Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.     

Epitope mapping and features of the epitope for monoclonal antibodies inhibiting enzymatic activity of Helicobacter pylori urease

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit.     

Specific degradation of H. pylori urease by a catalytic antibody light chain

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a K(m) of 1.6 x 10(-5) m and a k(cat) of 0.11 min(-1), suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.     

UreA2B2: a second urease system in the gastric pathogen Helicobacter felis

Urease activity is vital for gastric colonization by Helicobacter species, such as the animal pathogen Helicobacter felis. Here it is demonstrated that H. felis expresses two independent, and distinct urease systems. H. felis isolate CS1 expressed two proteins of 67 and 70 kDa reacting with antibodies to H. pylori urease. The 67-kDa protein was identified as the UreB urease subunit, whereas the N-terminal amino acid sequence of the 70-kDa protein displayed 58% identity with the UreB protein and was tentatively named UreB2. The gene encoding the UreB2 protein was identified and located in a gene cluster named ureA2B2. Inactivation of ureB led to complete absence of urease activity, whereas inactivation of ureB2 resulted in decreased urease activity. Although the exact function of the UreA2B2 system is still unknown, it is conceivable that UreA2B2 may contribute to pathogenesis of H. felis infection through a yet unknown mechanism.     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

Genetic immunization of ducks for production of antibodies specific to Helicobacter pylori UreB in egg yolks

Following genetic immunization of laying ducks with a plasmid expressing Helicobacter pylori UreB (large subunit of urease), IgY against UreB were obtained from egg yolks. These polyclonal and monospecific IgY antibodies are of higher-titer and specifically recognize recombinant H. pylori urease purified from Escherichia coli. To our knowledge this is the first report describing generation of IgY antibodies directed against antigens of H. pylori by DNA-based immunization.     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .     

H.pylori尿素酶B亚单位在保加利亚乳杆菌的表达与鉴定

目的构建表达幽门螺杆菌（Helicobacter pylori,H、pylori）尿素酶B亚单位（UreB）的基因工程乳杆菌,并对其进行初步的安全性评估。方法采用高保真PCR从H.pylori标准菌株NCTC 11637中扩增ureB基因,插入乳酸菌表达质粒pMG36e,将重组质粒电转入保加利亚乳杆菌L6032中,获得表达ureB的基因工程乳杆菌。在含乳糖的MRS培养基诱导目的蛋白表达,Western blot鉴定其免疫原性。连续传代培养60代,检测基因工程乳杆菌的稳定性、形态学与生理生化特性以进行初步的安全性评估。结果特异PCR、酶切和测序鉴定均证实ureB基因克隆入表达载体pMG36e,SDS-PAGE结果显示,重组质粒pMG36e-ureB电转入保加利亚乳杆菌所构建的基因工程乳杆菌能表达约64KD的蛋白,Western blot证明该蛋白能与抗H.priori ureB的兔血清反应。稳定性、形态学与生理生化特性检测结果表明,基因工程乳杆菌与原始菌株保加利亚乳杆菌完全一致。结论成功构建能表达H.pylori UreB的保加利亚乳杆菌L6032-UreB,该基因工程菌在形态与生理生化特性上未发生任何变异,从而为探索幽门螺杆菌感染的益生菌制剂调理疗法奠定了坚实的基础。     

Isolation of Helicobacter pylori genes that modulate urease activity

Helicobacter pylori urease, a nickel-requiring metalloenzyme, hydrolyzes urea to NH3 and CO2. We sought to identify H. pylori genes that modulate urease activity by constructing pHP8080, a plasmid which encodes both H. pylori urease and the NixA nickel transporter. Escherichia coli SE5000 and DH5alpha transformed with pHP8080 resulted in a high-level urease producer and a low-level urease producer, respectively. An H. pylori DNA library was cotransformed into SE5000 (pHP8080) and DH5alpha (pHP8080) and was screened for cotransformants expressing either lowered or heightened urease activity, respectively. Among the clones carrying urease-enhancing factors, 21 of 23 contained hp0548, a gene that potentially encodes a DNA helicase found within the cag pathogenicity island, and hp0511, a gene that potentially encodes a lipoprotein. Each of these genes, when subcloned, conferred a urease-enhancing activity in E. coli (pHP8080) compared with the vector control. Among clones carrying urease-decreasing factors, 11 of 13 clones contained the flbA (also known as flhA) flagellar biosynthesis/regulatory gene (hp1041), an lcrD homolog. The LcrD protein family is involved in type III secretion and flagellar secretion in pathogenic bacteria. Almost no urease activity was detected in E. coli (pHP8080) containing the subcloned flbA gene. Furthermore, there was significantly reduced synthesis of the urease structural subunits in E. coli (pHP8080) containing the flbA gene, as determined by Western blot analysis with UreA and UreB antiserum. Thus, flagellar biosynthesis and urease activity may be linked in H. pylori. These results suggest that H. pylori genes may modulate urease activity.     

