PATIENTS

H pylori strains were isolated from gastric biopsy specimens of 63 H pyloriinfected patients who had undergone endoscopy in Tokyo University Hospital. The patient population consisted of 40 men and 23 women with a mean age of 52.5 (range 22–78). The endoscopic findings were as follows: gastric ulcer (13); duodenal ulcer (12); non-ulcer dyspepsia (22); gastric cancer (16).

H PYLORI CULTURE

Biopsy specimens were cultured on Columbia agar with 5% (v/v) horse blood and Dent antibiotic supplement (Oxoid, Basingstoke, Hants, UK) at 37°C for five days under microaerobic conditions (Campy-Pak Systems; BBL, Cockeysville, Maryland, USA). The organisms were identified as H pylori by Gram stain morphology, colony morphology, and positive urease, catalase, and oxidase activities. The isolates were kept at −80°C in Brucella broth with 5% (v/v) fetal bovine serum (FBS) containing 16% (v/v) glycerol.

DETECTION OF CagA PROTEINS IN H PYLORI ISOLATES

Western blot analysis was performed to detect CagA proteins produced by H pylori strains as previously described.25 CagA protein was investigated usingH pylori whole cell lysates. The whole cell lysates were washed twice in normal saline, centrifuged, and boiled at 100°C for five minutes in Laemmli sample buffer27 before use. The whole cell lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% gel) and transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel Germany). The filter was blocked with buffer containing 5% (v/v) 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 CagA. After being washed, the membrane was incubated with ¹²⁵I-labelled goat anti-rabbit IgG, and then exposed to x ray film.

PREPARATION OF GENOMIC DNA

H pylori bacterial cells, suspended in 10 mM Tris/HCl, pH 7.5, containing 1 mM EDTA, were treated with 0.3% SDS and 0.6 mg/ml proteinase K at 60°C overnight. DNA was extracted with phenol, phenol/chloroform and chloroform, precipitated with ethanol, and suspended in TE. The DNA was stored at −20°C until use.

CLONING OF THE GENES OF THE CAG REGION

DNA was extracted from H pylori ATCC 43579 cells. Oligonucleotide primers were designed to amplify the genes of the cag region;cagA, cagB,cagC, cagE (picB), cagM,cagN, cagQ,cagT, cag15,cag13, cag12,cag10, cag6–7, IS605, and the left end of the cag region (LEC) which contained both inside and outside genes of the cag PAI according to the nucleotide sequences of the cag region previously reported (GenBank accession numbers AC000108 and U60176) (table 1, fig 1). PCR cycling conditions were 30 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for one to two minutes. The PCR products were cloned into pCRII using the original TA cloning kit (Invitrogen, San Diego, California, USA). The nucleotide sequence of the insert was determined by the dideoxy chain termination procedure.28 Cloned plasmids were digested withEcoRI and BstXI, which were able to digest the full length of the PCR products, and the digests electrophoresed on 1% agarose gel. The appropriate fragments were then excised from the gels and the DNA extracted with the gene clean kit to be used as probes (Bio 101, La Jolla, California, USA).

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Figure 1

Map of the cag pathogenicity island. The names of the genes are from GenBank accession number AC000108 and U60176. Cross bars indicate probes used in this study. Fifty nine of 63 strains isolated in Japan all had cagI and cagII. Large deletions were revealed in the remaining four. cagA negative strains isolated in Western countries lacked whole cagI and cagII.

SOUTHERN BLOT ANALYSIS

For each sample, 5 μg of the genomic DNA was digested withHindIII for 12 hours at 37°C, electrophoresed on 1% agarose gel, and transferred to a nylon membrane (Amersham International, Bucks, UK) as described by Sambrooket al.29 The DNA samples were hybridised to previously prepared DNA probes, which were digested from cloned plasmids and labelled with [³²P]dCTP with the Ready To Go DNA labelling kit (Pharmacia Biotech, Uppsala, Sweden). The membrane was prehybridised with ExpressHyb solution (Clontech, Palo Alto, California, USA) at 60°C for one hour, and hybridisation was carried out at 60°C for two hours. The membrane was washed four times with 2 × standard saline citrate (SSC)/0.05% SDS for 10 minutes at room temperature, twice with 0.1 × SSC/0.1% SDS for 20 minutes at 50°C, and then autoradiography was performed using a BAS2000 image analyser (Fuji Photo Film Co, Ltd, Tokyo, Japan).

