Vesicle production relative to bacterial growth. Electron micrographs of the H. pylori strain, CCUG17875, harvested after (A) 24 h; (B) 48 h; and (C) 72 h. Bar length represents 1 µm. Arrow points out vesicles.

Analysis of H. pylori vesicles isolated according to size. Crude H. pylori vesicle preparations were separated on a gradient according to size.
A–C. Electron micrographs of fractions 2, 4 and 9 that represent the differences in vesicle size achieved by separation. Leb-binding adhesins on the surface of the vesicles are visualized with immunogold labelling of Leb–receptor conjugates. Bar length represents 0.2 µm.
D. A bar graph representing the average size of vesicles isolated in fractions 2: 50 nm (n = 207); 4: 94 nm (n = 146); and 9: 108 nm (n = 188). Maximum and minimum ranges for each fraction are represented by the two ends of the vertical lines associated with each bar.
E. A bar graph representing the percentage of Leb-binding vesicles present in each fraction, i.e. fraction 2: 23% (n = 524); 4: 60% (n = 351); and 9: 81% (n = 183). The presented data represent one of three independent sets of experiments.

Identification of major protein components in H. pylori vesicles.
A. Protein extracts of H. pylori CCUG17875 vesicles (fractions 3–9) were separated by both 10% and 15% SDS-PAGE to resolve the protein bands present. Thirty-four of the most predominant bands (labelled to the right of each gel lane) were excised and subjected to nanoflow LC FT-ICR MS/MS. Molecular weights (kDa) are marked to the left of each gel lane.
B. The identified vesicle proteins were sorted into functional COG classes and outer membrane proteins respectively. Dark grey bar represents vesicle proteins in each class and light grey bars represent number of proteins of each class of the total H. pylori proteome. Out of the total H. pylori proteome, 77% of all OM proteins were found in the vesicles; 21% of cellular processes; 21% of metabolism; 14% of information, storage and processing; and 12% of poorly classified/not classified.
C. Comparison of MS I and MS II analyses presented as a Venn diagram. The two data sets are represented proportional to the size of the data sets. Approximately 38% of the proteins identified in the MS I analysis were found in the MS II analysis, whereas 93% of proteins from the MS II analysis were found in the MS I analysis.
D. OM proteins and vesicles were analysed by immunoblot using antibodies specific for the lipoprotein AlpB, the BabA adhesin, the SabA adhesin, the CagA and the HtrA proteins respectively. Signal densities were measured. Signals corresponding to OM were set to 1.0 to correlate the relative expression of signals obtained in vesicles. The ratio of OM versus vesicles were: AlpB: 1:0.7; BabA: 1:0.3; SabA: 1:0.3; CagA: 1:0.9; HtrA: 1:2.0. The immunoblots shown in the figure represent one for each protein of a series of blots.
E. H. pylori CCUG17875 were grown on Brucella blood agar and in Brucella broth. Protein extracts of OM and of vesicles obtained from the two different growth conditions were analysed for their protein expression patterns by SDS-PAGE. Molecular weights (kDa) are labelled on the far left.
F. The same volume from fractions 1–11 of vesicles separated by density were analysed by immunoblotting using antibodies specific for BabA adhesin, SabA adhesin, lipoprotein AlpB, CagA and VacA respectively.

Adherence of H. pylori vesicles to human gastric epithelium. Vesicle adherence to the epithelium of human gastric mucosa via BabA–Leb interactions and SabA–sLex interactions was detected using biotinylated anti-H. pylori antibody and streptavidin-conjugated Cy3. Vesicles isolated from H. pylori strains 17875/Leb (BabA+/SabA−) and 17875/sLex (BabA−/SabA+) were incubated with human gastric tissue sections as follows:
A. 17875/Leb vesicles.
B. 17875/Leb vesicles pre-incubated with Leb receptor conjugate prior to incubation with gastric tissue sections.
C. 17875/sLex vesicles.
D. 17875/sLex vesicles pre-incubated with sLex receptor conjugate prior to incubation with gastric tissue sections. Bar lengths represents 100 µm.

Isolation of H. pylori vesicles according to density. Crude H. pylori vesicle preparations were subjected to flotation and separated using density gradients.
A. The density and protein concentration of each fraction was measured. The densities are depicted in grey and protein concentrations in black.
B. The same volume from each fraction was analysed by SDS-PAGE. Fraction 1 corresponds to the top fraction and has the lowest density (1.135 g ml⁻¹).
C. Electron micrographs of H. pylori vesicles from strain CCUG17875, are shown including: crude fraction, fraction 3, 5 and 7. Bar length represents 0.5 µm.

Identification of major phospholipid components in H. pylori vesicles. H. pylori membranes were fractionated using sucrose density gradient centrifugation without detergents.
A. Equal volumes from the top five fractions were analysed by SDS-PAGE.
B. Fractions were probed in immunoblots for the IM protein ComB10, and the OM proteins BabA, SabA and OipA respectively.
C. Major phospholipids present in whole H. pylori bacterial cells (WC) versus inner membrane (IM, fraction 2), outer membrane (OM, fraction 3) and H. pylori vesicles (ves) were identified using high-resolution NMR. The displayed ³¹P projections of two-dimensional ³¹P, ¹H correlation spectra are scaled for equal intensity of the PE signals. Slight shifts in ³¹P positions are due to variations in solvent composition. The following phospholipids were identified: Phosphatidylglycerol (PG), phosphatidyl ethanolamine (PE), lyso PE (LPE), phosphatidylcholine (PC), lyso PC (LPC) and cardiolipin (CL).

Presence of receptor-binding adhesins on the surface of H. pylori vesicles.
A. Electron microscopy was used to detect the binding of biotinylated Leb–receptor conjugates and streptavidin-conjugated gold particles to BabA adhesins on the surface of H. pylori vesicles isolated from strain 17875/Leb.
B. Only background labelling was observed on vesicles isolated from strain 17875/sLex that is devoid of BabA expression. Bar length in A and B represents 0.5 µm.
C. sLex-binding adhesins were identified on 17875/sLex-derived vesicles using sLex–receptor conjugate and streptavidin-conjugated gold particles.
D. Only background labelling was observed on vesicles isolated from strain 17875/Leb, which lacks sLex-binding activity. Bar length in C and D represents 0.2 µm.
E. Electron micrographs of vesicles incubated with anti-BabA antibodies detected by 10 nm gold particles and sLex receptor conjugate detected by 5 nm gold particles. The larger 10 nm dots correspond to BabA adhesins and the 5 nm dots represent SabA adhesins.
F. A receptor displacement assay according to the Scatchard method was used to measure the affinity constant (K_a) of ¹²⁵I-Leb receptor conjugate to the BabA adhesin in H. pylori whole cells (WC) (K_a = 1.34E + 11) versus vesicles (ves) (K_a = 1.42E + 11) from strain 17875/Leb.

CagA is localized to the surface of H. pylori vesicles. Immunogold-labelling of CagA protein.
A. Electron micrographs of H. pylori strain P12.
B. Electron micrographs of H. pylori strain P12ΔcagA. Bar length in A and B represents 0.5 µm.
C. An enlargement of the indicated area in (A).
D. Vesicles of strain P12 treated with Trypsin (+), NP40 (++) or neither (-) as indicated. Samples were analysed by immunoblot using antibodies against BabA, CagA and VacA respectively.