Therapeutic effects of molecularly designed antigen UREB138 for mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori) is a bacteria that is well known as the principal cause of chronic gastritis and peptic ulcer disease in humans. Because no effective vaccine has yet been established, we designed a new biomolecule as a vaccination antigen capable of preventing the infection of H. pylori. The designed biomolecule involves a 138 stretch (aa 201-aa 338 of beta-subunit of H. pylori urease), which is the functionally important region for urease activity. The region was expressed as a recombinant protein, called UREB138. The therapeutic vaccination was performed using UREB138 in mice persistently infected with H. pylori. The subcutaneous administration of UREB138 remarkably reduced the number of bacteria in the mice stomach compared with the control. Immunization with UREB138 enhanced the urease-specific IgA and IgG1 in the serum. Immunohistochemical staining for IgA in gastric mucosa showed that the number of mice positively stained with IgA was significantly higher in UREB138-immunized mice than in control mice. Furthermore, the expression of interferon-gamma mRNA in the gastric tissues with eradicated bacteria was higher than in the non-eradicated group. The combination of Th1- and Th2-mediated immunity plays a role in reducing the colonization of bacterial numbers of H. pylori.     

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.

Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylori was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.     

Synthetic peptides mimicking antigenic epitope of Helicobacter pylori urease

Short peptides resembling the Helicobacter pylori urease antigen (UreB F8 Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe) with deleted aspartic acid and glutamic acid residues, anchored through a triazine linker via the N-terminal moiety to cellulose plate were prepared. The peptides were used for binding of antibodies from sera of patients with medically confirmed atherosclerosis. Recognition of the peptides was also tested with anti-Jack beans urease antibodies. The important role of a Gly-Gly spacer separating the peptides from the cellulose support was shown. Different patterns of binding of antibodies from H. pylori infected patients and anti-Jack bean urease antibodies were observed only in the case of pentapeptides. The peptide Gly-Gly-Leu-Val-Phe-Lys-Thr was recognized by most of the tested sera.     

Activities of Urease and Nickel Uptake of Helicobacter pylori Proteins are Media- and Host-Dependent

Background:  Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
Aim:  To compare nickel uptake and urease activity on an inter- and intraspecies level.
Results:  In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli . On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
Conclusion:  H. pylori took nickel much more efficiently up than E. coli . The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.     

Antibody enzymes in Helicobacter pylori-associated infection

The amidase activity of a fraction of IgG antibodies to H. pylori in cases of bacterial persistence in the antral section of the stomach and the duodenal bulb and the role of enzyme antibodies in the pathogenesis of this infection were evaluated. 113 patients were examined under clinical conditions. Diagnosis was made with the use of the morphological method, the rapid urease test (Jatrox-H.p.-Test, Germany) and ELISA (Diagnostic Automation Inc., USA). The amidase activity of serum IgG was determined. As proteolytic substrate benzoylarginine-p-nitroanilide (BAPNA) was used. The study revealed that in the blood serum of patients with chronic gastritis and/or duodenitis, caused by H. pylori, and the active persistence of H. pylori in the mucous membrane IgG antibodies to H. pylori having BAPNA-amidase activity could be detected.     

Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model

Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA(183-203)) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.     

Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease A subunit

Epitope vaccine based on urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In this study, we constructed an epitope vaccine with mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA_183-203) of H. pylori urease A subunit named CTB-UA. The CTB-UA fusion protein was expressed in Escherichia coli, and the purified protein was used for intraperitoneal immunization experiments in BALB/c mice. The experimental results indicated that anti-CTB-UA antibody could recognize both H. pylori urease A subunit (UreA) and urease B subunit (UreB). Besides, the CTB-UA epitope vaccine had good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies which showed effectively inhibitory effect on the enzymatic activity of H. pylori urease. CTB-UA is a promising molecule to be investigated as H. pylori vaccine antigen candidate.     

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.     

Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth

Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages.     

Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange.

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.     

pH-dependent protein profiles of Helicobacter pylori analyzed by two-dimensional gels

Background. Helicobacter pylori survives transient exposure to extreme acid prior to adherence and growth on the gastric epithelium at neutral pH.
Materials and Methods. The effect of pH stress on protein profiles of H. pylori was observed using two-dimensional gel electrophoresis (2-D gels). H. pylori 26695 was grown microaerobically in tryptone-yeast extract broth, 3% fetal bovine serum. Growth in acid alkalinized the medium, whereas growth in base caused acidification. For 2-D gel analysis of protein profiles, cultures were grown in media buffered at pH 5.7 and at pH 7.5.
Results. Under all pH conditions, the most abundant proteins observed were the urease structural subunit UreB and the chaperonin GroEL. Growth in acid significantly increased the abundance of UreB. Thus, urease expression is not completely constitutive, as reported previously, but shows regulation by pH. Another protein observed only at low pH was identified as mammalian apolipoprotein A-I, possibly taken up by H. pylori from bovine serum in the growth medium. This finding, if confirmed, suggests that uptake of high-density lipoprotein from the human host may facilitate acquisition of cholesterol, required for formation of the unique cholesteryl glucosides in the membrane of H. pylori. In growth above pH 7, three stress proteins were induced: GroES (HspA), GroEL (HspB), and the antioxidant AhpC homolog TsaA. In addition, N-terminal sequence analysis identified five additional proteins that had not previously been reported on 2-D gels of H. pylori (FMN, SodB, TrxB, TsaA, and Tsr).
Conclusions. In summary, our 2-D gel study reveals expression of several proteins dependent on growth pH.