Proteome analysis of highly immunoreactive proteins of Helicobacter pylori

Background. Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. Method. The total complement of protein from seven strains of H. pylori was resolved by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori‐infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme‐antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide‐mass fingerprinting using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results. Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease β‐subunit UreB; elongation factor EF‐Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. Conclusion. These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF‐Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.     

Structure Based Annotation of Helicobacter pylori Strain 26695 Proteome

The availability of complete genome sequences of H. pylori 26695 has provided a wealth of information enabling us to carry out in silico studies to identify new molecular targets for pharmaceutical treatment. In order to construe the structural and functional information of complete proteome, use of computational methods are more relevant since these methods are reliable and provide a solution to the time consuming and expensive experimental methods. Out of 1590 predicted protein coding genes in H. pylori, experimentally determined structures are available for only 145 proteins in the PDB. In the absence of experimental structures, computational studies on the three dimensional (3D) structural organization would help in deciphering the protein fold, structure and active site. Functional annotation of each protein was carried out based on structural fold and binding site based ligand association. Most of these proteins are uncharacterized in this proteome and through our annotation pipeline we were able to annotate most of them. We could assign structural folds to 464 uncharacterized proteins from an initial list of 557 sequences. Of the 1195 known structural folds present in the SCOP database, 411 (34% of all known folds) are observed in the whole H. pylori 26695 proteome, with greater inclination for domains belonging to α/β class (36.63%). Top folds include P-loop containing nucleoside triphosphate hydrolases (22.6%), TIM barrel (16.7%), transmembrane helix hairpin (16.05%), alpha-alpha superhelix (11.1%) and S-adenosyl-L-methionine-dependent methyltransferases (10.7%).     

Carcinogenic Helicobacter pylori Strains Selectively Dysregulate the In Vivo Gastric Proteome,Which May Be Associated with Stomach Cancer Progression,

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Highlights

• •H. pylori dysregulates the in vivo gastric proteome of gerbils in a strain-specific manner.
• •H. pylori increases RABEP2 and G3BP2 levels in cell culture.
• •H. pylori upregulates RABEP2 and G3BP2 in gerbil and human gastric epithelium.
• •Levels of RABEP2 and G3BP2 increase with severity of malignant lesions in vivo.

     

Analysis of Aztreonam-Inducing Proteome Changes in Nondividing Filamentous Helicobacter pylori

When stressed, bacteria can enter various nondividing states. In the present study, nondividing filamentous form in Helicobacter pylori was induced by a β-lactam antibiotic, aztreonam. In order to find possible cell division checkpoints in H. pylori, 2-DE was used to compare the proteomic profile of nondividing filamentous H. pylori with its spiral form. In total, 21 proteins involved in various cellular processes showed differential expression. One protein induced by aztreonam was a cell division inhibitor (minD), related to cell division. We then constructed the deletion mutant of minD in H. pylori 26695. Scanning electron microscope observation showed that the deletion of this protein provoked some bacteria to change into a short rod-shape and the viability of the mutant is lower than that of the wild type. Moreover, sequence comparison showed that minD of H. pylori and that of Escherichia coli share 50?% amino acid identity. This suggested that this protein possibly plays the similar part in H. pylori as in E. coli.     

Carcinogenic role of tumor necrosis factor-alpha inducing protein of Helicobacter pylori in human stomach

Helicobacter pylori is the definitive carcinogen for stomach cancer and is known to induce proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1(IL-1) in the stomach. Based on our findings that TNF-alpha is an endogenous tumor promoter, we identified the TNFalpha inducing protein (Tipalpha) gene family, and confirmed Tipalpha and HP-MP1 as new carcinogenic proteins of H. pylori.Tipalpha protein is unique to H. pylori, and this paper shows the strong tumor promoting activity of Tipalpha gene family, in cooperation with Ras protein and its mechanisms of action in relation to NF-kappaB activation, and discusses the carcinogenic role of Tipalpha in stomach cancer. Our recent finding showing that penicillin-binding proteins of other bacteria are weak homologues of Tipalpha is also discussed.     

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.     

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.     

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.     

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.     

Genetic relationship among isolates of Helicobacter pylori: evidence for the existence of a Helicobacter pylori species-complex

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'.     

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.