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Though Helicobacter pylori (H. pylori) has been classified as class I carcinogen, key virulence factor(s) generated by H. pylori that causes gastric cancer remains to be fully determined. Here, we show that deletion of peptidyl-prolyl cis-trans isomerase (PPIase) prevented H. pylori from stimulating human gastric epithelial cell (AGS) proliferation. Consistent with this observation, ectopic expression of H. pylori PPIase promoted AGS cell proliferation and anchorage-independent growth. To gain insight into the biochemical mechanism of PPIase-induced effect, early signal events involved in mitogenic signaling pathways were evaluated. Expression of H. pylori PPIase caused an increase in basal as well as EGF-stimulated phosphorylation of ERK and EGF receptor at Tyr1086. Treatment with MEK inhibitor completely blocked PPIase-induced cell proliferation. Our results suggest that H. pylori PPIase has the potential to activate mitogenic signaling pathway and to promote transformation of gastric epithelial cells. H. pylori PPIase may represent a novel target for therapeutic management of gastric cancer patients., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Helicobacter pylori, a microaerophilic Gram-negative bacterium, is known to cause chronic gastritis, peptic ulcer and gastric cancer. Genes that are present in certain isolates may determine strain-specific traits such as disease association and drug resistance. In order to understand the pathogenic mechanisms of gastric diseases, identify molecular markers of the diseases associated with H. pylori strains and provide clues for target treatment of H. pylori-related diseases, a subtracted DNA library was constructed from a gastric cancer-associated H. pylori strain and a superficial gastritis-associated H. pylori strain by suppression subtractive hybridization. The presence of gastric cancer-specific genes was identified by dot blot hybridization, DNA sequencing and PCR-based screening. Twelve gastric cancer-specific high-copy genes and nine low-copy genes were found in gastric cancer compared with the superficial gastritis strain. These genes were confirmed by PCR analysis of H. pylori isolates. Notably, peptidyl-prolyl cis-trans isomerase (PPIase) was detected positively in 11 out of 22 (50%) gastric cancer-associated H. pylori strains. In contrast, <24% of the H. pylori strains from superficial gastritis showed positive results. Given the potential role of PPIases in cell growth, apoptosis and oncogenic transformation, our results suggest that PPIase may represent a novel marker and potential therapeutic target for gastric cancer., (© 2011 The First Affiliated Hospital, China Medical University. FEMS Microbiology Letters © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.)

Helicobacter pylori infects the stomach of about half of the world's human population, frequently causing chronic inflammation at the origin of several gastric pathologies. One of the most remarkable characteristics of the species is its remarkable genomic plasticity in which homologous recombination (HR) plays a critical role. Here, we analyzed the role of the H. pylori homologue of the AddAB recombination protein. Bioinformatics analysis of the proteins unveils the similarities and differences of the H. pylori AddAB complex with respect to the RecBCD and AddAB complexes from Escherichia coli and Bacillus subtilis, respectively. Helicobacter pylori mutants lacking functional addB or/and addA show the same level of sensitivity to DNA-damaging agents such as UV or irradiation and of deficiency in intrachromosomal RecA-dependent HR. Epistasis analyses of both DNA repair and HR phenotypes, using double and triple recombination mutants, demonstrate that, in H. pylori, AddAB and RecOR complexes define two separate presynaptic pathways with little functional overlap. However, neither of these complexes participates in the RecA-dependent process of transformation of these naturally competent bacteria., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

In order to elucidate the role of polyphosphate kinase (PPK) during the course of an infection by Helicobacter pylori, PPK deficient mutants were constructed using two genetic backgrounds: Hp141v and X47-2AL. The efficiencies of the parental strains and the derivative mutants at colonizing the gastric mucosa of mice were compared. When animals received the Hp141v and the X47-2AL parental strains, 100% of the mice remained colonized for the duration of the 45 days experiment. In contrast, none of the mice that were given the PPK deficient X47-2AL derivative strain had a detectable bacterial load in their gastric mucosa, while the deficient Hp141v derivative strain was detected in 100%, 20% and 40% of the mice at days 3, 15 and 45 post-inoculation (p.i.), respectively. The absence of PPK expression did not impair the in vitro growth of the ppk mutants. However, the reduced ability of the ppk defective mutants to colonize mice was associated with a significant decrease in both motility and in an accumulation of polyP in the bacterial cells. These results are consistent with an essential role of PPK during the initial steps of colonisation of the mouse gastric mucosa and confirm that PPK may act on the virulence of H. pylori partly through an energy dependent mechanism.

