Isolation of a 3-deoxy-D-mannooctulosonic acid disaccharide from Salmonella minnesota rough-form lipopolysaccharides

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70 degrees C/3 h. After dialysis of the hydrolysates about one third of the total 3-deoxy-D-mannooctulosonic acid (dOclA) content but no neutral sugars were found in the dialysate. By high-voltage paper electrophoresis, a compound with the mobility of 1.2 relative to dOclA could be isolated from the dialysate. It was identified as a dOclA disaccharide by hydrolysis without or after reduction with sodium borohydride and by analysis with the thiobarbituric acid assay under different conditions. The ketosidic linkage in the disaccharide is assumed to be 2.4 or 2.5.     

Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.

The chemical structure of free lipid A isolated from rough- and smooth-form lipopolysaccharides (R-LPS and S-LPS, respectively) of the human gastroduodenal pathogen Helicobacter pylori was elucidated by compositional and degradative analysis, nuclear magnetic resonance spectroscopy, and mass spectrometry. The predominant molecular species in both lipid A components are identical and tetraacylated, but a second molecular species which is hexaacylated is also present in lipid A from S-LPS. Despite differences in substitution by acyl chains, the hydrophilic backbone of the molecules consisted of beta(1,6)-linked D-glucosamine (GlcN) disaccharide 1-phosphate. Because of microheterogeneity, nonstoichiometric amounts of ethanolamine-phosphate were also linked to the glycosidic hydroxyl group. In S-LPS, but not in R-LPS, the hydroxyl group at position 4' was partially substituted by another phosphate group. Considerable variation in the distribution of fatty acids on the lipid A backbone was revealed by laser desorption mass spectrometry. In tetraacyl lipid A, the amino group of the reducing GlcN carried (R)-3-hydroxyoctadecanoic acid (position 2), that of the nonreducing GlcN carried (R)-3-(octadecanoyloxy)octadecanoic acid (position 2'), and ester-bound (R)-3-hydroxyhexadecanoic acid was attached at position 3. Hexaacyl lipid A had a similar substitution by fatty acids, but in addition, ester-bound (R)-3-(dodecanoyloxy)hexadecanoic acid or (R)-3(tetradecanoyloxy)hexadecanoic acid was attached at position 3'. The predominant absence of ester-bound 4'-phosphate and the presence of tetraacyl lipid A with fatty acids of 16 to 18 carbons in length differentiate H. pylori lipid A from that of other bacterial species and help explain the low endotoxic and biological activities of H. pylori LPS.     

Comparative proteome analysis of Helicobacter pylori

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.     

Helicobacter spp. Other Than Helicobacter pylori

Non- H. pylori Helicobacter species (NHPHS) are associated with several important human and animal diseases. In the past year research into this group of bacteria has continued to gain attention, and novel species have been described in new niches owing to improvements in detection methods. Polymerase chain reaction and/or sequencing remain the gold standard for the detection of this genus. New insights into the pathogenesis of the NHPHS in hepatobiliary, gastric, and intestinal diseases were gained. In particular, data revealed interaction between hepatic steatosis and infectious hepatitis in the development of hepatocellular carcinoma. Evidence of an association between hepatitis C virus and Helicobacter spp. in hepatocarcinoma development was also provided; and male sex hormone signaling appeared to influence infectious hepatitis induced by Helicobacter hepaticus . More findings support an association between Helicobacter heilmannii and gastric adenocarcinoma; and in mice, mucins MUC4 and MUC5 but not MUC1 influence the colonization and pathogenesis of Helicobacter felis . Data indicated that the roles of the adaptive immune system in H. hepaticus -induced intestinal tumorigenesis are different in the small and large intestines, and environmental factors, such as bile acids may modulate H. hepaticus carcinogenic potential. New reports in the prevention and eradication of NHPHS showed a protective response against Helicobacter suis induced by vaccine administration, and a successful cross-foster rederivation method successfully eradicated Helicobacter spp. from contaminated mice litters. Overall, the studies provided insights into the pathophysiology of Helicobacter species other than Helicobacter pylori.     

Helicobacter pylori and hormones.

