Helicobacter Pylori Urease Research Articles

Synthesis, molecular docking, and activity of Schiff-base copper(II) complex with N-n-butylsalicylaldiminate as Helicobacter pylori urease inhibitor

A Schiff-base copper(II) complex, bis(N-n-butylsalicylaldiminato)copper(II), was synthesized and its solid-state structure was determined by X-ray crystallography. Its inhibitory activity against Helicobacter pylori urease was evaluated in vitro and it showed strong inhibitory activity against urease (IC50 = 0.95 µM), compared with acetohydroxamic acid (IC50 = 42.47 µM) as a positive reference. A docking analysis using the AUTODOCK 4.0 program was performed to explain the potent inhibitory activity of the complex against urease.

Surface Properties of Helicobacter pylori Urease Complex Are Essential for Persistence

The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche.

Synthesis, molecular docking, and inhibitory activity of a Ni Schiff-base complex against urease

A Ni(II) Schiff-base complex, Ni(C14H10NOBr2)2, was synthesized and structurally characterized by single-crystal X-ray analysis. Its inhibitory activity against Helicobacter pylori urease was evaluated in vitro and showed strong inhibitory activity against H. pylori urease compared with acetohydroxamic acid (IC50 = 42.12 µmol L−1), which is a positive reference. A docking analysis using the autodock 4.0 program could explain the inhibitory activity of the complex against urease.

Cross-reaction of antibody against Helicobacter pylori urease B with platelet glycoprotein IIIa and its significance in the pathogenesis of immune thrombocytopenic purpura

Many clinical investigations have suggested that Helicobacter pylori (H. pylori) infection might be associated with immune thrombocytopenic purpura (ITP), but its role in the pathogenesis of ITP is unsettled. In this study, we cultured H. pylori, produced recombinant H. pylori urease (ure) B, and then prepared monoclonal antibody (MoAb) against ureB, 1F11, both 1F11 and MoAb against human platelet glycoprotein (GP) IIIa, SZ21, could bind to the band of GP IIIa of normal platelet lysate, but not to that from a patient with Glanzmann thrombasthenia (GT) whose GP IIb-IIIa complex was absent. Flow cytometry showed that normal platelets were reacted with 1F11 and SZ21, while GT platelets were not. In immuno-radiometric assay, the binding of (125)I-labeled 1F11 to GP IIIa was higher than that to GP Ib, GP IIb, GP VI, and P-selectin. 1F11 could partly compete with SZ21 in a binding to platelet surface. In addition, 1F11 inhibited platelet aggregation induced by adenosine diphosphate, but had no effect on platelet P-selectin expression or Thromboxane B(2) production of platelets. These results indicate that H. pylori ureB antibody could cross-react with human platelet GP IIIa and partly inhibit platelet aggregation. UreB may be a crucial component of H. pylori involved in the pathogenesis of a subset of ITP.

Helicobacter pylori Urease May Exist in Two Forms: Evidence from the Kinetic Studies

Purified Helicobacter pylori urease displayed a sigmoid curve in the plot of velocity versus [S] at urea concentrations less than 0.1 mM. Under conditions where preservatives, glycerol, or polyethylene glycol (PEG), were added to the enzyme reaction, the substrate hydrolysis was consistent with Michaelis-Menten kinetics, with a Km of 0.21+/-0.06 mM and a Vmax of 1200+/-300 micromol min(-1) mg(-1). However, at saturating substrate concentrations, the kinetic parameters of H. pylori urease were unaffected by the presence of the preservatives, and enzyme catalysis conformed to Michaelis-Menten kinetics. The Hill coefficients of the enzyme-catalyzed urea hydrolysis in the presence and absence of PEG were 1 and 2, respectively. Based on these findings, we suggest that H. pylori urease may exist in aggregated and dissociated forms, each with intact function but differing kinetics that may be of importance in maximizing urea breakdown at varying urea concentrations in vivo.

Oral vaccination with liposome-encapsulated recombinant fusion peptide of urease B epitope and cholera toxin B subunit affords prophylactic and therapeutic effects against H. pylori infection in BALB/c mice

A new fusion peptide CtUBE of cholera toxin B subunit and Helicobacter pylori urease B subunit epitope was expressed in Escherichia coli. With this fusion peptide, an oral liposome vaccine against H. pylori infection was prepared and evaluated in BALB/c mice. Based on the results of urease tests, quantitation of culturable bacteria colonies in mice stomachs and histological identification of gastritis, the mice were protected significantly after intragastric vaccination with this CtUBE liposome vaccine, which increased the content levels of specific anti-urease serum IgG and mucosal IgA for both prophylactic and therapeutic vaccination protocols. These results showed that the fusion peptide CtUBE retained immunogenicity and could be used as antigen in the development of an oral vaccine against H. pylori infection.

