Enrichment of Low Abundance Proteins of Helicobacter pylori Strain 26695 by the Heparin Chromatography

Low-abundance cellular proteins normally invisible on the standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) map must be enriched appropriately in order to be visualized and identified in cells or tissues. We applied proteins of H. pylori strain 26695 to a immobilized heparin-affinity resin, which has an affinity for nucleic acid-binding proteins, protein biosynthesis factors, and growth factors. The whole cell extract of H. pylori strain 26695 was fractionated by the heparin-agarose chromatography, and was analyzed by 2-DE. The 2-DE SDS-PAGE displayed spots after silver staining, which were identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Among the ca. 150 spots that were processed, 79 proteins representing 57 genes were identified. Eleven proteins were determined to be nucleic acid-associated. Eighteen proteins were newly identified in this study, including DNA topoisomerase I. These results may provide guidance for enriching low abundance proteins of H. pylori and contribute to the construction of a master protein map of H. pylori.

RFLP Analysis of cag7 Gene of Helicobacter pylori

The cag7 gene of Korean H. pylori strains was analyzed by RFLP to develop a discriminatory tool for genotyping clinical isolates. For this study, a total of 82 H. pylori strains were isolated from the patients; 27 strains from the patients with chronic gastritis, 26 from duodenal ulcer, and 29 from gastric cancer. Genomic DNA was isolated and subjected to PCR targeting entire ORF or the repeat regions I and II of cag7 gene. PCR products from entire ORF or repeat region I of cag7 gene were divided into two types. However, there was no difference in the length of PCR products from the repeat region II. By the PCR genotyping of the entire cag7 gene, genotypes A and B were established, which showed approximately 5,100 and 5,500 bp PCR products, respectively. The repeat region I showed approximately 600 or 1,000 bp DNA fragments by PCR. The length of cag7 gene was determined by the size variation in the repeat region I. In addition, RFLP analysis of the PCR products of cag7 gene showed 11 subtypes, based on the major bands. These findings illustrate that the genetic diversity of the repeat region I would serve a reliable target for the genotyping of the cag7 gene.

Analysis of cag Pathogenicity Island of Helicobacter pylori Korean Isolate

It is commonly believed in the Western World that the more severe forms of gastroduodenal diseases like peptic ulcer are associated with infection by specific Helicobacter pylori strains classified as type I being considered to be more virulent than type II strains. However, in Korea, most of H. pylori isolates belong to type 1 strains regardless of virulence. Type I H. pylori strains differ from type II strains by the presence of the cag pathogenicity island (cag PAI) composed of a block of genes. In this study, the nucleotide sequence of cag PAI of the H. pylori Korean strain 51 was determined and compared with those of strains 26695 and J99 to assess the structural variation in the region and to evaluate its implication in the virulence of the H. pylori. The cag PAI of H. pylori strain 51 was smaller in size and in the number of constituting ORFs in comparison with 26695 and J99 strains. Although many cag orthologues were nearly identical one another with the similarity of 90% or more at the nucleotide and amino acid levels, there were some remarkable and significant differences in several cag genes among the three cag PAIs. Surprisingly, the percent similarities at amino acid level were lower than those at nucleotide level in one third of the ORFs. The two genes (cag7 and cagA) of strain 51 differed in sizes and deduced amino acid sequences from the corresponding genes of the other two strains. When comparing cagA ORF of H. pylori strain 51 with that of 8 non-Korean strains, phylogenetic tree revealed that the strain 51 formed a separate branch with the most far distances from the other strains except for a Japanese strain. The Cag7 protein of, strain 51 had a deletion in the repeat region II, suggesting a major change in the conformation and function of the protein.

Characterization of Type 2 Restriction Endonucleases (Hpy51) from Helicobacter pylori Strain 51

This study describes the purification and characterization of type II restriction endonuclease of Helicobacter pylori in order to understand the DNA restriction and modification of H. pylori. H. pylori cell extract was subjected to polyethyleneimine treatment, salt precipitation, heparine-sepharose column chromatography, and fast protein liquid chromatography (FPLC) using Resource Q column and Mono Q column to purify the type II restriction endonuclease. Hpy51-I was characterized to recognize the sequneces 5`-GT(G/C)AC-3`, yielding 5-base 5` protruding ends. The restriction sequence was identical to that of Tsp 45 I. The enzyme exhibited its maximal activity in the presence of 10-20 mM LaCl, but was inhibited completely in the presence of more than 80 mM NaCl. The enzyme showed its maximal activity in the presence of 1-10 mM MgC1(2). The optimal pH and temperature for enzyme activity was pH 9.0 and 37 degrees C, respectively. MnC1(2) could not substitute for MgC1(2) in reaction mixture. And addition of j3-mercaptoethanol and bovine serum albumin in reaction mixture led to loss of enzyme activity of Hpy51-I. The whole cell extract of H. pylori strain 51 was confirmed to carry the enzyme activity for methylation of Hpy51-I-recognised sequence. Hpy51-I digested genomic DNAs of enteric bacteria to less than I kb while it could not cut the genomic DNAs of H. pylori isolates. In this study, the type II restriction enzyme (Hpy51-I) of H. pylori was identified and characterized its biochemical properties, demonstrating that Hpy51-I might be one of the barriers for preventing the introduction of foreign DNAs into H. pylori.