BACTERIAL STRAINS AND GROWTH

H pylori strains were isolated from gastric biopsy samples of 68 patients (40 men and 28 women; age range 22–85 years; mean age 52.5) who underwent endoscopy in Tokyo University Hospital. The endoscopic findings were gastric ulcer in 16, duodenal ulcer in 17, non-ulcer dyspepsia in 25, and gastric cancer in 10 patients. Biopsy specimens were cultured on Columbia agar with 5% horse blood and Dent antibiotic supplement (Oxoid, Basingstoke, Hants, UK) at 37 °C for five days under microaerobic conditions. Isolates were identified asH pylori by Gram stain morphology and by positive urease, catalase, and oxidase activities. They were kept at −80°C in brucella broth with 5% fetal bovine serum (FBS) containing 16% glycerol. ATCC43526 cells were used as VacA and CagA positive controls, and strain Tx30a was used as CagA negative control.25 ,26

RECOMBINANT VAcA AND CAgA PROTEINS AND ANTISERA TO RECOMBINANT VAcA AND CAgA

Recombinant VacA25 and CagA26 proteins and the rabbit antiserum to each recombinant protein were prepared as described previously. In brief, the entire vacA gene (GenBank accession number AF001358) and cagA gene (GenBank accession number AF001357) of the ATCC43526 H pyloristrain were cloned, and partial fragments of vacA(encoding amino acids 1, which is the ATG start codon, to 744) andcagA (encoding amino acids 107–531) genes were subcloned in frame with the sequence coding for the histidine tag of pQE vectors (Qiagen, Chatsworth, CA, USA). The histidine-tagged VacA and CagA proteins expressed in Escherichia coli were purified on Ni/nitrilotriacetic acid resin (Qiagen). Antibodies against the recombinant proteins were raised in New Zealand White rabbits.

DETECTION OF VAcA AND CAgA PROTEINS IN H PYLORIISOLATES

Immunoblot analysis was performed to detect VacA and CagA proteins produced by H pylori strains. VacA protein was examined using H pylori surface proteins prepared by the method of Xiang et al.19 In brief, the cultured bacterial cells were washed twice with distilled water, and, to separate the surface protein from bacterial cells, vortex mixed with distilled water for one hour at room temperature. After centrifugation at 10 000 g for ten minutes, the supernatants were stored at −30 °C until use.

CagA protein was examined using whole cell lysates. The whole cell lysates were washed twice in normal saline and centrifuged, and boiled at 100 °C for 5 min in Laemmli sample buffer before use.28 The surface proteins and the whole cell lysates were separated by electrophoresis in sodium dodecyl sulphate/10% polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The filter was blocked with buffer containing 5% skimmed milk in 25 mM Tris/HCl, pH 7.4, containing 150 mM NaCl and 5 mM KCl. The membrane was incubated with a 1:100 dilution of rabbit antiserum against the recombinant VacA or CagA. After being washed, the membrane was incubated with¹²⁵I-labelled goat anti-rabbit immunoglobulin G, and then exposed to x ray film.

VACUOLATING ASSAY

H pylori strains were cultured at 37 °C in brucella broth containing 5% FBS under microaerobic conditions for 48 hours. The culture medium was centrifuged at 10 000 g for 20 min, and cell-free supernatant was sterilised by passage through a 0.2 μm pore size filter and concentrated 20-fold by using a 30 kDa ultrafiltration membrane (Amicon, Lexington, MA, USA), then stored at −80 °C until use for the vacuolating assay. HeLa cells were cultured in plastic flasks in Dulbeccos’ modified Eagle’s medium containing 10% FBS at 37 °C in 5% CO₂ atmosphere. Confluent cell cultures were treated with trypsin and adjusted to a concentration of 1 × 10⁴ cells per ml. At 24 hours before the addition of culture supernatant, 100 μl of cells were seeded into each well of a 96-well microtitre plate. A 10μl sample of concentrated supernatant obtained from the culture medium was incubated with the HeLa cells for 24 hours. The result of the vacuolating assay was defined as positive when vacuolation was visible in more than 50% of HeLa cells by light microscopy.

TYPING OF VAcA GENE BY POLYMERASE CHAIN REACTION (PCR)

The vacA gene encodes a precursor protein made up of amino-terminal signal sequence, the cytotoxin proper, and a membrane protein which is removed during transmembrane transport. Genotyping ofH pylori was proposed on the basis of the nucleotide sequence in the signal sequence (types s1a, s1b, and s2) and that in the cytotoxin region (m1, m2). We amplified the vacA gene by PCR using previously described genotype-specific primers.27 Thermal cycling was at 94 °C for 30 seconds, 50 °C for 15 seconds, and 72 °C for one minute, for a total of 35 cycles. PCR products were analysed by 1% agarose gel electrophoresis with ethidium bromide staining. ATCC43526 was used as a s1a/m1 control, and Tx30a was used as a s2/m2 control.

