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PubMed2004

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+.

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.

Algorithms↗

PubMed1998

Comparison of culture, histopathology and urease testing for the diagnosis of Helicobacter pylori gastritis and susceptibility to amoxycillin, clarithromycin, metronidazole and tetracycline.

Gastric biopsy specimens were taken from 737 patients undergoing upper gastrointestinal endoscopy and assessed for Helicobacter pylori infection. The diagnostic utilities of H. pylori culture (733 patients), detection of urease production (724 patients) and histopathological examination (469 patients) were compared. Since each of these techniques may fail to diagnose patients infected with H. pylori, an attempt was made to estimate the true rate of infection using a mathematical approach that combined the results of culture, histopathology and urease testing; 34% of the 733 patients were thought to be infected. Using this figure as a benchmark, the sensitivity, specificity, positive predictive value and negative predictive value of H. pylori culture were 73.2%, 100%, 100% and 86.3%, respectively, compared with 58.7%, 100%, 100% and 89.6%, respectively for urease production and 77.0%, 100%, 100% and 82.4%, respectively for histopathology. Thus, histopathological examination was the single most reliable test. A combination of histopathological examination and H. pylori culture diagnosed 99.5% of patients that were estimated to be truly infected. The minimum inhibitory concentrations of a number of antibiotics were measured for 135 isolates of H. pylori. All isolates were susceptible to amoxycillin and tetracycline whereas 5.2% were resistant to clarithromycin and 60% were resistant to metronidazole.

Amoxicillin↗

PubMed1987

Use of a urease-antibody conjugate in an enzyme immunoassay for the detection of blastomycosis.

A urease-antibody conjugate was used in an enzyme-linked immunoassay (ELISA) for the detection of antibodies in blastomycosis. A Blastomyces dermatitidis immunodiffusion antigen was used as the reference antigen in a solid phase indirect ELISA procedure and the endpoints were determined visually. Urease ELISA results on serum specimens from patients with blastomycosis compared favorably (90%) with results obtained spectrophotometrically by the alkaline phosphatase enzyme system. Specificity was evaluated with assays on sera from patients with histoplasmosis (20% cross reactivity), coccidioidomycosis (0% cross reactivity) and on Histoplasma capsulatum skin-test positive individuals (0% cross reactivity). The ease of performance of the urease ELISA combined with no requirements for specialized spectrophometric equipment are factors that favor the continued development of the test as an alternative serodiagnostic method. The assay may prove to be useful and a valuable adjunct to fungal antibody screening procedures.

Antibodies, Fungal↗

PubMed2000

Molecular mechanics evaluation of the proposed mechanisms for the degradation of urea by urease.

A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the NHis272-Ni1-O-Curea, and Ni1-O-Curea-Nurea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional "His320 acts as a base" mechanism is unlikely. while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.

Apoenzymes↗

PubMed2001

Inhibition of jack bean urease by N-(n-butyl) thiophosphorictriamide and N-(n-butyl) phosphorictriamide: determination of the inhibition mechanism.

N-(n-butyl)thiophosphorictriamide (NBPT) and its oxygen analogue N-(n-butyl)phosphorictriamide (NBPTO) were studied as inhibitors of jack bean urease. NBPTO was obtained by spontaneous conversion of NBPT into NBPTO. The conversion under laboratory conditions was slow and did not affect NBPT studies. The mechanisms of NBPT and NBPTO inhibition were determined by analysis of the reaction progress curves in the presence of different inhibitor concentrations. The obtained plots were time-dependent and characteristic of slow-binding inhibition. The effects of different concentration of NBPT and NBPTO on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships for a one-step enzyme-inhibitor interaction, qualified as mechanism A. The inhibition constants of urease by NBPT and NBPTO were found to be 0.15 microM and 2.1 nM, respectively. The inhibition constant for NBPT was also calculated by steady-state analysis and was found to be 0.13 microM. NBPTO was found to be a very strong inhibitor of urease in contrast to NBPT.

Amides↗

PubMed2004

Heavy metal ions inhibition of jack bean urease: potential for rapid contaminant probing.