RNA EXTRACTION AND NORTHERN BLOT ANALYSIS OFcagA TRANSCRIPTS

H pylori bacterial cells were washed with normal saline and suspended in TE. Total RNA was extracted using Rneasy Total RNA kit (Qiagen, Chatsworth, California, USA). For each sample, 10 μg total RNA was electrophoresed on formamide-agarose gel and transferred to a nylon membrane (Amersham International). The hybridisation was performed using the cagAgene as described above, except that the membrane was kept at 68°C instead of 60°C, and washed four times with 2 × SSC/0.05% SDS for 10 minutes at room temperature, twice with 0.1 × SSC/0.1% SDS for 20 minutes at 50°C, and then exposed to x ray film.

SEQUENCE ANALYSIS OF cagANEGATIVE STRAIN

The nucleotide sequence of cagA and the upstream transcript of cagA genes were analysed in one of the cagA negative strains, T-94. Preliminary analysis using 15 primers designed inside the cagA gene revealed that nucleotides from position 910 to the terminal codon were retained. Subsequently, a primer named cagA-20R (5’-TTGGTCTTTATAACCAACGG-3’ corresponding tocagA position 1020–1001) was designed. PCR was performed using cagA-20R and 10 previously described random primers.30 The amplicons were cloned into the pCRII vector using the original TA cloning kit (Invitrogen). The cloned samples were screened by colony hybridisation. The Escherichia coli colonies were transferred to a nylon membrane (Amersham International). The hybridisation was performed using the full length of thecagA gene as probe. Hybridysation and washing were performed as for the Southern blot described above. Plasmid DNA was extracted from positive colonies and the nucleotide sequence of the insert determined by the dideoxy chain termination procedure.28

STIMULATION OF IL-8 SECRETION IN GASTRIC EPITHELIAL CELL LINES

Experiments were performed as previously described.17 ,18 H pyloristrains were harvested in Brucella broth containing 7.5% FBS for 24 hours at 37°C. After centrifugation, they were resuspended at 8 × 10⁷cells/ml in RPMI 1640 containing 10% FBS and used immediately.

MKN-28 cells were routinely maintained in RPMI 1640 supplemented with 10% FBS. Confluent monolayers of MKN-28 cells in 24 well plates were co-cultured with H pylori strains for 16 hours in quadruplicate. Supernatants were then aspirated and stored at −70°C until assayed for IL-8 by enzyme immunoassay (PerSeptive Diagnostics, Framingham, Massachusetts, USA). Concentrations of IL-8 were determined from a standard curve of recombinant IL-8. Bacterial induced IL-8 secretion was expressed as ng/ml after subtraction of background unstimulated control culture values.

STATISTICAL ANALYSIS

Differences in categorised data were analysed with Fisher’s exact probability test, and differences in mean values by analysis of variance. p<0.05 was considered significant.

DETECTION OF CagA PROTEIN

CagA protein was detected by western blot analysis as previously described. Overall, 57 of 63 (90%) strains were positive for CagA protein. The prevalence of CagA protein positive strains was 12/13 for patients with gastric ulcer, 11/12 for those with duodenal ulcer, 18/22 for patients with non-ulcer dyspepsia, and 16/16 for gastric cancer patients.

SOUTHERN BLOT ANALYSIS OFcagA GENE

Southern blot analysis of chromosomal DNA derived from 63H pylori strains cleaved withHindIII revealed that all 63 (100%) strains isolated in Japan were positive for the cagAgene. Six strains that were negative for CagA protein, as determined by western blot analysis, also had the cagAgene. Some restriction fragment length polymorphism patterns were observed, but no relation was found between gastroduodenal disease and these patterns. We also analysed two cagApositive and eight cagA negative strains isolated in the Western countries as controls. Negative strains did not contain cagA gene, as expected.

NORTHERN BLOT ANALYSIS OFcagA TRANSCRIPTS

To determine whether CagA protein expression is inhibited at the transcriptional level, we analysed 17 CagA protein positive strains and six CagA protein negative strains isolated in Japan. All 17 positive strains had cagA transcripts. In the negative strains, two of six had cagAtranscripts, but the remaining four did not. ATCC 43526 and 43579 were used as positive controls, and Tx30a was used as a negative control (fig 2).

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Figure 2

Northern blot analysis of cagA transcripts from seven strains. cagA transcripts were present in T-57 (lane 2), T-1 (lane 5), and ATCC 43526 (lane 7), and absent from T-94 (lane 1), T-85, T-25 (lanes 3 and 4), and Tx30a (lane 6). The membrane was also hybridised to 23S rRNA to monitor the amount of RNA loaded.

SOUTHERN BLOT ANALYSIS OFcagI

The H pylori isolates were analysed for the cagB, cagC,cagE (picB),cagM, cagN, and cagQ genes of thecagI region. Overall, 59/63 (94%) strains had all the cagI genes, but the remaining four (6%) lacked all six cagI genes in spite of the presence of the cagA gene. Surprisingly, four strains that lacked cagI did not have cagA transcripts and CagA protein. In the strains derived from patients with non-ulcer dyspepsia, 4/22 (18%) strains did not have cagI, whereas all 41 strains from patients with peptic ulcer and gastric cancer had cagI. This difference was statistically significant (p<0.05; Fisher’s exact probability test). ATCC 43526 and 43579 had all the genes ofcagI, and eight CagA negative strains including Tx30a lacked cagI.

SOUTHERN BLOT ANALYSIS OFcagII

We analysed cagT,cag15, cag13,cag12, cag10, andcag6–7 of cagII, and LEC. All isolates with allcagI genes (59 strains) were positive for all six cagII genes. All clinical isolates (63 strains) hybridised with cag6–7irrespective of the presence of cagI. In the analysis of cagT andcag15, strains that had cagI all had these genes, but four strains without cagI did not have cagT andcag15 genes. In the analysis ofcag13, cag12, and cag10 genes, strains that hadcagI all hadcag13, cag12, andcag10 genes, in four strains withoutcagI, three strains hadcag13, cag12, andcag10 genes, and one strain did not have these genes. ATCC 43526 and 43579 had all thecagII genes and eight CagA negative Western strains including Tx30a lacked all the cagII genes (fig 1). All 73 strains used in this study hybridised with theLEC.

SOUTHERN BLOT ANALYSIS OF IS 605

Overall, 20/63 (31%) Japanese strains had IS605. The existence of IS605 was not related to the existence ofcag PAI nor to gastroduodenal status in the host. We also investigated IS605 in eight PAI negative strains isolated in Western countries, and three of the eight (38%) were positive (table 2).

JUNCTIONAL SEQUENCES OF THEcag PAI DELETED REGION

Sequence analysis of the T-94 strain revealed that thecagA gene was truncated at position 500, and the cag13 sequence was found directly upstream. About 25 kb of cag PAI including the cagA promoter region was deleted, and no insertional sequences were found in the junctional region (fig3).

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Figure 3

Junctional sequences of T-94, which has cag PAI deleted. cagII was present down to position 14 723 (GenBank accession number AC000108) in the middle of cag13. The cagA gene was present up to position 19 462 (GenBank accession number U60176). This position is equal to position 500 of the cagA gene. An approximately 25 kb segment of cag PAI was deleted. 1) indicates the position of GenBank accession number AC000108. 2) indicates the cagA position of GenBank accession number U60176. Asterisks denote identity.

H PYLORI STIMULATION OF IL-8 SECRETION IN GASTRIC EPITHELIAL CELL LINES

We analysed IL-8 stimulation by 16 strains: 10 clinical isolates and ATCC 43579, which were CagA protein and PAI positive; four clinical isolates (T-25, T-68, T-85, T-94), which were characterised as CagA protein negative with PAI deleted; and thecag PAI negative Tx30a. As shown in fig 4, IL-8 secretion induced by the PAI deleted strains and Tx30a was significantly lower than that induced by ATCC 43579 and the othercag PAI positive isolates (p<0.05; analysis of variance). These results indicate that intactcag PAI is necessary for the stimulation of IL-8 secretion, and the cagA gene is not an appropriate marker of IL-8 secretion.

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Figure 4

Secretion of interleukin-8 (IL-8) from MKN-28 cells over the 16 hours after stimulation. T-77, T-78, T-79, T-81, T-82, T-90, T-100, T-102, T-103, T-108 strains were clinical isolates with an intact cag pathogenicity island (PAI). 43579 indicates ATCC 43579. Tx30a is a strain with cag PAI completely deleted. In T-25, T-68, T-85, and T-94, cag PAI was partially deleted. Results are expressed as the mean and SD from four to six experiments.