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PubMed2000

Pilot study of phoP/phoQ-deleted Salmonella enterica serovar typhimurium expressing Helicobacter pylori urease in adult volunteers.

Attenuated Salmonella enterica serovar Typhi has been studied as an oral vaccine vector. Despite success with attenuated S. enterica serovar Typhimurium vectors in animals, early clinical trials of S. enterica serovar Typhi expressing heterologous antigens have shown that few subjects have detectable immune responses to vectored antigens. A previous clinical study of phoP/phoQ-deleted S. enterica serovar Typhi expressing Helicobacter pylori urease from a multicopy plasmid showed that none of eight subjects had detectable immune responses to the vectored antigen. In an attempt to further define the variables important for engendering immune responses to vectored antigens in humans, six volunteers were inoculated with 5 x 10(7) to 8 x 10(7) CFU of phoP/phoQ-deleted S. enterica serovar Typhimurium expressing the same antigen. Two of the six volunteers had fever; none had diarrhea, bacteremia, or other serious side effects. The volunteers were more durably colonized than in previous studies of phoP/phoQ-deleted S. enterica serovar Typhi. Five of the six volunteers seroconverted to S. enterica serovar Typhimurium antigens and had strong evidence of anti-Salmonella mucosal immune responses by enzyme-linked immunospot studies. Three of six (three of five who seroconverted to Salmonella) had immune responses in the most sensitive assay of urease-specific immunoglobulin production by blood mononuclear cells in vitro. One of these had a fourfold or greater increase in end-point immunoglobulin titer in serum versus urease. Attenuated S. enterica serovar Typhimurium appears to be more effective than S. enterica serovar Typhi for engendering immune responses to urease. Data suggest that this may be related to a greater stability of antigen-expressing plasmid in S. enterica serovar Typhimurium and/or prolonged intestinal colonization. Specific factors unique to nontyphoidal salmonellae may also be important for stimulation of the gastrointestinal immune system.

Administration, Oral↗
PubMed2000

Immunization with recombinant Helicobacter pylori urease in specific-pathogen-free rhesus monkeys (Macaca mulatta).

Immunization with urease can protect mice from challenge with Helicobacter pylori, though results vary depending on the particular vaccine, challenge strain, and method of evaluation. Unlike mice, rhesus monkeys are naturally colonized with H. pylori and so may provide a better estimate of vaccine efficacy in humans. The purpose of this study was to examine the effectiveness of H. pylori urease as a vaccine in specific-pathogen (H. pylori)-free rhesus monkeys. Monkeys raised from birth and documented to be free of H. pylori were vaccinated with orogastric (n = 4) or intramuscular (n = 5) urease. Two control monkeys were sham vaccinated. All monkeys were challenged with a rhesus monkey-derived strain of H. pylori, and the effects of vaccination were evaluated by use of quantitative cultures of gastric tissue, histology, and measurement of serum immunoglobulin G (IgG) and salivary IgA. Despite a humoral immune response, all monkeys were infected after H. pylori challenge, and there were no differences in the density of colonization. Immunization with urease therefore does not fully protect against challenge with H. pylori. An effective vaccine to prevent H. pylori infection will require different or more likely additional antigens, as well as improvements in the stimulation of the host immune response.

Animals↗
PubMed2006

The UreEF fusion protein provides a soluble and functional form of the UreF urease accessory protein.

Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.

Bacterial Proteins↗
PubMed1991

Macromolecular structure and aggregation states of Helicobacter pylori urease.

Urease purified from Helicobacter pylori by differential ultracentrifugation and fast pressure liquid chromatography was composed of subunits with apparent molecular weights (MrS) of 66,000 and 30,000. Electron microscopy of this purified material demonstrated that it formed disc-shaped macromolecular aggregates that were approximately 13 nm in diameter and 3 nm thick. Images of both negatively stained and shadowed preparations indicated that the discs tended to stack to form pairs and then these pairs further aggregated to form four-disc stacks. This stacking of subunits explains the heterogeneity observed previously in the molecular weight of urease preparations. In some negatively stained preparations there were also some smaller (approximately 8-nm-diameter) annular units present, which may represent individual urease units or possibly an aggregate of one of the two subunits from which urease is constructed.