The kinetics of heavy metal ions inhibition of jack bean urease was studied by progress curve analysis in a reaction system without enzyme-inhibitor preincubation. The inhibition was found to be biphasic with an initial, small inhibitory phase changing over the time course of 5-10 min into a final linear steady state with a lower velocity. This time-dependent pattern was best described by mechanism B of slow-binding inhibition, involving the rapid formation of an EI complex that subsequently undergoes slow conversion to a more stable EI* complex. The kinetic parameters of the process, the inhibition constants Ki and Ki* and the forward k5 and reverse k6 rate constants for the conversion, were evaluated from the reaction progress curves by nonlinear regression treatment. Based on the values of the overall inhibition constant Ki*, the heavy metal ions were found to inhibit urease in the following decreasing order: Hg2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > Pb2+ > Co2+ > Fe3+ > As3+. With the Ki* values as low as 1.9 nM for Hg2+ and 7.1 nM for Cu2+, 100-1000 times lower than those of the other ions, urease may be utilized as a bioindicator of the trace levels of these ions in environmental monitoring, bioprocess control or pharmaceutical analysis.

Biological Assay↗

PubMed2006

Piperidines: a new class of urease inhibitors.

A variety of piperidines (2-12, 14-26) with variable substituents at N-atoms have been synthesized and evaluated as urease inhibitors. The synthesized compounds showed varying degree of urease inhibitory activity ranging from 31.97 to 254 microM. The size and electron-donating or -withdrawing effects of substituents influence the activity, which lead to the formation of urease inhibitors.

Enzyme Inhibitors↗

PubMed1997

Suppression of Helicobacter pylori urease activity by sucralfate and sulglycotide.

The effect of gastroprotective agents, sucralfate and sulglycotide, on the in vitro activity of H. pylori urease was investigated. The bacterium was subjected to sonication, centrifuged, and the supernatant used as an enzyme source. The assays revealed that the rate of urea degradation was proportional to enzyme protein up to 100 micrograms and remained constant with time for 10 min. Introduction of sucralfate or sulglycotide to the assay system led to the reduction in the rate of ammonia production. With both drugs the optimal inhibition was attained at 10 micrograms/ml, at which dose a 63.1% decrease in urease activity occurred with sucralfate and a 70.2% inhibition was obtained with sulglycotide. The findings demonstrate that the inhibitory action of sucralfate and sulglycotide on H. pylori urease activity may be of value in the treatment of gastric disease associated with H. pylori infection.

Ammonia↗

PubMed1998

Recombinant Helicobacter pylori urease activates primary mucosal macrophages.

Helicobacter pylori urease is absorbed into the gastric mucosa at sites of inflammation, but whether the enzyme activates mucosal macrophages is not known. Because mucosal macrophages differ phenotypically and functionally from blood monocytes, whether recombinant H. pylori urease (rUrease) activated purified lamina propria macrophages in vitro was investigated. rUrease (1-10 microgram/mL) induced primary mucosal macrophages to produce interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha but not IL-8 proteins in a dose-dependent manner (P<.05 to P<.001). Quantitative reverse transcriptase-polymerase chain reaction using capillary electrophoresis laser-induced fluorescence showed that rUrease (0.1-10 microgram/mL) also induced dose-dependent expression of IL-1beta, IL-6, and TNF-alpha but not IL-8 mRNA (P<.05), suggesting that rUrease-induced production of certain cytokines is regulated at the level of gene transcription. These findings indicate that the ability of H. pylori urease to activate mucosal macrophages, resulting in production of proinflammatory cytokines, may be involved in the pathogenesis of H. pylori-associated mucosal inflammation.

Helicobacter pylori↗

PubMed2005

Oral immunization of mice with lactic acid bacteria producing Helicobacter pylori urease B subunit partially protects against challenge with Helicobacter felis.

