Journal of Bacteriology, August 2004, p . 5182-5185, Vol . 186, No . 15

Evolution of the Helicobacter pylori Vacuolating Cytotoxin in a Human Stomach

Francisco Aviles-Jimenez,¹ Darren P . Letley,¹ Gerardo Gonzalez-Valencia,² Nina Salama,³ Javier Torres,² and John C . Atherton^1*

Wolfson Digestive Diseases Centre and Institute of Infection, Immunity and Inflammation, University of Nottingham, University Hospital, Nottingham NG7 2UH, United Kingdom,¹ Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico,² Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington³

Received 5 March 2004/ Accepted 27 April 2004

 
We describe two subclones of Helicobacter pylori, isolated contemporaneously from a human stomach, which differ markedly in the vacuolating cytotoxin gene, vacA, but whose near identity in sequences outside this locus implies a very recent common origin . The differences are consistent with homologous recombination with DNA from another strain and result in a changed vacA midregion and, importantly, in changed toxicity .

 
The stomach-colonizing bacterium Helicobacter pylori is the main cause of peptic ulceration and gastric cancer, although most colonized people remain asymptomatic . One important virulence factor is the vacuolating cytotoxin, VacA, a secreted pore-forming toxin that causes epithelial cell vacuolation . Virtually all H . pylori strains express VacA, but those expressing active forms are more frequently associated with disease (8) . The basis of differential toxicity is that the toxin gene, vacA, is polymorphic (4) . vacA varies most markedly in its midregion, which encodes the toxin-cell binding domain (4, 23): type m1 VacA binds more extensively to cells and is more closely associated with disease than is type m2 (14, 21, 23) . vacA also varies in its signal region, encoding the signal peptide and the N terminus of the mature toxin (4, 6): type s1 VacA is toxic, but type s2 has a short N-terminal extension on the mature toxin which abolishes vacuolating activity (16, 18) . Strains with all four possible combinations of the vacA signal and midregions have been found, implying previous homologous recombination within vacA between H . pylori strains (4, 17) .

H . pylori exhibits pronounced genetic diversity, and nucleotide sequence comparisons between loci from different strains show that a common source of variation in vacA and other genes is past recombination events between homologous genes from different strains (25) . Indeed, H . pylori exhibits evidence of more frequent recombination events with heterologous strains than any other known bacterial species (25), and mathematical analysis and microarray and nucleotide sequence analysis of strains isolated longitudinally from the same patient imply that this recombination is ongoing (7, 11, 15) . A previous study has shown that otherwise identical isolates in a single stomach differed in the presence of another locus that is important in H . pylori virulence, the cag pathogenicity island, and sequence analysis showed that this difference likely arose through recombination with another strain (13) . A different study, in which H . pylori was reisolated from a series of patients after a period of time, showed recombination in several genes including, in one case, vacA (7) . The recombination in vacA resulted in a stop codon and lack of VacA expression—a rare finding . From these data, it might be predicted that rapid evolution of vacA may occur to rearrange the gene's mosaic structure and result in the different commonly observed signal and midregion combinations (and thus changed toxicity) . However, despite the important implications this occurrence would have for pathogenicity and patient management, it has not previously been demonstrated .

As part of another study looking at the association between H . pylori vacA types and virulence in Mexico, we identified both s1/m1 and s1/m2 vacA isolates from a single stomach . Cocolonization by different H . pylori strains is not rare (12, 24), and it has been shown previously that it is particularly common in Mexico (10, 22) . We and others have repeatedly shown that such strains are easily distinguished by a variety of DNA fingerprinting methods . However, to our surprise, on this occasion the s1/m1 and s1/m2 isolates had identical fingerprints on initial testing, implying a very recent common origin . In the present study, we definitively confirm the near identity of these isolates, show that the sequence of vacA alleles suggests recent homologous recombination with another strain, and show that this has resulted in a marked difference in toxicity between the isolates . If this phenomenon proves to be widespread, it has potentially important implications for H . pylori virulence and for infection management strategies .