Helicobacter pylori↗
PubMed1993

Characterization of a plasmid-encoded urease gene cluster found in members of the family Enterobacteriaceae.

Plasmid-encoded urease gene clusters found in uropathogenic isolates of Escherichia coli, Providencia stuartii, and Salmonella cubana demonstrated DNA homology, similar positions of restriction endonuclease cleavage sites, and manners of urease expression and therefore represent the same locus. DNA sequence analysis indicated that the plasmid-encoded urease genes are closely related to the Proteus mirabilis urease genes.

Amino Acid Sequence↗
PubMed2004

Motility of urease-deficient derivatives of Helicobacter pylori.

Early studies of a ureB mutant derivative of Helicobacter pylori had suggested that urease is needed for motility and that urease action helps energize flagellar rotation. Here we report experiments showing that motility is unaffected by deletion of ureA and ureB (urease genes) or by inactivation of ureB alone, especially if H. pylori strains used as recipients for transformation with mutant alleles are preselected for motility. This result was obtained with the strain used in the early studies (CPY3401) and also with 15 other strains, 3 of which can colonize mice. We conclude that urease is not needed for H. pylori motility.

Helicobacter pylori↗
PubMed1967

Occurrence of urease in T strains of Mycoplasma.

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.

Culture Media↗
PubMed1982

Urease testing of mycobacteria with BACTEC radiometric instrumentation.

A total of 140 mycobacterial isolates from patients treated at Fitzsimons Army Medical Center or the National Jewish Hospital and Research Center and from animal specimens submitted to the National Veterinary Services Laboratory were tested by using a urease procedure modified for use with a BACTEC model 301. Mycobacterial suspensions were prepared by using Middlebrook 7H10 Tween broth. Of the 98 mycobacteria isolates which were urease positive utilizing standard methodology, all were positive using the radiometric procedures. Similarly, all 42 urease-negative isolates were also negative employing the new methodology. Although maximum radiometric readings were observed at 48 h, all positive strains were readily identified 24 h after inoculation without sacrificing either test sensitivity or specificity. Thus, urease testing of mycobacteria, using the modified BACTEC radiometric methodology, was as sensitive, as specific, and more rapid than conventional methods.

Bacteriological Techniques↗
PubMed1984

Simple spot test for rapid detection of urease activity.

A spot test for the detection of urease activity was developed and evaluated with 761 strains of gram-negative bacteria. The test was compared with the conventional Christensen urea agar slants and urease test on the Vitek Enterobacteriaceae card (Vitek Systems, Inc., St. Ana, Mo.). Of the 348 strains of the Proteus-Providencia-Morganella group that were urease positive, 327 (94%) yielded positive results within 1 min, and all strains yielded positive results within 2 min. All these organisms also gave a positive urease reaction on the Vitek Enterobacteriaceae card test within 5 h and on the Christensen urea agar slants in 4 to 48 h. All the bacteria that did not hydrolyze urea by these two tests also remained negative by the spot test.

Bacteria↗
PubMed1987

Urease activity of Proteus penneri.

Ten strains of Proteus penneri isolated from geographically diverse laboratories were tested for urease activity. Cell lysates from urea-induced cells had a mean activity of 4.9 +/- 4.1 mumol of NH3 per min per mg of protein. On nondenaturing 6% polyacrylamide activity gels, the enzymes of P. penneri had very similar electrophoretic mobilities within species and within the Proteus genus but were distinct from the ureases of Providencia and Morganella species. On lower-percentage polyacrylamide, differences in mobilities of the ureases could be detected between the Proteus species. From representative strains, the P. penneri urease was found to be inducible by growth in urea and had an apparent molecular weight of 246,000 +/- 9,000, an isoelectric point of 5.1, and a Km for urea of 14 mM and was inhibitable by acetohydroxamic acid, hydroxyurea, and EDTA. In an in vitro model of struvite formation, a P. penneri strain produced abundant crystals on a glass rod submerged in synthetic urine in the absence but not presence of acetohydroxamic acid (500 micrograms/ml).

Chromatography, Gel↗
PubMed1988

Evaluation of a rapid urease test to detect Campylobacter pylori infection.