BACKGROUND: The development of an efficacious vaccine against infection with Helicobacter pylori, the causative agent of chronic gastritis, peptic ulcer disease, and gastric adenocarcinoma, remains a challenge. Since the use of mucosal adjuvants is limited in human application, we have evaluated the potential of recombinant Lactobacillus strains producing H. pylori urease B (UreB) subunit to deliver this antigen to the gastrointestinal tract. METHODS: Mice were injected orally 3 times with a triple dose of recombinant Lactobacillus plantarum NCIMB8826, the recombinant isogenic cell-wall mutant (alr(-) MD007 strain) expressing UreB, or a mixture of recombinant UreB and cholera toxin (rUreB/CT) as a control. Urease-specific seric immunoglobulin (Ig) G and IgA were measured by use of an enzyme-linked immunosorbent assay. After challenge with Helicobacter felis, stomach infection was examined by use of the rapid urease test and by polymerase chain reaction detection of Helicobacter genomic DNA. RESULTS: Intragastric immunization with both recombinant Lactobacillus strains and rUreB/CT elicited UreB-specific antibodies. After challenge, reduction of H. felis load in the stomachs of mice was observed only after immunization with the recombinant mutant strain MD007 or with rUreB/CT. CONCLUSIONS: This is the first report of successful induction of partial protection against H. felis with a mucosal prime-boost regimen in which recombinant Lactobacillus strains were used as antigen-delivery vehicles.

Adjuvants, Immunologic↗

PubMed1975

Role of urease in pyelonephritis resulting from urinary tract infection with Proteus.

The role of urease in induction of pyelonephritis was studied by treatment of proteus-infected rats with acetohydroxamic acid, a potent inhibitor of urease. Infection was produced by introduction of Proteus mirabilis into the bladder along with a zinc disk. Controls were treated identically but received no acetohydroxamic acid. The number of bacteria per milliliter of urine was the same in both groups. The number of bacteria in the kidneys and the extent of renal damage was much greater in controls. Common enterobacteraceal antigen was not detected in the renal parenchyma of rats treated with acetohydroxamic acid. Treatment with acetohydroxamic acid thus prevented invasion of and damage to kidney tissue without reduction of urinary infection. Thus new evidence was found that the invasive properties of Proteus in the urinary tract are dependent on alkalinization of urine by urease and the resulting damage to the renal epithelium.

Acute Disease↗

PubMed1985

Specific inhibition of urease by N-acylphosphoric triamides.

Benzoyl- and isopentenoyl phosphoric triamides (BPA and IPA) strongly inhibited urease activities from jack bean, soybean, watermelon seed, Proteus mirabilis, P. rettgeri, P. vulgaris, Mycobacterium smegmatis, and Ureaplasma urealyticum. Their I50 values (the final concentration causing 50% inhibition), independent of enzyme source, were 2-21 nM, which are about 1,000-fold lower than that of caprylohydroxamic acid, one of the most potent urease inhibitors. ATP-urea amidolyase activity was inhibited 50% by BPA at a higher concentration of 0.28 mM, but was not affected by IPA even at 1.3 mM. Thirteen kinds of hydrolases (trypsin, chymotrypsin, thermolysin, leucine aminopeptidase, papain, lipase, alpha-amylase, glucuronidase, asparaginase, arylsulfatase, alkaline phosphatase, acid phosphatase, and true cholinesterase), two oxidoreductases (catalase and alcohol dehydrogenase), three transferases (glutamic-oxaloacetic aminotransferase, gamma-glutamyl transpeptidase, and arylsulfotransferase) and two kinases (pyruvate kinase and creatine kinase) were not affected at all even at 1 mM BPA and IPA. Exceptionally, pseudo-cholinesterase from human serum was inhibited by BPA and IPA, whose I50 values were 70 nM and 10 muM, respectively, using acetylthiocholine as a substrate. These values increased to 0.55 muM and 54 muM, respectively, when acetylcholine was used as a substrate. These results show that N-acylphosphoric triamides potently and specifically inhibit urease activity at concentrations of nM order.

Amidohydrolases↗

PubMed1998

A comparison of new rapid urease tests for detection of Helicobacter pylori: test characteristics and factors affecting positivity and time to positivity.

