6(ureB::TnKm). We conclude that the urease of H. pylori does not play a role in the adherence of the organism to gastric cells.

Adhesins, Bacterial↗
PubMed1998

Use of green fluorescent protein to assess urease gene expression by uropathogenic Proteus mirabilis during experimental ascending urinary tract infection.

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made with gfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33-38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5alpha and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression of P. mirabilis virulence genes in experimental urinary tract infection.

Animals↗
PubMed1998

Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection.

To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 microg p.o. or rectally or 10 microg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H. pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls. Quantitative H. pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H. pylori. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40. 89 ng/ml), which was detectable prior to challenge with H. pylori. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4(+) T cells showed levels of recruitment to be dependent upon challenge with H. pylori and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.

Administration, Intranasal↗