STATISTICAL ANALYSIS

Fisher’s exact probability test and χ² test were used to test the independence on contingency tables. p<0.05 was considered to be statistically significant.

DETECTION OF VAcA PROTEIN BY IMMUNOBLOT

VacA protein was detected among the cell surface proteins obtained from 59 of 68 (87%) H pylori isolates (fig 1A, tables 1and 2). The positive rate was 14/16 (88%) in the isolates from patients with gastric ulcer, 15/17 (88%) from those with duodenal ulcer, 21/25 (84%) from those with non-ulcer dyspepsia, and 9/10 (90%) from gastric cancer patients. Statistically, the positive rate of VacA protein was not dependent on the gastroduodenal disease of the host (table 2).

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Figure 1

(A) Immunoblot analysis of VacA. Surface proteins obtained from seven H pylori strains were probed with antiserum against the recombinant VacA protein. VacA protein was expressed in T-1 (lane 1), T-5, T-6 (lanes 3 and 4), T-24 and T-39 (lanes 6 and 7), but not in T-48 (lane 2) or T-25 (lane 5). (B) Immunoblot analysis of CagA. Total lysates obtained from seven H pylori strains were probed with antiserum against the recombinant CagA protein. CagA protein was expressed in T-1, T-48, T-5, T-6 (lanes 1–4), T-24 and T-39 (lanes 6 and 7), but not in T-25 (lane 5). Molecular mass is shown in kDa.

The immunoblot analysis using whole cell lysates detected VacA protein even in the isolates that were negative by immunoblot analysis using cell surface proteins, suggesting that VacA protein was produced by all isolates but its secretion was disturbed in some of them.

VACUOLATING ASSAY

The vacuolating assay was positive in 54 of 59 (92%) VacA positive isolates and three of nine (33%) VacA negative ones (tables 1and 2). Although discordant results were found in 8 of 68 (12%) isolates, the correlation between the two assays was strong enough (p<0.001) to assume that most isolates positive for VacA protein by the immunoblot analysis did secrete active cytotoxin.

Five strains were positive for VacA protein by immunoblot but negative for the vacuolating assay. These strains may be secretors of inactive cytotoxin similar to the Tx30a strain. Another three isolates were positive for the vacuolating assay but negative by the immunoblot analysis. The reason for this discrepancy is not known, but it may be that substances other than VacA induced vacuolation.

GENOTYPE OF VAcA GENE

The genotype of the vacA signal sequence was determined in 45 isolates, including six that were negative for VacA by both immunoblot and vacuolating assay. Interestingly, the signal sequence in all isolates was of type s1a, which is supposed to represent high vacuolating activity.27 The genotype of the mid region was also investigated in 45 isolates, as well as type m1 in five (11%) and type m2 in two (4%). PCR failed to amplify the mid region in the remaining 38 (84%) isolates (table3).

DETECTION OF CAgA PROTEIN BY IMMUNOBLOT

The immunoblot analysis detected CagA protein in 61 of 68 (90%)H pylori isolates (fig 1B, tables 1 and 2). The positive rate was 15/16 (94%) in the isolates from patients with gastric ulcer, 15/17 (88%) from those with duodenal ulcer, 21/25 (84%) from those with non-ulcer dyspepsia, and 10/10 (100%) from gastric cancer patients. As in the case of the VacA protein described above, the positive rate of expression of CagA protein was high irrespective of the gastroduodenal disease of the host (table 2).

The estimated molecular mass of the CagA protein visualised by immunoblot varied from 120 to 140 kDa. This may indicate its heterogeneity among H pylori isolates, although we could find no correlation between its molecular mass and the gastroduodenal status of the host.

CORRELATION BETWEEN VAcA AND CAgA

The expression of CagA protein correlated strongly with that of VacA, as determined by immunoblot (p = 0.005), and with vacuolating cytotoxin activity in vitro (p = 0.002). Overall, 54 of 68 (79%) isolates were positive for cytotoxin activity and CagA protein. These isolates are defined as Type I strains in the following text. Type II strains were negative for these assays, and intermediate strains were neither Type I nor Type II.

The prevalence of Type I strains was 14/17 (82%) in the isolates from patients with duodenal ulcer, 13/16 (81%) from those with gastric ulcer, 18/25 (72%) from those with non-ulcer dyspepsia, and 9/10 (90%) from gastric cancer patients. The prevalence did not differ significantly between the groups.

Three isolates were negative for VacA protein, vacuolating activity, and CagA protein (Type II). The prevalence of these all-negative isolates was 3/25 (12%) in patients with non-ulcer dyspepsia and 0/43 (0%) in patients with peptic ulcer or gastric cancer. This difference was statistically significant (p = 0.046).