We assessed two new rapid urease tests, the Helicobacter Urease Test (HUT, Astra, Sweden) and the Polish test, for accuracy, reaction time, and the effect of biopsy site and bacterial density on test characteristics and time to positivity. A prospective study was conducted in two groups of patients: 64 consecutive patients undergoing upper endoscopy for dyspepsia and 61 consecutive patients with duodenal ulcer on upper endoscopy. In the first group, test accuracy, time to positivity, and possible associations with biopsy site and bacterial density were assessed between the tests. In the second group, the two new tests were compared with the CLOtest for time to positivity and effect of bacterial density on test outcome. The Polish and HUT test had similar specificities (97%), but the Polish test was more sensitive (90.3% vs. 80.7%). The Polish test was positive within 10 minutes in 55% of the positive patients compared with 10% for the HUT test. There was no association between bacterial density (by histologic count) and reaction time in either test or in the CLOtest in the second group. The Polish test was more accurate and had a quicker time interval to positivity than the HUT. There was no significant association between bacterial density and reaction time in any of the urease tests assessed, and the biopsy site did not affect test accuracy in the HUT test.

Adult↗

PubMed1998

Effect of simethicone on the accuracy of the rapid urease test.

OBJECTIVE: Simethicone, which is a principal ingredient in the defoaming agent used during gastroscopy, can inhibit the growth of Helicobacter pylori (MIC, 64-128 mg/l). This prospective study was designed to evaluate the in vivo and in vitro effects of simethicone on the accuracy of the rapid urease test (RUT). METHODS: In the in vivo study, three sets of gastric biopsies (two from the antrum, and one from the corpus) were taken from 75 patients. The first set was examined histologically, and the second set was used for the RUT (pre-simethicone RUT). Then, 25 ml simethicone (1200 mg/l) was introduced into the stomach for a contact time of 1 min. A third set of gastric biopsies was taken for the RUT (post-simethicone RUT). In the in vitro clinical study, 41 patients were recruited. The first set of gastric biopsies was used for the RUT (pre-incubation RUT). The second set was incubated in 1 ml of simethicone for 5 min before being used for the RUT (post-incubation RUT). In the spectrophotometric study, urease activity before and after incubation in simethicone for 5 min was quantified in 12 patients by measuring the absorbance at 560 nm. RESULTS: Reading at 15 min, the concordance rate between the pre-simethicone and post-simethicone RUT was 98%. In the in vitro clinical study, the concordance rate between the pre-incubation and post-incubation RUT was 97%. The spectophotometric study showed a significant reduction of 43% in urease activity after incubation in simethicone. CONCLUSION: The application of a defoaming agent containing simethicone does not affect the accuracy of the RUT. However, simethicone modestly suppresses urea hydrolysis.

Diagnostic Techniques, Digestive System↗

PubMed1980

Nickel requirement of a urease-deficient mutant in Aspergillus nidulans.

The addition of nickel ions restored urease activity in vivo and ability to grow on urea in a mutant strain of Aspergillus nidulans otherwise unable to utilize urea. This train carries a mutation in the ureD locus, one of four loci involved in urea utilization. No other urease-deficient strains tested responded to the presence of nickel ions. The analogous characteristics of the ureD mutant and the nitrate reductase and xanthine dehydrogenase associated cnxE mutants in Aspergillus nidulans are discussed. It is postulated that the ureD locus is in some way involved in the production or incorporation of a nickel cofactor essential for urease activity.

Aspergillus nidulans↗

PubMed1989

The urease of Ureaplasma urealyticum.

The urease from Ureaplasma urealyticum (serotype 8) has been purified by immuno-affinity column chromatography. Two active nickel-containing forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single active peak of apparent molecular mass 190 kDa was shown by FPLC. Total inactivation and denaturation of the enzyme to give three subunit polypeptides (one of 72 kDa containing nickel, one of 14 kDa and one of 11 kDa) was achieved by treatment with SDS and boiling. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The enzyme displayed a pH optimum of 6.9 and pI values were determined. Storage of the purified enzyme at -70 degrees C followed by thawing to 20 degrees C caused a partial breakdown to inactive subunits. Anti-urease monoclonal antibodies bound both to the active enzyme and to the inactive 72 kDa subunit, and the antibodies cross-reacted with ureases from all of the other human serotypes. Competition assays with the antibodies revealed four distinct epitopes of the enzyme, all distinct from its active site.

