Comparison of Gene Expression Patterns between Helicobacter pylor 26695 and its Superoxide Dismutase Isogenic Mutant

Helicobacter pylori, a causative agent of gastroduodenal diseases, is a Gram-negative microaerophilic bacterium. Although H. pylori locates in the microaerophilic mucous layer, the bacteria would come into contact harmful reactive oxygen species generated by host immune system. It has been reported that H. pylori harbors various defense mechanisms which can protect bacterial cells from oxygen exposure. The change of the gene expression profile of sodB-negative isogenic mutant of H. pylori 26695 was analyzed by high resolution 2-DE followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS and microarray analysis. Eighteen genes and 41 genes were upregulated and downregulated respectively, either transcriptionally or translationally. Expression levels of three genes including trxB, yxjE and ribE that were changed both on a mRNA level and on a protein level were confirmed by RT-PCR analysis. However, change of expression levels of other major antioxidants such as KatA, AhpC and NapA were not detected, which means Sod is regulated by different way from that of KatA and AhpC. Mutant study of other antioxidant proteins may give us better understanding for the regulation of stress response in H. pylori.

Proteomic Analysis of Thiol-active Proteins of Helicobacter pylori 26695

Helicobacter pylori are a capnophilic bacterium, which colonize gastric mucosa and are resistant to acidic and oxidative damage. Thiol-active proteins subserve redox functions in tolerating oxidative stress and environmental toxicants, such as hydrogen peroxide and hypochlorous acid. We analyzed disulfide-containing proteins of H. pylori strain 26695. Active disulfide-containing proteins were separated by thiol-affinity chromatography, displayed with two-dimensional electrophoresis (2-DE), and identified by MALDI-TOF-MS. Thirty-five putative disulfide proteins, including AhpC (HP1563), GroEL (HP0011), and FrdB (HP0191), were identified in this study. In addition, 4 disulfide proteins of HypB, FusA, TufB, and AhpC showed enhanced intensities in the periplasmic space when compared with the pellet, suggesting that these proteins might play roles in the first redox system against environmental oxidative stresses. Disulfide-containing proteins identified in this study will provide the standard landscape for constructing the proteome components responsible for redox regulation of H. pylori.

Purification and Characterization of Helicobacter pylori gamma-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) was purified to electrophoretic homogeneity from the cell extract of H. pylori. The purified enzyme consisted of heavy and light subunits with molecular weights of 38 kDa and 21 kDa, respectively. N-terminal amino acid sequence of heavy and light subunits revealed that H. pylori GGT was processed into 3 parts for a signal peptide of 27 amino acid residues, a heavy subunit of 352 residues, and a light subunit of 188 residues during translation. The reaction rate for hydrolysis of gamma-GpNA was 84.4 micromol/min per milligram of protein, and that for the gamma-glutamyl transfer from gamma-GpNA to gly-gly was 23.8 micromol/min per milligram of protein. The apparent Km values of H. pylori GGT for gamma-glutamyl compounds were on the order of 10-3 to 10-4 M and those for acceptor peptides and amino acids were on the order of 10-1 to 10-2 M. The GGT protein kept approximately 80% of the initial enzymatic activity on incubation at 60degrees C for 15 min. The optimum temperature and pH for reactions of both hydrolysis and transpeptidation were 40degrees C and 9.0, respectively. The transpeptidation and hydrolysis reactions catalyzed by H. pylori GGT were strongly inhibited by L-Gln and moderately inhibited by L-Ala, L-Ser, beta-chloro-L-Ala, and L-Glu. These results demonstrated that the biochemical properties of H. pylori GGT are different from those of other bacterial GGTs. Further, H. pylori GGT might degrade glutathione in the gastric mucous layer of humans if the enzyme could be secreted in the bacterial niches.

Comparison of Proteome Components of Helicobacter pylori Before and After Mouse Passage

The mouse model is alleged to be a useful tool for understanding of pathophysiological roles of Helicobacter pylori in the development of gastric disorders. However, it has been observed that H. pylori strains significantly differed in their fitness in mice and even mouse strains differed in their susceptibilities to a H. pylori strain. Bacterial components of H. pylori which could affect on its fitness in mice have to be elucidated for the establishment of the mouse model for H. pylori infections. In the comparison of colonization ability between two H. pylori Korean isolates, 51 (isolated from a patient with duodenal ulcer) and 52 (isolated from a patient with gastric cancer), 52 could colonize better than 51 on the gastric mucosa of mouse. Proteome components of H. pylori 52, as a good colonizer and H. pylori 51, as a poor one were quantitatively compared each other. Five bacterial proteins including catalase, urease subunit alpha/beta, enolase and ferritin, were up-regulated in 52. In addition, the respective proteome components of the two strains were also compared with their mouse-passaged homologous strains. Seven and five proteins, which included catalase, flagellin A/B in common, were up-regulated in mouse-adapted 51 and 52, respectively. Among the fourteen identified proteins, urease subunit alpha/beta, flagellin A/B, catalase, ferritin, superoxide dismutase and neutrophil-activation protein have been previously known to be necessary to gastric colonization of H. pylori in animal models. The other up-regulated proteins including enolase, elongation factor Tu and fructose-bisphosphate aldolase have been reported to be associated with acid tolerance of H. pylori. These data provide confirmatory evidence for the importance of those proteins in the development of H. pylori-associated gastric disorders.

Analysis of Low Molecular Weight Proteome from H. pylori Cell Extract Using the High Performance Liquid Chromatography

Low molecular proteins (LMPs) which are smaller than 20 kDa are difficult to visible on a standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-D SDS-PAGE) map. LMPs must be enriched appropriately to be analyzed. We isolated LMPs of Helicobacter pylori 26695 from 1-D polyacrylamide gel and digested by pepsin. Pepsin-digested LMPs were separated by HPLC and each fraction was analyzed by hybrid tandem mass spectrometer. Seventy nine peptides, representing 27 genes, including copper ion binding protein (CopP, 7 kDa), thioredoxin (TrxA, 11.9 kDa) and ribosomal protein L23 (Rpl23, 10.5 kDa) were identified. Some proteins larger than 40 kDa including Omp2, Omp21, Omp27, Omp30, Omp32, catalase and HP1083 were also identified. This work may give researchers a useful way to analyse the expressed LMPs which could not be identified on the conventional 2-D SDS-PAGE.