Seventy-five consecutive patients referred for upper gastrointestinal tract endoscopy were evaluated for Campylobacter pylori infection by pathology, culture, and a biochemical test to detect bacterial urease. Forty-eight patients (64%) had C. pylori present based on pathology or culture or both. Thirty-two urease tests were positive after 1 h, all in patients with C. pylori detected by the two other methods (specificity, 100%; sensitivity, 67%). After 24 h, 47 urease tests were positive, but only 40 had C. pylori present (specificity, 74%; sensitivity, 83%). When read after 1 h, the urease test was highly specific and led to rapid presumptive diagnosis.

Campylobacter↗
PubMedDate not supplied

[Flavonoids as effective protectors of urease from ultrasonic inactivation in solutions].

Inactivation of soybean urease in aqueous solution at pH 5.4, 36 degrees C, and high-frequency sonication (2.64 MHz, 1.0 W/cm2) is substantially reduced in the presence of seven structurally different flavonoids. A comparative kinetic study of the effect of these flavonoids on the effective first-order rate constants that characterize the total (thermal and ultrasonic) inactivation k(i), thermal inactivation k(i)*, and ultrasonic inactivation k(i)(US) of 25 nM enzyme solution was carried out. The dependences of the three inactivation rate constants of the urease on the concentrations of flavonoids within the range from 10(-11) to 10(-4) M were obtained. The following order of the efficiency of the flavonoids used in respect of the urease protection from ultrasonic inactivation was found: astragalin > silybin > naringin > hesperidin > quercetin > kaempferol > morin. The results confirm a significant role in the inactivation of the urease of HO* and HO2*, free radicals, which are formed in the ultrasonic cavitation field.

Flavanones↗
PubMed1991

Is Helicobacter pylori associated hypergastrinaemia due to the bacterium's urease activity or the antral gastritis?

Eradication of Helicobacter pylori is associated with a fall in serum gastrin but the way in which the infection raises the serum gastrin concentration is not clear. It may be related to the ammonia produced by the bacterium's urease stimulating gastrin release by the antral G cells. Alternatively, the antral gastritis induced by the infection may modify the regulation of gastrin release. We have examined serum gastrin in 10 patients before and 24 hours after starting triple anti-H pylori treatment consisting of tripotassium dicitrato bismuthate 120 mg four times daily, metronidazole 400 mg three times daily, and amoxycillin 500 mg three times daily. The urease activity, assessed by the 20 minute value of the 14C-urea breath test, fell from a median of 176 (range 116-504) kg% dose/mmol CO2 x 100 pretreatment to 5 (2-15) at 24 hours (p less than 0.005). The median antral gastritis score was 6 (4-6) pretreatment and fell to 3 (2-5) at 24 hours (p less than 0.02), and this was due to resolution of the polymorphonuclear component. Despite this complete suppression of bacterial urease activity and partial resolution of antral gastritis the median basal gastrin concentration remained unchanged, being 57 ng/l (45-77) pretreatment and 59 ng/l (45-80) at 24 hours and the median integrated gastrin response to a standardised meal was also unaltered, being 4265 ng/l/min (range 1975-8350) and 4272 ng/l/min (range 2075-6495) respectively. These findings do not support a causal association between H pylori urease activity and hypergastrinaemia and show rapid improvement of antral gastritis after starting anti-H pylori treatment.

Adult↗
PubMed1991

Effect of inhibition of Helicobacter pylori urease activity by acetohydroxamic acid on serum gastrin in duodenal ulcer subjects.

The mechanism of the hypergastrinaemia associated with Helicobacter pylori infection is unknown. It may be an effect of the ammonia produced by the bacterium near the antral epithelial surface. We have examined the effect on serum gastrin of inhibiting H pylori urease activity with acetohydroxamic acid in six duodenal ulcer patients. On day 1 the fasted patients received placebo tablets at 8 am, a peptide meal at 10 am, and a 14C urea breath test at 11.30 am. The next day 750 mg acetohydroxamic acid was administered orally in place of the placebo. The median (range) 30 minute breath test value (dose/mmol CO2 X kg body wt X 100) was 152 (111-335) on day 1, but only 22 (14-95) the next day (p less than 0.03). Further studies performed in one subject confirmed that acetohydroxamic acid lowered the ammonium concentration and raised the urea concentration in gastric juice. The inhibition of urease activity and ammonia production did not result in a fall in the basal gastrin concentration or in the median integrated gastrin response to the peptide meal, which was 78 ng/1.h (range 21-222) on day 1 and 79 ng/1.h (33-207) the next day. Ten days after acetohydroxamic acid, the urea breath test values were similar to those before treatment. This study shows that the raised gastrin concentration in patients with H pylori infection is not directly related to the organism's urease activity. It also shows that temporary suppression of H pylori urease activity does not clear the infection.