In a local ethically approved study, H . pylori was cultured from endoscopic biopsy specimens from the stomach of a 71-year-old Mexican male with duodenal ulceration . DNA was extracted from six single-colony isolates from the gastric antrum and five from the corpus . PCR-based typing, performed as previously described (5) (Table 1), showed that all of the corpus isolates were midregion type m2, but that four antral isolates were m1 and two were m2 . All of the isolates were signal region type s1 .

 
To assess whether isolates with different vacA types represented different strains of H . pylori, we compared the genomic fingerprints of the m1 and m2 isolates by two methods, random amplified polymorphic DNA (RAPD)-PCR and amplified fragment length polymorphism (AFLP) analysis, as previously described (2, 9) (Table 1) . Both methods gave identical fingerprints for all isolates, implying a single clonal origin (Fig . 1) . Isolates from six other Mexican patients all gave obviously different fingerprints by the two methods . To assess more sensitively whether the isolates represented different genomic types, we performed a microarray analysis (11) of these isolates . As expected, and exactly as has been shown previously for single-colony isolates of the same strain from another patient (11), this analysis showed some differences at other loci (data not shown) . However, hierarchical cluster analysis revealed that m1 and m2 isolates were related to each other just as closely as m1 isolates were related to other m1 isolates and m2 isolates were related to other m2 isolates, showing that these were not distinct, separate genomic groupings (Pearson correlation coefficients were 0.81 to 0.82 for m1 versus m2, 0.81 to 0.82 for m1 versus m1, and 0.82 to 0.88 for m2 versus m2) . Finally, we selected one isolate each from vacA types m1 and m2 and PCR amplified and sequenced regions from two unrelated genes, 471 bp of HP0142 (mutY, an adenine glycosylase gene) and 1,051 bp of HP0834 (yphC, a GTPase gene) (26) . In published comparisons, the mean nucleotide substitution rates between alleles from different strains at these loci were 24 and 19%, respectively, with minimum rates of 7 and 6% (1) . For our isolates, mutY homologues were identical and yphC homologues had a single base substitution, confirming the recent clonal origins of our isolates .

 
To analyze differences in vacA between the essentially clonal m1 and m2 isolates, we sequenced the vacA genes of isolates of each type by automated sequencing of overlapping PCR products . Differences were found in two regions, one of 439 bp (with 35 different base pair substitutions) and one of 378 bp (with 48 bp substitutions and a 75-bp insertion) (Fig . 1) . The second was in the midregion, explaining the difference in PCR-based vacA typing . Outside these regions, the vacA sequences were identical, except for a single base substitution in the untranslated mRNA leader (bp 719), again confirming the sequences' near clonality . A partial vacA sequence analysis of one additional isolate each of types m1 and m2 showed complete identity with the originals .

To show that the m1 and m2 vacA sequences were not acquired from elsewhere in the genome, we designed specific PCR primers internal to the differing m1 and m2 midregions . Using the m1-specific primers, as expected, we amplified a product of the predicted size from chromosomal DNA from the m1 isolates but not from the m2 isolates . We found the converse to be true when we used the m2-specific primers . We did not isolate plasmid DNA from isolates despite successful isolation from a control strain with a plasmid . This result implies that the midregion was acquired from another strain either simultaneously or previously colonizing the stomach . Extensive searching, by picking individual colonies from frozen stored biopsy specimens, failed to identify this parent strain, implying either that it had been selected against or that it colonized a nonsampled part of the stomach .

To assess whether the evolution of vacA had resulted in a changed toxin phenotype, we next assessed the vacuolating cytotoxin activity of broth culture supernatants from the vacA m1 and m2 isolates, as previously described (4, 6, 19) . We found that vacA m1 isolates caused extensive vacuolation of the gastric epithelial cell line AGS, whereas m2 isolates caused no vacuolation (Fig . 1); this finding was expected, since other m2 strains do not induce AGS cell vacuolation (18) . For a control, we assessed the vacuolation of RK13 cells which bind both m1 and m2 VacA (23), and these cells were vacuolated with both isolates . We confirmed that different effects on AGS cells were not due to different levels of VacA by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and VacA immunoblotting with culture supernatants . These results, showing a changed phenotype, were as we would predict based on previous mechanistic studies of the vacA sequence-function relationship: the exchange of the vacA m1 midregion for an m2 midregion confers cell line specificity on the vacuolating phenotype of vacA, and the opposite exchange, replacing an m2 midregion with an m1 midregion, removes that cell line specificity (18) . Thus, it seems likely that in the strain we describe here, the recombination in the midregion rather than that in the more-5' region is responsible for the change in toxin phenotype .

