The complex methylome of the human gastric pathogen Helicobacter pylori

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.     

The complete genome sequence of Helicobacter pylori strain G27

Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the world's population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research.     

The secE gene of Helicobacter pylori

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

The vacuolating cytotoxin of Helicobacter pylori

Helicobacter pylori, the causative agent of chronic superficial gastritis and duodenal ulcer disease in humans, produces a unique cytotoxin (VacA) that induces cytoplasmic vacuolation in eukaryotic cells. The structural organization and processing of the vacuolating cytotoxin are characteristic of a family of proteins exemplified by Neisseria gonorrhoeae IgA protease. Although only 50% of H. pylori isolates produce detectable cytotoxin activity in vitro, vacA homologues are present in virtually all isolates. Several families of vacA alleles have been identified, and there is a strong correlation between presence of specific vacA genotypes, cytotoxin activity, and peptic ulceration. Experiments in a mouse model of H. pylori-induced gastric damage indicate that the cytotoxin plays an important role in inducing gastric epithelial necrosis.     

The thioredoxin system of Helicobacter pylori

This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

Helicobacter pylori catalase

Helicobacter pylori is the major aetiological agent of gastroduodenitis in humans. Due to the potential importance of catalase in the growth and survival of Helicobacter pylori on the surface of inflamed mucosae, we have characterized catalase from H. pylori as a prelude to further studies on the function of the enzyme in vivo. The catalase activity of H. pylori was significantly affected by the presence of blood, serum or erythrocytes in the growth medium: the greatest activity was expressed when the bacterium was grown on medium containing serum. H. pylori catalase is a tetramer with a subunit Mr of 50,000. The enzyme had a pI of 9.0-9.3, was active over a broad pH range and was stable at 56 degrees C. It was non-competitively inhibited by sodium azide, and had no detectable peroxidase activity. The Km for the purified catalase was measured as 43 +/- 3 mM-H2O2 and the V as 60 +/- 3 mmol H2O2 min-1 (mg protein)-1. The native catalase has absorption maxima at 280 nm and 405 nm with further minor shoulders or peaks at 510 nm, 535 nm and 625 nm, consistent with the presence of an iron-porphyrin prosthetic group.     

Helicobacter pylori motility

Motility is essential for Helicobacter pylori colonization. This review discusses the biochemistry, genetics and genomics of the H. pylori flagellum, and compares these features with well-characterized bacteria.     

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.     

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.     

Genomics of Helicobacter pylori

During this review period, we have definitely entered into the genomic era. The Helicobacter pylori studies reported here illustrate the use of most of the technologies currently available to globally interrogate the genome of a pathogen. Global analysis of the gene content of H. pylori strains gives insight into the extent of its genetic diversity and its in vivo evolution. Our understanding of the particularities of H. pylori as a gastric pathogen colonizing a unique niche has been improved by studies aimed at: (i) the identification of H. pylori-specific genes; (ii) the establishment of correlations between the presence of one or a group of genes (or proteins) with clinical outcome; and (iii) the analysis of global regulatory circuits or responses to the extracellular signals. The response of host cells to H. pylori infection will be developed in the chapter 'H. pylori and gastric malignancies' by Sepulveda and Coehlo. Despite our knowledge of the H. pylori genome, the function of about one third of its total proteins is still unknown. Functional genomics are straightforward approaches for the identification of new gene functions or metabolic pathways as well as for the understanding of cellular processes and the detection of new virulence factors. In silico studies combined with experimental work will undoubtedly continue to develop. To date, the expansion of proteomics with refinements in mass spectrometry technology has illustrated that through immunoproteomics and comparative studies, relevant novel antigens can be identified. Genomics not only provides invaluable information on H. pylori but also opens new perspectives for diagnostic or therapeutic applications.