Cross Reaction of Antibody Against Helicobacter Pylori Urease B with Platelet Glycoprotein IIIa and Its Significance in the Pathogenesis of ITP.

The data about association of Idiopathic Thrombocytopenic Purpura (ITP) with Helicobacter pylori (H pylori) infection are always contradictory, and the factors that incite ITP remain uncertain. Several hypotheses have been suggested about the striking discrepancies between the reported results, one of which is variable effects of genetically diverse H pylori strains in remote geographic regions. The sequence of gene for H pylori urease B is relatively constant, and its protein production is believed to be a crucial factor resulting in some diseases. We therefore conducted a study to assess the role of H pylori in the pathogenesis of ITP, and to investigate its immune mechanisms. Firstly, bacteria H pylori were isolated from gastric mucosal tissue of a patient with peptic ulcer and cultured. H pylori urease B gene was amplified, then ligated into expression plasmid. The latter was transfected into E coli. The recombinant urease B was used to develop monoclonal antibody (MoAb), 1F11, against urease B of H pylori. In Western blot analysis, both 1F11 and SZ21 (MoAb against GP IIIa) bound to the band of protein 95 KDa of normal platelet lysate, but not to that of platelet lysate prepared from a patient with Glanzmann thrombobasthenia (GT), in whom platelet membrane GP IIb/IIIa were absent. Flow cytometry analysis also showed that normal platelets were reacted with both 1F11 and SZ21, whereas platelets of the GT patient showed a low reactivity with 1F11 or SZ21. In immunoradiometric assay, in which four MoAbs against different platelet membrane GPs (GP Ib, GP IIb, GP IIIa and GP IV) were each coated on microtiter plates, and platelet lysate and then 125I-labeled 1F11 were separately added, the radioactivity value (count per minute) in plates coated with SZ21 was the highest among all coated plates. All the three different experimental results showed that there exists a cross reaction between MoAb 1F11 against Helicobacter pylori urease B and platelet glycoprotein IIIa. In addition, MoAb 1F11 could inhibit ADP induced platelet aggregation in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 levels induced by ADP, because platelet P-selectin expression and TXB2 synthesis could occur before aggregation, the “final step” of platelet aggregation, just like what reported in Glanzmann thrombocytopenia. Finally, we performed a prospective clinical study on H pylori infection by means of immuno-radiometric assay of urease B in 164 ITP patients, 88 (54%) were found H pylori-positive. platelet counts in 15 of 38 H pylori-positive patients were significantly increased after eradication treatment, and were completely recovered in 7 patients. In conclusion, our results first indicate that antibidy against H pylori urease B could cross-react with platelet GP IIIa, and inhibit platelet aggregation. H pylori eradication may improve the platelet counts in some chronic ITP patients.

A catalytic antibody heavy chain HpU-2 degrading its epitope peptide and H. pylori urease

The HpU-2 monoclonal antibody (mAb) raised against Helicobacter pylori urease mainly recognized the α-subunit of the urease. On the other hand, the heavy chain of HpU-2 mAb (HpU-2-H) isolated from the parent mAb recognized both the α- and β-subunit, in which the β-subunit was recognized more strongly than the β-subunit. HpU-2-H cleaved a peptide, SVELIDIGGNRRIFGFNALVD, which is the epitope sequence recognized by HpU-2 mAb, showing a double-phase reaction profile at 25 °C in a phosphate buffer. After an induction time of 24 h, the cleavage of the peptide was initiated by HpU-2-H at a high rate and it was completed at 80 h of incubation. By mass spectroscopy, two main fragmented peptides, SVELIDIGGNRR and SVELIDIGGNRRIFG, were identified. In addition, many small peptide fragments were produced by successive cleavage of the fragmented peptides. Cleavage tests for H. pylori urease by HpU-2-H revealed that the β-subunit of the urease was cleaved first and completely decomposed at 20 h of incubation. Cleavage of the α-subunit started after the complete decomposition of the β-subunit. These cleavage results were in good agreement with the immunological features of HpU-2-H. The irrelevant proteins, BSA and HSA, were hardly cleaved by HpU-2-H.

