Protein glycosylation has been considered as a fundamental phenomenon shared by all domains of life. In Helicobacter pylori, glycosylation of flagellins A and B with pseudaminic acid have been rigorously confirmed and shown to be essential for flagella assembly and bacterial colonization. In addition to flagellins, several other proteins including RecA, AlpA/B, and BabA/B in H. pylori have also been reported to be glycosylated and to be dependent on the lipopolysaccharide (LPS) biosynthetic pathway. However, these proteins have not been purified for sugar-specific staining or structural analysis to confirm the existence of carbohydrate motifs. Here, using a combined approach of genetics, protein purification, and sugar-specific staining, we demonstrate that RecA is not a glycoprotein. Moreover, using LPS-protein reconstitution experiments, we demonstrate that the presence of O-antigen containing full-length LPS interferes with the electrophoretic mobility of H. pylori RecA and many other proteins including AlpA/B on SDS-PAGE. Finally, we demonstrate that full-length LPS extracted from E. coli affects electrophoretic migration of H. pylori proteins, while full-length LPS extracted from H. pylori similarly influences the electrophoretic migration of E. coli proteins. The impact is more subtle with E. coli LPS compared to H. pylori LPS, indicating that the magnitude of effect of LPS effects on protein mobility is dependent on bacterial source of the LPS. These findings suggest that the effects of full-length LPS on protein electrophoresis may represent a more general phenomenon. As LPS is a unique component of virtually all Gram-negative bacteria, our data suggest that when observing protein electrophoretic mobility shifts between wild-type and LPS mutant strains or between subcellular fractionation samples, the influence of LPS on protein electrophoretic migration should be considered first, rather than interpreting it as potential protein glycosylation that is dependent upon LPS biosynthetic pathway.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.

Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.

Through advances in analytical methods to detect glycoproteins and to determine glycan structures, there have been increasing reports of protein glycosylation in bacteria. In this review, we summarize the known pathways for bacterial protein glycosylation: lipid carrier-mediated 'en bloc' glycosylation; and cytoplasmic stepwise protein glycosylation. The exploitation of bacterial protein glycosylation systems, especially the 'mix and match' of three independent but similar pathways (oligosaccharyltransferase-mediated protein glycosylation, lipopolysaccharide and peptidoglycan biosynthesis) in Gram-negative bacteria for glycoengineering recombinant glycoproteins is also discussed.

Background: The knowledge of pathological features of gastric inflammation due to Helicobacter pylori (HP) infection is mostly derived from chronically infected, symptomatic patients. Inflammatory patterns described in HP infection include active chronic gastritis (ACG), chronic gastritis (CG), lymphocytic gastritis (LG) and focal enhancing gastritis (FEG). None of these patterns are pathognomonic without demonstrable organisms. LG pattern (LGP) is defined as intraepithelial lymphocytosis of gastric mucosa (≥25 per 100 gastric epithelial cells), which is also seen in other diseases, particularly coeliac disease. Aim: To describe the evolution of HP related LGP, based on a unique group of subjects who were voluntarily infected with HP as a part of an ethically approved trial. Method: Of 36 H. pylori sero-negative individuals with normal endoscopic and biopsy findings, 30 subjects were infected with 5 different H. pylori strains, the rest given placebo. Biopsy, serology and culture results were obtained from all subjects at 2 and 12 weeks and were independently assessed by a blinded pathologist. Results: Of the challenged group, 29/30 showed active inflammation over the 12 weeks. LGP was restricted to 2 HP strains. HP was demonstrated in only 1/16 biopsies from the 8 recruits that showed LGP, compared to 33/44 biopsies of 22 recruits without the LGP. All LG cases showed activity. Conclusions: LG is associated with specific strains of H. Pylori, appears to be self-limiting and organisms are demonstrated very infrequently. LGP may be responsible for a significant proportion of ‘non HP gastritis’ in clinical practice. The presence of activity may help differentiate LGP due to HP from coeliac disease.

This review covers the current knowledge and gaps in Helicobacter pylori lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram-negative bacterium which colonizes the luminal surface of the human gastric epithelium. Both a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry of the Lewis antigen decorated O-antigen of H. pylori LPS promote immune escape and chronic infection. To date, the complete structure of H. pylori LPS is not available, and the proposed model is a linear arrangement composed of the inner core defined as the hexa-saccharide (Kdo-LD-Hep-LD-Hep-DD-Hep-Gal-Glc), the outer core composed of a conserved trisaccharide (-GlcNAc-Fuc-DD-Hep-) linked to the third heptose of the inner core, the glucan, the heptan and a variable O-antigen, generally consisting of a poly-LacNAc decorated with Lewis antigens. Although the glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O-antigen chains have been identified and characterized, there are many gaps in regard to the biosynthesis of the core LPS. These limitations warrant additional mutagenesis and structural studies to obtain the complete LPS structure and corresponding biosynthetic pathway of this important gastric bacterium.

