Uncoupling the enzymatic and autoprocessing activities of Helicobacter pylori gamma-glutamyltranspeptidase

Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.     

Biofilm formation by Helicobacter pylori

Helicobacter pylori NCTC 11637 produces a water-insoluble biofilm when grown under defined conditions with a high carbon:nitrogen ratio in continuous culture and in 10% strength Brucella broth supplemented with 3 g l-1 glucose. Biofilm accumulated at the air/liquid interface of the culture. Light microscopy of frozen sections of the biofilm material showed few bacterial cells in the mass of the biofilm. The material stained with periodic acid Schiff's reagent. Fucose, glucose, galactose, and glycero-manno-heptose, N-acetylglucosamine and N-acetylmuramic acid were identified in partially purified and in crude material, using gas chromatography and mass spectrometry. The sugar composition strongly indicates the presence of a polysaccharide as a component of the biofilm material. Antibodies (IgG) to partially purified material were found in both sero-positive and sero-negative individuals. Treatment of the biofilm material with periodic acid reduced or abolished immunoreactivity. Treatment with 5 mol l-1 urea at 100 degrees C and with phenol did not remove antigenic recognition by patient sera. The production of a water-insoluble biofilm by H. pylori may be important in enhancing resistance to host defence factors and antibiotics, and in microenvironmental pH homeostasis facilitating the growth and survival of H. pylori in vivo.     

Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice

Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.     

Crystal structure of Helicobacter pylori formamidase AmiF reveals a cysteine-glutamate-lysine catalytic triad

Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75A resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an alpha-beta-beta-alpha four-layer architecture characteristic of the nitrilase superfamily. One exterior alpha layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight alpha-beta-beta-alpha-alpha-beta-beta-alpha dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 A, respectively. These structures reveal a small formamide-binding pocket that includes Cys(166), Glu(60), and Lys(133) catalytic residues, in which Cys(166) acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl d-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.     

Characterization of monospecies biofilm formation by Helicobacter pylori

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.     

Helicobacter pylori does not mediate the formation of carcinogenic N-nitrosamines

Background. Both N‐nitroso compounds and colonization with Helicobacter pylori represent known risk‐factors for the development of gastric cancer. Endogenous formation of N‐nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N‐nitroso compounds. Based on experiments with a noncarcinogenic N‐nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N‐nitrosamines. Materials and Methods. Bacteria were grown in the presence of 0–1000 µM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3. Results. Incubation of Neisseria cinerea (positive control) with 500 µM morpholine and 500 µM nitrite, resulted in a significant increase in formation of N‐nitrosomorpholine, but there was no significant induction of N‐nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions. Conclusion. H. pylori does not induce formation of the carcinogenic N‐nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficultly nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.     

Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.     

Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad.

Leader peptidase (LP) is the enzyme responsible for proteolytic cleavage of the amino acid leader sequence from bacterial preproteins. Recent data indicate that LP may be an unusual serine proteinase which operates without involvement of a histidine residue (M. T. Black, J. G. R. Munn, and A. E. Allsop, Biochem. J. 282:539-543, 1992; M. Sung and R. E. Dalbey, J. Biol. Chem. 267:13154-13159, 1992) and that, therefore, one or more alternative residues must perform the function of a catalytic base. With the aid of sequence alignments, site-specific mutagenesis of the gene encoding LP (lepB) from Escherichia coli has been employed to investigate the mechanism of action of the enzyme. Various mutant forms of plasmid-borne LP were tested for their abilities to complement the temperature-sensitive activity of LP in E. coli IT41. Data are presented which indicate that the only conserved amino acid residue possessing a side chain with the potential to ionize, and therefore with the potential to transfer protons, which cannot be substituted with a neutral side chain is lysine at position 145. The data suggest that the catalytic activity of LP is dependent on the operation of a serine-lysine catalytic dyad.     

Cloning of a cholesterol-alpha-glucosyltransferase from Helicobacter pylori

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.     

Choosing a Benchtop Sequencing Machine to Characterise Helicobacter pylori Genomes

The fully annotated genome sequence of the European strain, 26695 was first published in 1997 and, in 1999, it was directly compared to the USA isolate J99, promoting two standard laboratory isolates for Helicobacter pylori (H. pylori) research. With the genomic scaffolds available from these important genomes and the advent of benchtop high-throughput sequencing technology, a bacterial genome can now be sequenced within a few days. We sequenced and analysed strains J99 and 26695 using the benchtop-sequencing machines Ion Torrent PGM and the Illumina MiSeq Nextera and Nextera XT methodologies. Using publically available algorithms, we analysed the raw data and interrogated both genomes by mapping the data and by de novo assembly. We compared the accuracy of the coding sequence assemblies to the originally published sequences. With the Ion Torrent PGM, we found an inherently high-error rate in the raw sequence data. Using the Illumina MiSeq, we found significantly more non-covered nucleotides when using the less expensive Illumina Nextera XT compared with the Illumina Nextera library creation method. We found the most accurate de novo assemblies using the Nextera technology, however, extracting an accurate multi-locus sequence type was inconsistent compared to the Ion Torrent PGM. We found the cagPAI failed to assemble onto a single contig in all technologies but was more accurate using the Nextera. Our results indicate the Illumina MiSeq Nextera method is the most accurate for de novo whole genome sequencing of H. pylori.     

Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.     

Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin

Helicobacter pylori secretes a toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxR-VacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. Subsequent experiments examined the capacity of each mutant toxin to form membrane channels. In a planar lipid bilayer assay, VacA proteins containing G13A, G22A, and G26A mutations formed anion-selective membrane channels, whereas VacA proteins containing P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A, and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. These data indicate that an intact proline residue and an intact G(14)XXXG(18) motif within the amino-terminal hydrophobic region of VacA are essential for membrane channel formation, and they also provide strong evidence that membrane channel formation is essential for VacA cytotoxicity.     

LPIAT1/MBOAT7 contains a catalytic dyad transferring polyunsaturated fatty acids to lysophosphatidylinositol

Human membrane bound O-acyltransferase domain-containing 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase 1 (LPIAT1), is an enzyme involved in the acyl-chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 rs641738 variant has been associated with the entire spectrum of fatty liver disease (FLD) and neurodevelopmental disorders, but the exact enzymatic activity and the catalytic site of the protein are still unestablished.Human wild type MBOAT7 and three MBOAT7 mutants missing in the putative catalytic residues (N321A, H356A, N321A + H356A) were produced into Pichia pastoris, and purified using Ni-affinity chromatography. The enzymatic activity of MBOAT7 wild type and mutants was assessed measuring the incorporation of radiolabeled fatty acids into lipid acceptors.MBOAT7 preferentially transferred 20:4 and 20:5 polyunsaturated fatty acids (PUFAs) to lysophosphatidylinositol (LPI). On the contrary, MBOAT7 showed weak enzymatic activity for transferring saturated and unsaturated fatty acids, regardless the lipid substrate. Missense mutations in the putative catalytic residues (N321A, H356A, N321A + H356A) result in a loss of O-acyltransferase activity.Thus, MBOAT7 catalyzes the transfer of PUFAs to lipid acceptors. MBOAT7 shows the highest affinity for LPI, and missense mutations at the MBOAT7 putative catalytic dyad inhibit the O-acyltransferase activity of the protein. Our findings support the hypothesis that the association between the MBOAT7 rs641738 variant and the increased risk of NAFLD is mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling. Taken together, the increased knowledge of the enzymatic activity of MBOAT7 gives insights into the understanding on the basis of FLD.     

Diagnosis of Helicobacter pylori

The different invasive and noninvasive diagnostic tests for Helicobacter pylori have been applied mainly in emerging countries. Molecular methods have been developed, especially a test for detection of H. pylori and its clarithromycin resistance directly from stools. The long-term effects of eradication on histologic lesions have been studied in a meta-analysis and the prognostic value of post-treatment in gastric mucosa-associated lymphoid tissue lymphoma has been assessed. An operating link for gastritis assessment (the OLGA staging) has also been published. Attempts to simplify the urea breath test protocol have been made, and new stool antigen tests have been proposed and compared to those previously available.     

Transmission of Helicobacter pylori

Helicobacter gastroduodenitis is a serious chronic infectious disease that is responsible for widespread morbidity and mortality. An understanding of the way in which it spreads is fundamentally important when considering measures for its control. Its prevalence is highest in the developing world and in individuals with a disadvantaged socio-economic childhood. The disease is believed to be contracted during the early years of life.A faeco-oral mode of transmission is considered by many to be the most likely mode of spread, however, the organism is difficult to culture both from faeces and from the environment and unlike other enteric organisms Helicobacter does not give rise to a diarrhoeal illness that would facilitate its transmission. An orooral route of spread has also been suggested, however, Helicobacter cannot be cultured from saliva, and if it was spread orally there is no reason why childhood should be the most frequent age for its acquisition.A third possibility is that the bacterium is transmitted gastro-orally. In favor of this hypothesis, the infection is easily acquired following gastric intubation with inadequately disinfected equipment. Children have a greater tendency to vomit than adults, and tend to explore with their fingers and place foreign objects in their mouths. Initial Helicobacter infection causes a dyspeptic illness characterised by mucousy vomiting, which may provide a vehicle for transmission. Furthermore, during the acute infection the organism induces achlorhydria in the host, possibly enabling the organism to survive longer in vomited mucus in the absence of acid. This theory fits best with the epidemiological data. Those most at risk are children living in an overcrowded environment who share beds with one another and live in houses that do not possess a fixed hot water supply (thus making cleaning up of vomit more difficult). It is also commoner in institutionalized children and is associated with school catchment areas.