Helicobacter pylori affects gastric acid secretion via several mechanisms. One of these is by changing gastric regulatory physiology. The infection elevates plasma gastrin levels and decreases gastric mucosal expression of the inhibitory peptide somatostatin. These changes may be due to products of H. pylori itself or inflammatory cytokines released in H. pylori infection: acid secretion is inhibited less by a low intra-gastric pH, infusions of cholecystokinin and gastric distention in infected persons. Eradication of H. pylori rapidly decreases basal acid secretion and gastrin-releasing, peptide-stimulated acid secretion. There are now reports that maximally-stimulated acid secretion, a measure of the parietal cell mass, falls significantly six and 12 months after eradication of H. pylori from duodenal ulcer patients. This might be due to withdrawal of the trophic effect of gastrin. However H. pylori can also decrease gastric acid secretion, both through the mechanisms described in Dr. Cave''s paper and by causing gastric mucosal atrophy with loss of parietal cells. The net effect on acid presumably depends on which mechanism predominates. The processes involved may be crucial determinants of clinical outcome. For example, infection with little atrophy and high acid secretion is associated with duodenal ulcers, while infection with atrophy and low acid secretion increases the risk of gastric cancer of the intestinal-type.     

Comparative genome analysis of Helicobacter pylori strains

DNA macroarrays were used to characterize 17 Helicobacter pylori strains isolated in four geographic regions of Russia (Moscow, St. Petersburg, Kazan, and Novosibirsk). Of all genes, 1272 (81%) proved to occur in all strains and to constitute a functional core of the genome, and 293 (18.7%) were strain-specific and greatly varied among the H. pylori strains. Most (71%) of the latter had unknown functions; the remainder included restriction-modification genes (3-9%), transposition genes (2-4%), and genes coding for outer membrane proteins (2-4%). The Russian H. pylori strains did not differ in genome organization or in the number and distribution of strain-specific genes from strains isolated in other countries.     

Helicobacter pylori and apoptosis.

In an attempt to understand the diverse effects of infection with Helicobacter pylori on epithelial mucosal mass and consequent clinical outcome, the relationship between H. pylori infection and gastric epithelial cellular turnover has been investigated. Our results indicate that H. pylori increases epithelial cell proliferation and apoptosis in vivo, but that infection with bacteria of the cagA genotype leads to relatively more proliferation than apoptosis. This review explores the causes of the induction of apoptosis in gastric epithelial cells by H. pylori and the consequences of alterations in apoptosis to the maintenance of gastric mucosal homeostasis.     

Alterations in Helicobacter pylori outer membrane and outer membrane vesicle-associated lipopolysaccharides under iron-limiting growth conditions

Outer membrane vesicles (OMVs) shed from the gastroduodenal pathogen Helicobacter pylori have measurable effects on epithelial cell responses. The aim of this study was to determine the effect of iron availability, and its basis, on the extent and nature of lipopolysaccharide (LPS) produced on H. pylori OMVs and their parental bacterial cells. Electrophoretic, immunoblotting and structural analyses revealed that LPSs of bacterial cells grown under iron-limited conditions were notably shorter than those of bacteria and OMVs obtained from iron-replete conditions. Structural analysis and serological probing showed that LPSs of iron-replete cells and OMVs expressed O-chains of Lewis(x) with a terminal Lewis(y) unit, whereas Lewis(y) expression was notably reduced on bacteria and OMVs from iron-limiting conditions. Unlike the O-chain, the core oligosaccharide and lipid A moieties of iron-replete and iron-limited bacteria and their OMVs were similar. Quantitatively, shed OMVs from iron-replete bacteria were found to be LPSenriched, whereas shed OMVs from iron-limited bacteria had a significantly reduced content of LPS. These differences were linked to bacterial ATP levels. Since iron availability affects the extent and nature of LPS expressed by H. pylori, host iron status may contribute to H. pylori pathogenesis.     

Immunology of Helicobacter pylori infection.

We have tried to answer several questions in this article, dealing with: ontogenesis of the immune response, presentation of H. pylori antigens to immune cells, systemic vs local immune response, cytokine Th1/Th2 configuration, the role of cytokines, especially represented during H. pylori infection, mimicry phenomena, extragastroduodenal sites and manifestations and finally, vaccine development. The new achievements in the vaccine field, also of Polish groups, were underlined.     

Mutational analysis of the Helicobacter pylori carbonic anhydrases

In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.     

Helicobacter pylori in Barrett's esophagus.