Modulating the adjuvanticity of alum by co-administration of muramyl di-peptide (MDP) or Quil-A

The characterization of the immunological cascades of the innate immune system activated by pathogen associated molecular patterns (PAMP) recognized by pattern recognition receptors (PRR) have allowed the elucidation of the mechanisms underlying the immunomodulatory properties of adjuvants. Thus, the combinatorial use of adjuvants with specific, complementary functions is investigated to achieve tailored immune responses to subunit vaccines. We have previously shown how combinatorial administration of chitosan and cholera toxin B or muramyl-di-peptide (MDP) intranasally, but not intramuscularly, can allow small doses of MDP which, when administered alone cannot adjuvantise Helicobacter pylori urease (rUre), achieve an immunomodulatory effect through the specific physiological effect of chitosan. The aim of this study was to investigate if in the context of rUre the adjuvantising effect of MDP could be realized via the intramuscular route by combination with aluminium hydroxide, as compared with the routinely used veterinary adjuvant combination of alum and Quil-A. Serum IgG kinetics were comparable between the two adjuvant combination groups. However, the alum + MDP combination afforded higher antigen-specific recall responses in splenocyte cultures, associated with elevated release of the type I immune response cytokines IFN-gamma and IL-2. This data suggests that the adjuvanticity of MDP can be modulated in the context of alum in a manner dissimilar to that of Quil-A, achieving a balancing effect on the responses elicited by alum adjuvantisation.

In VitroInhibitory Effect of Flavonoids on Growth, Infection and Vacuolation ofHelicobacter pylori

Flavonoids, which are main constituents of herbal medicines, have been reported to inhibit the growth of Helicobacter pylori (HP). Therefore, to evaluate the anti-HP activity of some flavonoids (flavanols, flavones, flavonols and isoflavonoids), their effects on the growth and vacuolation of HP as well as the infective properties of HP against HeLa cells were investigated. Catechins, quercetin and naringenin weakly inhibited the growth of HP, but all tested compounds did not inhibit HP infection into KATO III cells and HP urease activity. Quercetin and naringenin inhibited HP VacA vacuolation in HeLa cells with IC (50) values of 0.046 and 0.36 mM, respectively. Quercetin also inhibited procaspase-3 activation to caspase-3 in HeLa cells induced by HP VacA toxin, which may induce cell death via the proteolytic activation of a cascade of caspases. However, quercetin did not affect Bax and Bcl-2 protein levels. Based on these findings, quercetin may improve gastric cell death by inhibiting apoptotic signaling by HP VacA toxin. Abbreviations. HP: Helicobacter pyloriBSA:bovine serum albumin ESL:enhanced chemiluminescence MIC:minimum inhibitory concentration MTT:methylthiazolyldiphenyl-tetrazolium bromide PBS:phosphate-buffered saline VacA:Vacuolating cytotoxin.

복분자(Rubus coreanus Miquel)로부터 Helicobacter pylori Urease Inhibitor의 분리 및 특성

위염과 위궤양의 일차적 발병인자로 알려진 Helicobacter pylori의 생육을 억제하고 urease 산물인 암모니아의 축적에 의한 위점막 손상을 완화시킬 목적으로 복분자로부터 H.pylori urease 저해물질을 분리정제하고 소재화와 관련된 일부 성질을 규명하였다. 양념채소류, 차류, 죽류, 건강채소류 등의 식용식물, 약용식물, 허브 및 해조류, 총 173종으로부터 극성도에 따라 계통추출한 수용성인 냉수추출물(Fr. 1), methanol 추출물(Fr. 4), 열수추출물(Fr. 5) 519점을 대상으로 H.pylori urease 저해활성을 검색하였다. 1차 및 2차 저해활성 검색 결과 복분자의 70% acetone추출물이 약 24%의 가장 높은 저해활성을 나타내었으며, 이 추출물을 ethyl acetate와 butanol을 사용하여 ethyl acetate/DW layer(RCE/RCW1)와 butanol/DW layer(RCB/RCW2)로 순차 분획한 후 활성획분인 RCW2 내의 활성본체를 확인할 목적으로 periodate oxidation과 pronase digestion을 실시한 결과 펩타이드 또는 단백질성 물질로 판명되었다. 저해활성물질은 DEAE-Toyopearl 650C, Butyl-Toyopearl 650M 및 Sephadex LH-20 순의 column chromatography에 의해 분리정제되었다. 분리urease 저해물질, RCW2-Ⅲcα는 HPLC의 gel permeation chromatography에 의해 비교적 순도 높은 분자량 약 13 kDa의 단일 물질임이 확인되었다. 저해활성물질은 열에 안정성을 보이는 내열성 단백질임을 알 수 있었고 위내 단백분해효소인 pepsin에도 가수분해 저항성을 나타냄으로써 기능성식품의 소재로 높은 소재화 적성을 보였다.