Adult↗
PubMed1993

Histology compared with chemical testing for urease for rapid detection of Helicobacter pylori in gastric biopsy specimens.

Gastric biopsy specimens from 283 patients with ulcer and non-ulcer dyspepsia attending five gastroenterology clinics in the northern region of the United Arab Emirates (UAE) were tested by the agar gel test (n = 115) or the ultra-rapid endoscopy room test (n = 168) for the presence of Helicobacter pylori urease. Results were compared with a histological technique using the Romanowsky type (Diff-3) stain for detecting H pylori in both antral and body type gastric mucosa. A sensitivity of 94% and specificity of 100% using the agar gel test compared with 87% sensitivity and 99.3% specificity for the ultra-rapid endoscopy room test. Grading of H pylori in gastric biopsy specimens showed that the higher the histological grade, the more likely that the urease test would be positive. Both forms of urease tests have high specificity for detecting H pylori in gastric biopsy specimens, although the urea agar test has a higher sensitivity than the ultra-rapid test. Low numbers of H pylori in gastric biopsy specimens are the most important determinant of a false negative urease test.

Adolescent↗
PubMed1998

Omeprazole may exert both a bacteriostatic and a bacteriocidal effect on the growth of Helicobacter pylori (NCTC 11637) in vitro by inhibiting bacterial urease activity.

AIMS: To assess the potential antibacterial effect of omeprazole, a benzimidazole proton pump inhibitor, on the growth of Helicobacter pylori in vitro and to evaluate the effect of this compound on bacterial urease activity. METHODS: The growth of H pylori was observed in liquid culture in the presence and absence of omeprazole (0.8 mg/ml). Urease activity was evaluated in aliquots removed from two hour cultures by monitoring the initial change in absorbency at 560 nm in the presence of 0.02% phenol red. RESULTS: The minimum inhibitory concentration of omeprazole against H pylori was 0.8 mg/ml. The concentration of omeprazole required to inhibit growth was dependent on inoculum density: omeprazole (0.8 mg/ml) prevented growth from a 1 x 10(6) cfu/ml inoculum, but not from the higher inocula of 10(7) or 10(8) cfu/ml. This is the first study to demonstrate that omeprazole exerts a bacteriocidal effect against low bacterial densities and a bacteriostatic effect when bacterial density is high. When used at the onset of growth, this concentration of omeprazole has a bacteriocidal effect after four hours, although it exerts a bacteriostatic effect when added to cultures after the exponential phase. Bacterial urease activity is competitively inhibited by omeprazole in a dose dependent manner. CONCLUSION: The results suggest that omeprazole exerts both a bacteriocidal and a bacteriostatic effect against H pylori and competitively inhibits bacterial extracellular urease activity.

Anti-Ulcer Agents↗
PubMed1976

Urease-negative strains of Clostridium sordellii.

Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.

Antarctic Regions↗
PubMed1986

Properties of urease from Ureaplasma urealyticum: kinetics, molecular weight, and demonstration of multiple enzyme isoelectric point forms.

In the several strains of Ureaplasma urealyticum that we examined, all originally isolated from human sources, the ureases were found to have a pH optimum between 7.2 and 7.5, and the Km was approximately 2.5 mM urea. Using nonreducing, nondenaturing conditions for polyacrylamide gel electrophoresis, the molecular weight of the holoenzyme was determined to be approximately 380 000. Treatment with reducing agents did not affect the electrophoretic mobility and, therefore, the molecular weight of ureaplasma urease. Immunoblot analysis (using antiserum to U. urealyticum urease) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing, denaturing conditions revealed two antigenically reactive bands of molecular weight 174 000 and 179 000. Under reducing, denaturing conditions, a single band of molecular weight approximately 179 000 was detected. Multiple forms of urease were detected by isoelectrofocusing but not by zonal electrophoresis.

Electrophoresis, Polyacrylamide Gel↗