Barrett's esophagus is an anatomicoclinical state in which, due to the prolonged action of gastroesophageal reflux, the squamous epithelium is replaced by columnar epithelium. Helicobacter pylori has been implicated in the pathogenesis of various gastrointestinal disorders and has occasionally been observed in Barrett's esophagus. The aim of this study is to determine the incidence of H. pylori in Barrett's esophagus and try to establish its role in the pathogenesis of this disorder. H. pylori was observed in 31 biopsies (44.3%) of the 70 studied, mainly when the epithelium is of the gastric atrophic-fundic type (p less than 0.01). Its presence shows no relation to the degree of inflammatory activity and does not seem, therefore, to play an important role in the pathogenesis of the lesion.     

Helicobacter pylori: therapeutic targets.

Helicobacter pylori is now considered a major pathogen of the upper gastrointestinal tract. It is seen as an important cause of peptic ulceration not associated with NSAID use. It is also increasingly linked to other diseases of the GI tract, although the relationship between the organism and conditions such as gastric cancer, non-ulcer dyspepsia and gastroesophageal reflux disease is not as clear as is the case in peptic ulcer disease. This is probably because of a lack of well-performed, statistically powerful, prospective therapeutic trials that indicate that H. pylori eradication is of benefit in these diseases. The high infection rate without overt disease seen in many populations, especially from developing countries, probably contributes to this "credibility gap." While we have excellent therapeutic regimens available at this time, rational targeting requires that the objective evidence in favor of therapeutic intervention in upper GI disease, as well as the local H. pylori epidemiology, needs to be considered.     

Helicobacter pylori associated gastric pathology.

Helicobacter pylori (HP), undoubtedly, the most common world-wide infection plays an important role in pathogenesis of peptic ulcer. Proof for a causal role for HP in peptic ulcer rests in two major points; 1) the majority of ulcer patients are HP infected and the prevalence of this infection for both gastric ulcer (GU) and duodenal ulcer (DU) is much higher than for gender- and age-adjusted controls and 2) the cure of HP infection dramatically reduces ulcer recurrence. Conclusions regarding the mechanisms by which HP induces peptic ulcer are restricted mainly to studies observing the consequences of its eradication by antibiotics combined with gastric inhibitors or bismuth agents. Several specific virulence factors such as cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) as well as other noxious substances including ammonia, lipopolysaccharide (endotoxin), platelet activating factor (PAF), nitric oxide (NO) and others have been implicated in gastritis and were found to be significantly more frequent in gastric cancer than in gender- and age-matched controls, especially in younger generation. Chronic inflammation, atrophic gastritis, intestinal metaplasia, impaired defense mechanisms combined with hypergastrinemia, deficiency of vitamin C in the stomach , excessive oxygen metabolites and epithelial cell proliferation have been associated with gastric cancer. This multi-step pathway originally proposed by Correa and his colleagues, long before the HP was discovered in the stomach, leads to cancer but may be reversed by eradication of HP. This is, however, a controversial issue because gastric atrophy and intestinal metaplasia may be also caused by other factors such as bile reflux, dietary irritants, and autoimmunity. The implication of HP in MALT-lymphoma is based on the observations that eradication of HP in early stage of low-grade of this tumor leads to complete remission. The significance of HP in non-ulcer dyspepsia remains questionable and requires further studies.     

Immunological aspects of Helicobacter pylori infection.

Host defence against Helicobacter pylori infection is a complex system of both specific and non-specific reactions. Among the non-specific defense mechanisms acting on bacteria before they reach the mucus layer in the stomach are digestive enzymes, lyzozyme, lactoferrin and other components with antimicrobal activity. The mucus layer is the final non-specific barrier against the bacteria reaching the gastric mucosa cells. On reaching the gastric mucosa Helicobacter pylori adheres to epithelial cells and bacterial antigens, chemotaxins and other components are liberated. Helicobacter pylori antigens are presented to immunate B lymphocytes, which interact with T-helper lymphocytes to become mature IgA-, IgD-, IgE-, IgG and Ig-M producing plasma cells. IgA dimers of secretory IgA are secreted through the gastric epithelium. IgE antibodies bind to basophils, which mature to histamine-producing mast cells. Histamine activates the acid production in the stomach and contributes to the chronic inflammation and tissue destruction. In addition, T lymphocytes are possible activated by Helicobacter pylori and contribute to the chronic inflammation.     

Inactivation of Helicobacter pylori by chlorination.

Three strains of Helicobacter pylori were studied to determine their resistance to chlorination. The organisms were readily inactivated by free chlorine and should therefore be controlled by disinfection practices normally employed in the treatment of drinking water.