Super Catalytic Antibody and Antigenase

By immunizing ground-state peptides or proteins, we can produce super catalytic antibodies possessing serine protease-like characteristics. The unique feature of super catalytic antibodies is their ability to decompose a target molecule that is being killed. The authors have succeeded in preparing super catalytic antibodies that destroy (i) the HIV-1 envelope protein gp41, (ii) chemokine receptor CCR5 peptide, and (iii) Helicobacter pylori urease, etc. Some of them can degrade antigens at high catalytic reaction rates. Regarding their Km and kcat, super catalytic antibodies show intermediary values between that of enzymes (high Km and kcat) and that of antibodies (low Km and kcat [=0]). The catalytic function of an antibody mostly resides in its light chain. From mouse Vκ germline analysis, it became clear that super catalytic antibodies are generated from some discrete germlines such as bb1, cr1, cs1, bl1, bj2 and bd2. In these Vκ germlines, at least one catalytic triad composed of three amino acid residues, namely, Asp1, Ser27a and His93, is encoded. Namely, the antibody light chains (super catalytic antibodies) generated from the germlines are inherently able to enzymatically decompose antigens. Thus, such antibody light chains can be referred to as antigenase (antigen-decomposing enzyme) and may have arisen during the evolution of antibodies to acquire a higher ability than that of enzymes for developing a sophisticated self-defense system for survival.

Prevention of Ammonia‐induced Gastric Lesions in Rats by Natural Honey

Purpose: Honey is widely used all over the world in complementary and alternative medicine in various disorders, including gastrointestinal lesions. Unfortunately, the therapeutic potential of honey has so far been neglected in modern medicine owing to a lack of systematic scientific studies. Recent studies using different models of experimentally induced acute and chronic gastric lesions in rats have demonstrated the gastric protection effects and molecular mechanisms of honey. As ammonia is highly cytotoxic, its production by urease of Helicobacter pylori makes it an important aggressive factor in mucosal injury. The present work was undertaken to evaluate potential gastric protection by honey and its possible mechanism(s) against ammonia‐induced gastric lesions in rats.Materials and Methods: Twenty‐four hour fasted rats were given 1 ml of ammonium hydroxide 1% intragastrically and were killed 1 hour later under deep ether anaesthesia. The gastric lesion index was calculated according to the method of T...

Suppression of Helicobacter pylori-induced gastritis by green tea extract in Mongolian gerbils

Since urease of Helicobacter pylori is essential for its colonization, we focused attention on foodstuffs which inhibit the activity of this enzyme. Among plant-derived 77 foodstuff samples tested, some tea and rosemary extracts were found to clearly inhibit H. pylori urease in vitro. In particular, green tea extract (GTE) showed the strongest inhibition of H. pylori urease, with an IC 50 value of 13 μg/ml. Active principles were identified to be catechins, the hydroxyl group of 5 ′-position appearing important for urease inhibition. Furthermore, when H. pylori-inoculated Mongolian gerbils were given GTE in drinking water at the concentrations of 500, 1000, and 2000 ppm for 6 weeks, gastritis and the prevalence of H. pylori-infected animals were suppressed in a dose-dependent manner. Since the acquisition by H. pylori of resistance to antibiotics has become a serious problem, tea and tea catechins may be very safe resources to control H. pylori-associated gastroduodenal diseases.