Helicobacter pylori is a human gastric pathogen which is extremely well adapted to its unique habitat. Crucial for the survival under the acidic conditions prevailing in the stomach is the enzyme urease. Transcriptome analysis has shown that transcription of a large number of genes responds to the exposure of H. pylori to acid including the genes encoding the urease subunits UreA and UreB as well as several genes which have been previously identified as target genes of the two-component system HP166-HP165. Here, we provide genetic evidence that a stimulus perceived by the histidine kinase HP165 is pH since increased transcription at acidic pH from the promoters controlling the expression of the ORFs hp119 and hp1432 which belong to the HP166-HP165 regulon is strictly dependent on the presence of histidine kinase HP165. Furthermore, we show that the basal transcription from the promoter of the ureA gene is modulated by the HP166-HP165 two-component system in response to acidic pH. On the other hand, the acid-induced increase in transcription of the promoter directing the expression of the orphan response regulator HP1021 is not controlled by the HP166-HP165 two-component system, nor is it mediated by HP1021 itself.

Helicobacter pylori induces a severe inflammatory response in the gastric mucosa. It is able to withstand the inflammatory response by producing proteins such as KatA and KapA. The C-terminus of KatA possesses a unique tetra-lysine motif not found in other catalases or other known protein sequences. Mutants deficient in this motif were constructed by site-directed mutagenesis. Cytoplasmic and periplasmic catalase activities were measured for the parental strain, a truncated KatA mutant (deficient in the unique C-terminal tetra-lysine motif) and a previously constructed KapA-deficient mutant (confirming previous observations regarding the possible periplasmic localisation of KatA). No differences were observed in the cytoplasmic catalase activities, however, the KapA-deficient mutant had approximately 5.5 times less catalase activity in the periplasmic extract when compared to the periplasmic preparations of either parental strain or KatA truncated mutant. N-terminal sequencing of KatA revealed no cleaved N-terminal signal peptide, indicating Sec-independent transport. These findings support previous reports that there is some form of interaction between KatA and KapA of H. pylori, an interaction which still needs to be characterised.

Urease is an essential virulence factor of the human gastric pathogen Helicobacter pylori, and is expressed to very high levels. The promoter of the urease operon contains sequences resembling the canonical -10 and extended -10 motifs, but no discernible -35 motif. To establish the role of different motifs and regions in the urease promoter, we fused the urease promoter to a genomic lacZ reporter gene in H. pylori, made substitutions in the aforementioned promoter motifs, and also made deletions in the upstream sequences removing regulatory sequences. Substitutions in the -10, extended -10 and predicted -35 motifs all significantly altered expression of the lacZ reporter gene, demonstrating their importance in transcription of the H. pylori urease operon. In contrast, sequential deletions upstream of the -35 region did not affect expression of the lacZ reporter gene. This demonstrates the modular structure of the H. pylori urease promoter, where basal levels of transcription are initiated from a typical sigma(70) promoter, which requires -10 and extended -10 motifs, and also its -35 motif for efficient transcription. Upstream sequences are not involved in basal levels of urease transcription, but play an important role in responses to environmental stimuli like nickel.

The Helicobacter pylori hpyIM gene encodes a type II DNA methyltransferase that is highly conserved among strains. To investigate the potential role of M.HpyI methyltransferase activity in controlling gene expression in H. pylori, we analyzed gene transcription profiles in wild-type strain J166 and an isogenic hpyIM mutant strain using gene arrays. This analysis showed that the expression of a majority of genes was unaffected by hpyIM mutation, especially in exponential phase cultures. However, in stationary phase cultures and in cells adherent to AGS gastric epithelial cells in vitro, loss of hpyIM function altered the expression of the stress-responsive dnaK operon. Complementation of the hpyIM mutation using a shuttle plasmid encoding a wild-type copy of the gene re-established the wild-type pattern of dnaK operon expression. These data suggested that hpyIM, encoding a DNA methyltransferase, may have a role in H. pylori physiology that supersedes its original function in a type II restriction-modification system.