Antibodies, Monoclonal↗

PubMed2002

cis-Acting elements that regulate the low-pH-inducible urease operon of Streptococcus salivarius.

Differential expression of the Streptococcus salivarius 57.I urease operon in response to pH is effected by repression of transcription from a proximal promoter, PUREI: To localize the cis-acting elements involved in the regulation of the urease operon, the intact promoter region and its derivatives were generated and fused to a promoterless chloramphenicol acetyltransferase (cat) gene. The promoter-cat fusions were established in the lacZ gene of S. salivarius by using a newly constructed integration vector. CAT-specific activities were examined in batch-grown cells at pH 7.5 and 5.5. The results indicated that a 21 bp region immediately 5' to the -35 element was required for efficient repression of PureI at neutral pH and that the 39 bp (-57 to -95) 5' to this region contained sequences required for optimal expression of PUREI: A potential secondary repressor-binding site was tentatively identified further upstream of the -35 element (-96 to -115). To further analyse the cis-acting elements, base changes were introduced into two AT-rich repeats within the primary repressor-binding site. One such derivative, S. salivarius M1, with five base substitutions immediately 5' to the -35 element, expressed 20-fold more CAT-specific activity at neutral pH than the strain carrying wild-type PureI-cat. Also, the pH sensitivity of strain M1 was greatly reduced, suggesting that this AT-rich region is crucial for repression of the urease operon. Deletion of three consecutive 15- or 16-base segments from -52 to -96 in the S. salivarius M1 background resulted in lower activities compared to strain M1, confirming the presence of sequences required for optimal expression of the operon. All of the PureI-cat fusions were also integrated into the gtfG gene of Streptococcus gordonii DL1, a non-ureolytic oral Streptococcus sp. Repression of PureI was observed at neutral pH in S. gordonii and the effects of the various mutations of the repressor-binding site largely paralleled those seen in S. salivarius, suggesting that the cis-elements may be a target for a global regulatory circuit that controls gene expression in streptococci in response to pH.

5' Flanking Region↗

PubMed1994

Pre-formed urease activity of Helicobacter pylori as determined by a viable cell count technique--clinical implications.

The pre-formed urease activity of three NCTC reference strains and five clinical isolates of Helicobacter pylori was determined at room temperature (21 degrees C) and 37 degrees C by a viable cell count technique with a conventional urea slope test (Christensen's agar) as well as the commercial CLO-test. The urease activity of two gastroduodenal commensals, Proteus mirabilis and Klebsiella pneumoniae, was also tested. H. pylori strains produced positive reactions with viable cell counts of 10(6)-10(8) cfu within 30 min and with counts of 10(3)-10(6) cfu within 2 h. For some strains, smaller numbers of organisms were needed with the CLO-test than with the conventional test, and incubation of the CLO-test strips at 37 degrees C slightly decreased the number of organisms required for positive results. P. mirabilis produced a positive result on urea slopes with an initial inoculum of 10(7)-10(8) cfu at 2 h, but no positive reaction occurred for K. pneumoniae at 12 h, even with an initial inoculum of 10(11) cfu. However, both P. mirabilis and K. pneumoniae gave a positive result after incubation for 24 h with initial inocula of < 10(1) cfu and 10(3)-10(4) cfu respectively. Incubation at 37 degrees C significantly reduced the inoculum size of these organisms required for a positive result after incubation for 4 h when tested with the slopes, but not with the CLO-test. These findings indicate that H. pylori possesses much greater pre-formed urease activity than P. mirabilis and K. pneumoniae. False negative results for clinical detection of H. pylori in gastroduodenal biopsies may be due to small numbers of organisms, especially after treatment with antimicrobial agents, and false positive results may arise from gastroduodenal commensals or contaminants.

Colony Count, Microbial↗