Design and Synthesis of Heterocyclic Hydroxamic Acid Derivatives as Inhibitors of Helicobacter pylori Urease

Helicobacter pylori produces ammonia to help counter the acidic environment in the human stomach. The production of ammonia, essential for the microorganism's survival and virulence, is the product of enzymatic conversion of urea by the H. pylori's urease. Inhibition of urease activity by dipeptide hydroxamic acids has previously been demonstrated using a variety of fluorides, thiols and hydroxamic acids. Studies employing computer-aided drug design techniques have been utilized to suggest a novel series of heterocyclic hydroxamic acid derivatives as potential as urease inhibitors. The heterocyclic compounds 7a,b, 10b, 12b, 16b, and 19b have been designed, synthesized, and preliminarily tested as dipeptide mimics which offer a structure that is more biologically stable than that of the reported dipeptide inhibitors.

How Helicobacter pylori urease may affect external pH and influence growth and motility in the mucus environment: evidence from in-vitro studies.

Survival of Helicobacter pylori is dependent upon urease in the cytoplasm and at the bacterial surface. We have sought to clarify how alkaline ammonium salts, released from urea by this enzyme, might alter mucus pH and so affect growth and motility of the bacterium in the gastric mucus environment. Experiments were conducted in vitro to determine how the growth and motility of H. pylori are affected by changes in external pH, and how the bacterium, by hydrolysing urea, alters the pH of the bicarbonate buffer that occurs at the gastric mucosal surface. These data were fitted into experimental models that describe how pH varies within the mucus layer in the acid-secreting stomach. H. pylori was motile between pH 5 and 8, with optimal motility at pH 5. It grew between pH 6 and 8, with optimal growth at pH 6. The bacterium had urease activity between pH 2.7 and 7.4, as evidenced by pH rises in bicarbonate-buffered solutions of urea. Changes in buffer pH were dependent upon initial pH and urea concentration, with the greatest rate of pH change occurring at pH 3. Modelling experiments utilizing these data indicated that (1) in the absence of urease, H. pylori growth and motility in the mucus layer would be restricted severely by low mucus pH in the acid-secreting stomach, and (2) urease will sometimes inhibit H. pylori growth and motility in the mucus layer by elevating the pH of the mucus environment above pH 8. Urease is essential to the growth and motility of H. pylori in the mucus layer in the acid-secreting stomach, but, paradoxically, sometimes it might suppress colonization by raising the mucus pH above 8. This latter effect may protect the bacteria from the adverse consequences of overpopulation.

Activity of helicobacter pylori urease and nixa-mediated nickel uptake is strongly affected by culture conditions

Activity of helicobacter pylori urease and nixa-mediated nickel uptake is strongly affected by culture conditions

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-γ-secretion, IFN-γ-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.

Immunity against Helicobacter pylori: significance of interleukin-4 receptor alpha chain status and gender of infected mice.

Vaccination of interleukin-4 (IL-4) receptor alpha (IL-4Ralpha) chain-deficient BALB/c mice with Helicobacter pylori urease and cholera toxin or with urease-expressing, live attenuated Salmonella enterica serovar Typhimurium cells revealed that protection against H. pylori infection is independent of IL-4- or IL-13-mediated signals. A comparison of male and female mice suggests a sexual dimorphism in the extent of bacterial colonization that is particularly evident in the absence of the IL-4Ralpha chain.

Acid-dependent adherence of Helicobacter pylori urease to diverse polysaccharides

Background & Aims: The significance of acid-primed recognition of ligands by Helicobacter pylori urease is unknown. This study aimed to further characterize the specificity of urease adherence in vitro and verify whether specific inhibition will translate into in vivo suppression of colonization. Methods: A highly sensitive competitive enzyme-linked ligand capture assay was used to quantify the capacity of each test inhibitor to compete with labeled mucin for binding sites on immobilized native urease. A model polymer that strongly bound urease was used in an in vivo trial using euthymic hairless mice as an infection model. Results: The blockage of urease–gastric mucin interaction by certain inhibitors revealed an acid-functional lectin-like activity by urease, specifically recognizing bacterial lipopolysaccharides and certain species of polysaccharides, nonbacterial glycolipids, and glycoproteins. Dextran sulfate significantly (P < 0.01) suppressed colonization of mice by H. pylori when given before and/or after challenge. Conclusions: The acid-driven high-affinity adherence of H. pylori urease to mucin and lipopolysaccharides contributes to gastric mucosal colonization by the bacterium based on in vivo targeting experiments using specific polysaccharides in a mouse model with acute infection. Acid-functional urease-homing polysaccharides that can interfere with urease-mucin or H. pylori whole cell–mucin interaction in vitro can significantly interfere with colonization by the bacterium in vivo.GASTROENTEROLOGY 2000;119:358-367