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.

Three electromorphs of iron superoxide dismutase (FeSOD) were identified among 29 Helicobacter pylori isolates by native gel electrophoresis and activity staining. The electromorphs designated isoforms A, B, and C are characterized by slow, intermediate and fast electrophoretic migration, respectively, which was not observed under denaturing conditions. The isoforms were not associated with virulence determinants and with the outcome of disease. Sequence analysis of the sodB gene in strains producing different FeSOD isoforms and comparison of deduced protein sequences revealed that differences in the electric migration behavior are associated with exchange of charged amino acids, suggesting that faster migration is caused by a more negative total charge of the proteins. Electrophoretic migration of native FeSOD was not influenced by changes in the iron cofactor concentration, oxidative stress, and different media, indicating that FeSOD isoforms represent stable strain-specific markers.

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.

Based on the predictive analysis of the cellular protein content from the complete genome sequence of Helicobacter pylori, discrepant results were previously reported concerning the occurrence of a protein kinase in this bacterium. To solve this ambiguity, we have directly assayed cellular extracts for their capacity of phosphorylating endogenous proteins. At least eight different proteins, ranging from 24 to 200 kDa, were found to be phosphorylated to a varying extent. Individual measurement of their phosphoamino acid composition showed that they all were modified at serine residues. These data indicate that H. pylori does contain a protein-serine kinase activity.

Acid resistance is considered an important virulence factor of the human pathogen Helicobacter pylori. The enzyme urease plays an important role in this acid resistance, but there are indications that other systems are present. We set out to establish the relevance of these urease-independent acid-resistance systems for growth at low pH. Four mutants out of a total of 1000 UV-mutants were urease positive, grew identical to wild-type on pH 7 plates, but did not grow on pH 5 plates. Whereas transformation of a mutant with its own chromosomal DNA did not restore growth at pH 5, transformation with wild-type DNA or DNA of one of the other mutants did restore the growth. From these complementation studies, we conclude that in H. pylori a urease-independent acid-resistance system, probably depending on the expression of more than one gene, is involved in the growth at low pH.

Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism. We have identified a gene in H. pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species. An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits. The majority (> 99%) of mutant cells were completely aflagellate. These results suggest that FliI is a novel ATPase involved in flagellar export in H. pylori.

We investigated the population genetics of 23 isolates of H. pylori by allozyme electrophoresis using 16 enzyme loci. Isolates were obtained from adult patients of whom 48% were of Greek extraction. Eight patients (35%) had an active duodenal ulcer. Allelic variation per loci ranged from 2 to 11 alleles. Four major genetic clusters were apparent, having > 75% fixed genetic differences. There was no distinct clustering (clonal structure) on the basis of the geographical origin of the persons from whom isolates were obtained, indicating that this bacterium has not recently jumped a species barrier into humans. Isolates associated with ulcer disease were not monophyletic, with isolates from ulcer patients being found in phylogenetically diverse branches of the dendogram derived from the data. Based on the genetic diversity of H. pylori isolates, we propose that isolates should be classified as belonging not to a single species but to a 'Helicobacter pylori species-complex'.

The targeted genomic strategy of random fusions to a partial gene encoding a signal sequence-deficient fragment of bacterial alkaline phosphatase was utilized to screen for secreted proteins in Helicobacter pylori. The rationale for targeting extracytoplasmic proteins was based on the hypothesis that most virulence factors and vaccine candidates are secreted or exported proteins. In addition, extracytosolic proteins represent good potential targets for drug intervention since they are in general more accessible to drugs than are cytoplasmically localized proteins. The application of this strategy to H. pylori allowed the identification of putative virulence factors and novel targets for drug intervention including four putative antibiotic efflux genes. The strategy used here is rapid and technically simple, relatively inexpensive, adaptable to a wide variety of microbes and genetic systems, and selects for expressed and accessible proteins.

