Figure 1.
H. pylori wecA, waaL and wzk mutant cells display no Lewis antigens.
Fluorescence microscopy of H. pylori G27 was performed using monoclonal antibodies against Lewis antigens. Panels A, C, E, G, I and K show phase contrast images, whereas panels B, D, F, H, J and L show bacteria displaying Ley based on reaction with the anti-Ley antibody. Equivalent results were obtained with the anti-Lex antibody (not shown). Lewis antigens are not present on all wild type H. pylori cells due to phase variable fucosyltransferases. The bars in the lower right corners of the pictures indicate 10 µm. The images are representative for the results obtained from three independent experiments.
Figure 2.
LPS of H. pylori wecA and waaL mutant strains contains no O antigens.
Purified LPS of (1) H. pylori G27 wild type, (2) wecA mutant, (3) wecA complemented and (4) waaL mutant strains were analyzed by (A) silver staining, and Western blotting using (B) monoclonal anti-Lex and (C) monoclonal anti-Ley antibodies. Protein marker standards were included for reference. Unlike the smooth LPS of the wild type and complemented strains, mutants expressed lipid A-core without O antigens. Comparable results were obtained with purified LPS from H. pylori J99 strains (not shown).
Figure 3.
Demonstration of WecAHP activity in E. coli.
Purified LPS from (1) E. coli parental strain W3110 transformed with pMF19, encoding a functional rhamnosyltransferase, (2) E. coli wecA mutant strain CLM37 transformed with pMF19 and empty vector pEXT20 and (3) CLM37 transformed with pMF19 and pIH22 containing wecAHP, were analyzed by (A) silver staining and (B) Western blotting using an antibody recognizing the E. coli O16 antigen. Protein marker standards were included for reference.
Figure 4.
Demonstration of WaaLHP activity in vitro.
Mixtures with purified WaaLHP, H. pylori lipid A-core (acceptor) and C. jejuni UndPP-linked heptasaccharide (LLO; substrate) were incubated overnight. (1) WaaLHP only, (2) substrate only, (3) acceptor only, (4) all components, (5) no WaaLHP, (6) no substrate, (7) no acceptor. Reaction products were detected by (A) silver staining and (B) Western blotting using an antibody reactive against the C. jejuni heptasaccharide (HR6). (C) Western blotting after removal of the substrate by mild acid hydrolysis confirmed the presence of heptasaccharide attached to lipid A-core, as this reaction product is not affected by the acid treatment. Arrows indicate the reaction product. Contaminating E. coli lipid A-core co-purified with WaaLHP (A, lane 1, 4, 6, 7). Note that the E. coli and H. pylori lipid A-cores as well as the C. jejuni LLO present similar electrophoretic mobilities. Protein marker standards were included for reference.
Figure 5.
H. pylori has a flippase able to translocate diverse UndPP-linked glycans.
Activity of the H. pylori translocase was examined with complementation assays in flippase deficient E. coli. (A, B) Glycosylation of the acceptor protein AcrA in the N-glycosylation pathway of C. jejuni was analyzed by Western blotting with (A) anti-AcrA antibody and (B) an antibody (HR6) recognizing the C. jejuni glycan. C. jejuni pglK was (1) functional, (2) mutated, or (3) mutated and complemented by H. pylori wzk. (C, D) Purified LPS of E. coli strains containing (1) a functional flippase Wzx, (2) a wzx mutation or (3) a wzx mutation complemented by wzk, was visualized by silver staining and Western blotting using an antibody recognizing the E. coli O16 antigen. Protein marker standards were included for reference.
Figure 6.
Wzk is the H. pylori O antigen flippase and can be replaced by C. jejuni PglK.
Purified H. pylori LPS was analyzed by (A) silver staining and Western blotting using (B) anti-Lex and (C) anti-Ley monoclonal antibodies. Lane 1 shows smooth LPS extracted from wild type H. pylori G27 cells; lane 2 LPS from the G27 wzk mutant strain not containing O antigens; smooth LPS production is restored in the wzk complemented strain shown in lane 3. Shown in lane 4: the C. jejuni flippase PglK can functionally complement the H. pylori Wzk, and restore a smooth LPS phenotype in the H. pylori G27 wzk mutant. Protein marker standards were included for reference.
Figure 7.
CagA translocation is functional in O antigen negative H. pylori strains.
Human gastric epithelial AGS cells were infected with H. pylori G27 (1) wild type, (2) wecA mutant, (3) wzk mutant, or (4) waaL mutant strains. (5) Non-infected AGS cells served as negative control. AGS membranes were examined for translocation and tyrosine phosphorylation of CagA by Western blotting using (A) anti-CagA antibody and (B) anti-phosphotyrosine (pY) antibody. (C) The host membrane marker h-Met was detected with an anti-h-Met antibody for a loading control. Arrows indicate full length phosphorylated CagA. A protein marker standard was included for reference.
Figure 8.
Novel LPS biosynthesis pathway in H. pylori.
The figure shows a simplified illustration of the H. pylori LPS biosynthesis pathway. The H. pylori O chains are assembled in the cytoplasm onto a polyisoprenoid membrane anchor. The O antigen synthesis is initiated by the UDP-GlcNAc: undecaprenyl-phosphate GlcNAc-1-phosphate transferase WecA. Processive glycosyltransferases alternately add Gal and GlcNAc residues, producing the linear O chain backbone. Fucosyltransferases then attach Fucose residues on selected locations of the O antigen backbone, generating Lewis antigens. The flippase Wzk transfers the O polysaccharide to the periplasm, where it is attached onto the lipid A-core by the action of the O antigen ligase WaaL. The LPS molecule can then be transported to the outer leaflet of the H. pylori outer membrane.