Normal 0 false false false MicrosoftInternetExplorer4 Helicobacter pylori bacterium causing gastritis, duodenitis, non-ulcer dyspepsia, adenocarcinoma, MALT lymphoma and now-a-days causing hepatocellular carcinoma and cholengiocarcinoma has been studied for its surface antigens. There are many virulence factors which have been studied since many years. Cag-PAI is the site for virulence and causes mixed infections. Sialic acid is a type of glycan which is a surface lectin associated with adherence to cell lines and other virulence factors studied in detail in H. pylori. Sialic acid dependency is been studied by performing heamagglutination studies. Sialidase activity was carried out by haemagglutination assay on H. pylori. Some strains were found Sia-dependent and many were found Sia-independent. CagA, VacA, VirD4 genes were identified by PCR in some strains and picB gene was not detected in the 19/20 strains analysed. Thus, it was found that Sia-dependent and Sia-independent strains of H. pylori were correlating with PCR strains analysed for virulent factors. H. pylori strains where in total 9/54 strains were Sia-dependent belonged to class I category and others to class II category. Haemagglutination and PCR analysis revealed pathogenecity in some strains. Therefore, if broad spectrum studies are carried out on a large scale, it will be very useful in the diagnosis and prognosis of the diseases caused by H. pylori. Keywords: H. pylori , haemagglutination, sialic acid, PCR, cag PAI Cite this Article Rupa Mulchandani, Hans-Olof Nilsson, Torkel Wadstrom. Helicobacter pylori Sialic Acid-Specific Surface Lectin Associated with pathogenecity Island Cag PAI. Research & Reviews: Journal of Microbiology and Virology . 2015; 5(3): 10–17p.

Helicobacter pylori is a Gram negative bacterium found on the luminal surface of the gastric epithelium. Infection is generally acquired during childhood and persists life-long in the absence of antibiotic treatment. H. pylori has a long period of co-evolution with humans, going back at least since human migration out of Africa about 60, 000 years ago [1, 2]. This coevolution is reflected in DNA sequence signatures observed in H. pylori strains of different geographic origin and has enabled the mapping of human migration out of Africa. This prolonged and intimate relationship is likely to have shaped the large and diverse repertoire of strategies which H. pylori employs to establish robust colonization and persist in the gastric niche. Key challenges that H. pylori encounters are fluctuation of acidic pH of the gastric lumen, peristalsis of the mucus layer leading to washout in the lower intestine, nutrient scarcity, and the innate and adaptive immune responses promoting local inflammation or gastritis [3-8]. These challenges, particularly host immune responses, are likely to represent the selective pressure driving H. pylori micro-evolution during transmission leading to persistence in the human host.

BACKGROUND:One mechanism utilized by bacterial pathogens for host adaptation and immune evasion is the generation of phenotypic diversity by the phasevarion that results from the differential expression of a suite of genes regulated by the activity of a phase-variable methyltransferase within a restriction modification (RM) system. Phasevarions are active in Helicobacter pylori, however there have been no studies investigating the significance of phase-variable RM systems on host colonization.METHODS:Two mutant types incapable of phase variation were constructed; a clean deletion mutant ('DEL') and a mutant ('ON') where the homopolymeric repeat was replaced with a non-repeat synonymous sequence, resulting in expression of the full-length protein. The resulting mutants were assessed for their colonisation ability in the mouse model.RESULTS:Five phase-variable genes encoding either methyltransferases or members of RM systems were found in H. pylori OND79. Our mutants fell into three categories; 1, those with little effect on colonization, 2, those where expression of the full-length protein was detrimental, 3, those where both mutations were detrimental.CONCLUSIONS:Our results demonstrated that phase-variable methyltransferases are critical to H. pylori colonization, suggesting that genome methylation and generation of epigenetic diversity is important for colonization and pathogenesis. The third category of mutants suggests that differential genome methylation status of H. pylori cell populations, achieved by the phasevarion, is essential for host adaptation. Studies of phase-variable RM mutants falling in the two other categories, not strictly required for colonization, represent a future perspective to investigate the role of phasevarion in persistence of H. pylori.