We previously reported that a gene hpcopA isolated from Helicobacter pylori is associated with copper transport. In the present study, the DNA upstream of the hpcopA was cloned and the nucleotide sequence analyzed. An open reading frame coding for 124 amino acids was predicted, which was connected in frame to the hpcopA. The deduced protein sequence exhibits striking homology with known copper-transporting P-type ATPases. Disruption of this ORF renders H. pylori hypersensitive to copper present in growth media, indicating that it encodes the N-terminal amino acid sequence of the hpCopA protein. Measurement of copper content in the wild-type and hpcopA-disrupted cells showed that the copper content was increased in the mutant cells, further supporting that the previous proposal that the gene hpcopA is involved in copper export. In addition, the cop operon consisting of the genes hpcopA and hpcopP was identified by primer extension and reverse transcription-polymerase chain reaction. Our results indicate that the genes for copper import and export are located in separate operons within H. pylori.

The peptic ulcer-causing bacterium Helicobacter pylori was found to contain an H2-uptake hydrogenase activity coupled to whole cell (aerobic) respiration. The activity was localized to membranes which functioned in the H2-oxidizing direction with a variety of artificial and physiological electron acceptors of positive redox potential. Immunoblotting of H. pylori membrane components with anti (B. japonicum) hydrogenase large and small subunit-specific antisera identified H. pylori hydrogenase peptides of approximately 65 and 26 kDa respectively, and H. pylori genomic DNA fragments hybridizing to the (B. japonicum) hydrogenase structural genes were identified. The membrane-bound activity was subject to anaerobic activation, like many NiFe hydrogenases. Difference absorption spectral studies revealed absorption peaks characteristic of b and c-type cytochromes, as well as of a bd-type terminal oxidase in the H. pylori H2-oxidizing membrane-associated respiratory chain.

The common mucosal immune system was stimulated by oral immunisation with jack bean urease and the adjuvant cholera toxin. A high level of local antibody and serum antibody was induced in mice following hyperimmunisation with this combination. No cross-reacting antibody was found against either Helicobacter pylori or Helicobacter felis. No protection was observed against oral challenge of immunised mice with living H. felis thus disproving the interesting hypothesis of Pallen and Clayton that plant urease might induce a protective immunity against helicobacter infection.

Genetic diversity amongst 21 human gastric isolates of Helicobacter pylori was investigated by polymerase chain reaction amplification and HaeIII digest (restriction fragment length polymorphism) analysis of an internal 2.4-kb segment of the urease A and urease B genes. H. pylori from 11 independent individuals yielded nine distinct restriction fragment patterns but only one pattern was common to H. pylori from two individuals. By contrast, multiple isolate sets of H. pylori from two patients each had common urease gene patterns. Most strains with the same urease gene patterns were distinguishable in their ribosomal RNA gene patterns. The study demonstrated diversity amongst H. pylori and established that PCR analysis of urease genes provided a novel method of identifying isolates. The profiles were reproducible and convenient to obtain and analyse, and were almost as discriminatory as HaeIII ribopatterns.

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.

Biotype, genome, protein and plasmid profile diversity amongst 40 epidemiologically unrelated strains of Helicobacter pylori was studied. Strains were API Zym biotypes II, III and IV but most (87%) were biotype II. Four subsets of strains were defined on a combination of motility (56% positive) and cytotoxin production (44% positive). A close association (P = 0.45) between these two features was observed for 69% of strains. Each strain of H. pylori had a unique DNA type defined by either HaeIII or HindIII total digest patterns and by ribopatterns, except for DNA of the rare strains not cut by these endonucleases. Strain diversity was confirmed by one-dimensional SDS-PAGE electrophoretic protein patterns. No consistent associations between cytotoxin activity and overall ribopattern or band subsets within a ribopattern were detected. Some strains (39%) contained a plasmid but the presence of plasmids was not consistently associated with either cytotoxin activity, biotype, motility or ribopattern. We conclude that the cytotoxin-producing strains of H. pylori were genomically as diverse as the non-cytotoxin producing strains.

Fast protein liquid chromatography and SDS-PAGE have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH.