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. 1997 Oct;41(4):463–468. doi: 10.1136/gut.41.4.463

High prevalence of cytotoxin positive Helicobacter pylori in patients unrelated to the presence of peptic ulcers in Japan

K Ogura 1, F Kanai 1, S Maeda 1, H Yoshida 1, M Ogura 1, K Lan 1, K Hirota 1, T Kawabe 1, Y Shiratori 1, M Omata 1
PMCID: PMC1891544  PMID: 9391243

Abstract

Background—It has been reported that infection with vacuolating cytotoxin positive Helicobacter pylori strains is associated with gastroduodenal disease in Western countries. 
Aims—To evaluate the prevalence of cytotoxin producing strains among patients with H pylori infection in relation to gastrointestinal diseases in Japan. 
Patients—Ninety seven patients undergoing endoscopy. 
Methods—A Western blot assay was conducted to detect serum antibodies against the cytotoxin using recombinant cytotoxin (VacA protein) as an antigen. To obtain a purified recombinant cytotoxin, the vacA gene (2233 nucleotides) was cloned into an expression vector to produce the protein (744 amino acids), which was expressed in Escherichia coli. 
Results—Serum IgG antibodies to the cytotoxin were present in 85%, 95%, 95%, and 100% of infected patients with gastric ulcer (n=26), duodenal ulcer (n=21), chronic gastritis (n=19), and endoscopically normal mucosa (n=14), respectively. 
Conclusion—The western blot method using recombinant VacA protein is simple and useful for detecting antibody to vacuolating cytotoxin. This method showed antibodies against cytotoxin were highly prevalent, even in subjects with endoscopically normal mucosa in Japan, indicating that the cytotoxin may not be an independent cause of gastrointestinal diseases induced by H pylori infection. 



Keywords: stomach; Helicobacter pylori; vacA; ulcer; gastritis

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Figure 1 .

Figure 1

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: Schematic representation of plasmid constructs used for sequence analysis of the vacA gene and expression of cytotoxin protein in E coli. P, promoter; 6× His, histidine hexamer tag; LP, leader peptide; OM exp, outer membrane exporter. 


Figure 2 .

Figure 2

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: Alignment of the deduced VacA amino acid sequences between Tox+ strain 43526 (sequenced in this study), Tox+ strain 60190, and Tox- strain Tx30a. The 3873 bp vacA ORF encodes 1291 amino acids. Colons denote identity, and asterisks indicate conserved amino acids. 


Figure 3 .

Figure 3

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: Expression of the vacA gene product in E coli. SDS-PAGE with Coomassie brilliant blue staining shows the recombinant VacA protein with the expected molecular weight of about 80 kDa (arrow). The migration of protein molecular mass standards is shown. 


Figure 4 .

Figure 4

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: Western blotting analysis of VacA in H pylori. Culture supernatants from H pylori strains were probed with control rabbit serum (lanes 1 and 2), rabbit serum raised against the recombinant VacA protein expressed in E coli (lanes 3-4, 6-8), and rabbit serum raised against VA-1 (lane 5). The size variability of VacA protein in Tox+ strain 43526 (lanes 1, 3, and 5) or Tox- Tx30a (lanes 2 and 4) is shown. The position of protein molecular mass standards is indicated. H pylori clinical isolate culture supernatants were also blotted with antirecombinant VacA rabbit sera (lane 6, Tox+; lane 7, Tox+; lane 8, Tox-). 


Figure 5 .

Figure 5

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: Western blotting with human sera or rabbit anti-VA-1 antiserum of the purified recombinant VacA protein. The recombinant VacA proteins were electrophoresed on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with human sera (lane 2, negative; lane 3, positive) or rabbit antiserum raised against VA-1 (lane 4, control; lane 5, immunised by VA-1). Gold staining of the cellulose membrane to which purified recombinant VacA protein was electroblotted is also shown (lane 1). The migration of protein molecular mass standards is indicated. 


Selected References

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