Lipopolysaccharide O-antigen profiles of Helicobacter pylori strains from Southwest China.

BACKGROUND: Helicobacter pylori lipopolysaccharide (LPS) structures vary among strains of different geographic origin. The aim of this study was to characterize the LPS O-antigen profiles of H. pylori strains isolated from Southwest China, and to further analyze the association of Lewis antigen expression with clinical outcomes and antibiotic resistance. RESULTS: A total of 71 H. pylori isolates from Southwest China were included for LPS profiling by silver staining and Western blotting after SDS-PAGE electrophoresis. We demonstrated that all the clinical isolates had the conserved lipid A and core-oligosaccharide, whereas the O-antigen domains varied significantly among the isolates. Compared with the common presence of the glucan/heptan moiety in LPS O-antigen structure of European strains, the clinical isolates in this study appeared to lack the glucan/heptan moiety. The expression frequency of Lex, Ley, Lea, and Leb was 66.2% (47/71), 84.5% (60/71), 56.3% (40/71), and 31.0% (22/71), respectively. In total, the expression of type II Lex and/or Ley was observed in 69 (97.2%) isolates, while type I Lea and/or Leb were expressed in 49 (69.0%) isolates. No association of Lewis antigen expression with clinical outcomes or with antibiotic resistance was observed. CONCLUSIONS: H. pylori strains from Southwest China tend to produce heptan-deficient LPS and are more likely to express type I Lewis antigens as compared with Western strains. This may suggest that H. pylori evolves to change its LPS structure for adaptation to different hosts.

Association of serum pepsinogens and gastrin-17 with Helicobacter pylori infection assessed by urea breath test.

Background: Association of gastric atrophy or cancer with levels of serum pepsinogens, gastrin-17 and anti-Helicobacter pylori IgG antibody have been extensively studied. However, the association of serum pepsinogen and gastrin-17 with H. pylori infection has not been studied in a large population. Aim: To investigate the impact of H. pylori infection on serum levels of pepsinogens and gastrin-17. Methods: A total of 354, 972 subjects who underwent health check-ups were included. Serum levels of pepsinogens and gastrin-17 were measured using the enzyme-linked immunosorbent assay. H. pylori infection was detected using 14C-urea breath test (UBT). Multivariable logistic regression analysis was used to investigate the association of serum pepsinogen and gastrin-17 with H. pylori infection. Results: H. pylori prevalence was 33.18% in this study. The mean levels of pepsinogens and gastrin-17 were higher, while the mean pepsinogen-I/II ratio were lower among H. pylori-positive than -negative subjects. In H. pylori-positive subjects, pepsinogen and gastrin-17 levels correlated positively, whereas the pepsinogen-I/II ratio correlated negatively with UBT values (e.g., the mean serum level of pepsinogen-I in subjects with UBT values in the range of 100-499dpm, 500-1499dpm, and ≥1500dpm was 94.77 ± 38.99, 102.77 ± 43.59, and 111.53 ± 47.47 ng/mL, respectively). Compared with H. pylori-negative subjects, the adjusted odds ratio (aOR) of having pepsinogen-I ≤ 70 ng/mL in the three H. pylori-positive but with different UBT value groups was 0.31 (p<0.001), 0.16 (p<0.001), and 0.08 (p<0.001), respectively; while the aOR of having G-17>5.70 pmol/L was 4.56 (p<0.001), 7.43 (p<0.001), and 7.12 (p<0.001). This suggested that H. pylori-positive subjects with higher UBT values were less likely to have pepsinogen-I ≤70 ng/mL (a serum marker for gastric atrophy), but more likely to have gastrin-17 >5.70 pmol/L (a marker for peptic ulcer). Conclusions: H. pylori-positive subjects with higher UBT values are unlikely to have gastric atrophy, but may have greater risk of severe gastritis or peptic ulcers. Our study suggests that H. pylori-positive patients with high UBT values may benefit the most from H. pylori eradication.

Primary antibiotic resistance of Helicobacter pylori among a Chinese Tibetan population.

Aim: To evaluate the primary antibiotic resistance in Helicobacter pylori strains isolated from a Chinese Tibetan population. Methods & materials: Gastric biopsies from 400 H. pylori treatment-naive Tibetan patients were collected for H. pylori isolation. Susceptibility to amoxicillin (AML)/clarithromycin (CLR)/levofloxacin (LEV)/metronidazole (MTZ)/tetracycline (TET)/rifampicin (RIF)/furazolidone (FZD) was determined by E-test or a disk diffusion assay. Results: Biopsies from 117 patients were H. pylori culture positive (29.3%). The primary resistance rates to MTZ, CLR, LEV, RIF, AML, TET and FZD were 90.6, 44.4, 28.2, 69.2, 7.7, 0.8 and 0.8%, respectively. Interestingly, 42.7% of the strains had simultaneous resistance to CLR and MTZ. Conclusion: Among Tibetan strains, primary resistance rates were high for CLR/MTZ/LEV, whereas primary resistance rates to AML/TET/FZD were low. The high resistance to RIF is a concerning finding.

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

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.

Insights from the redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

H. pylori is a Gram-negative extracellular bacterium, first discovered by the Australian physicians Barry Marshall and Robin Warren in 1982, that colonises the human stomach mucosa. It is the leading cause of peptic ulcer and commonly infects humans worldwide with prevalence as high as 90% in some countries. H. pylori infection usually results in asymptomatic chronic gastritis, however 10-15% of cases develop duodenal or gastric ulcers and 1-3% develop stomach cancer. Infection is generally acquired during childhood and persists for life in the absence of antibiotic treatment. H. pylori has had a long period of co-evolution with humans, going back to human migration out of Africa. This prolonged relationship is likely to have shaped the overall host-pathogen interactions and repertoire of virulence strategies which H. pylori employs to establish robust colonisation, escape immune responses and persist in the gastric niche. In this regard, H. pylori lipopolysaccharide (LPS) is a key surface determinant in establishing colonisation and persistence via host mimicry and resistance to cationic antimicrobial peptides. Thus, elucidation of the H. pylori LPS structure and corresponding biosynthetic pathway represents an important step towards better understanding of H. pylori pathogenesis and the development of novel therapeutic interventions.

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

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.

Xer recombinase and genome integrity in Helicobacter pylori, a pathogen without topoisomerase IV.

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.

The secreted Helicobacter cysteine-rich protein A causes adherence of human monocytes and differentiation into a macrophage-like phenotype.

Helicobacter pylori genomes typically contain 8 or 9 genes that code for secreted and highly disulfide-bridged proteins designated Helicobacter cysteine-rich proteins (Hcp). Here we show that HcpA (hp0211) but not HcpC (hp1098) triggers the differentiation of human myeloid Thp1 monocytes into macrophages. Small amounts of HcpA cause the transition of round-shaped monocytes into cells with star-like morphologies, adherence to the culture dish surface, phagocytosis of opsonized fluorescent microspheres, and expression of the surface marker protein CD11b, all of which are indicative of a macrophage-like phenotype. We conclude that HcpA acts as a bacterial immune modulator similar to a eukaryotic cytokine.

SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast.

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Delta cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Delta and slc4Delta cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Delta and slc4Delta cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Delta and slc4Delta cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.

Phg2, a kinase involved in adhesion and focal site modeling in Dictyostelium.

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.