We have shown that the VacA toxin can evolve in vivo to alter its toxicity, presumably through recombination with another, unidentified, H . pylori strain . Because only two strains were identified, we cannot be certain which is the daughter, but both acquisition and loss of toxin activity within the stomach have important potential implications for pathogenesis and future clinical management strategies . For example, if H . pylori pathogenicity changes, disease expression may change, conceivably contributing to phenomena such as the waxing and waning of ulcers . One reason for developing typing systems for H . pylori based on virulence determinants such as vacA has been the hope that such strains could be identified and treated before they cause disease (3, 20) . If rapid evolution in vivo as demonstrated here is widespread, such a strategy would be illogical . One challenge now is to assess whether the evolution of virulence determinants such as vacA and cag is a common phenomenon, as would be predicted from the observed extent and pattern of DNA sequence diversity at other loci (1, 7, 25) . That it has been demonstrated by chance in vacA in this study and in cag in a previous study (13) would imply that such evolution is not rare .

Nucleotide sequence accession numbers GenBank accession numbers for new DNA sequence data referred to in this paper are AY663830 to AY663835 .

 
John Atherton is funded by a Senior Clinical Fellowship from the Medical Research Council, London, United Kingdom . Francisco Aviles is funded by a scholarship from the CONACyT Foundation, Mexico City, Mexico .

 
* Corresponding author . Mailing address: Wolfson Digestive Diseases Centre, University Hospital, Nottingham NG7 2UH, United Kingdom . Phone: 44 115 9249924 . Fax: 44 115 9422232 . E-mail: john.atherton@nottingham.ac.uk.

1. Achtman, M., T . Azuma, D . E . Berg, Y . Ito, G . Morelli, Z . J . Pan, S . Suerbaum, S . A . Thompson, A . van der Ende, and L . J . van Doorn. 1999 . Recombination and clonal groupings within Helicobacter pylori from different geographical regions . Mol . Microbiol . 32:459-470.
2. Akopyanz, N., N . O . Bukanov, T . U . Westblom, S . Kresovich, and D . E . Berg. 1992 . DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting . Nucleic Acids Res . 20:5137-5142.
3. Atherton, J . C. 1997 . The clinical relevance of strain types of Helicobacter pylori . Gut 40:701-703.
4. Atherton, J . C., P . Cao, R . M . Peek, Jr., M . K . Tummuru, M . J . Blaser, and T . L . Cover. 1995 . Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori . Association of specific vacA types with cytotoxin production and peptic ulceration . J . Biol . Chem . 270:17771-17777 .
5. Atherton, J . C., T . L . Cover, R . J . Twells, M . R . Morales, C . J . Hawkey, and M . J . Blaser. 1999 . Simple and accurate PCR-based system for typing vacuolating cytotoxin alleles of Helicobacter pylori . J . Clin . Microbiol . 37:2979-2982 .
6. Atherton, J . C., R . M . Peek, Jr., K . T . Tham, T . L . Cover, and M . J . Blaser. 1997 . Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori . Gastroenterology 112:92-99.
7. Falush, D., C . Kraft, N . S . Taylor, P . Correa, J . G . Fox, M . Achtman, and S . Suerbaum. 2001 . Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age . Proc . Natl . Acad . Sci . USA 98:15056-15061 .
8. Figura, N., P . Guglielmetti, A . Rossolini, A . Barberi, G . Cusi, R . A . Musmanno, M . Russi, and S . Quaranta. 1989 . Cytotoxin production by Campylobacter pylori strains isolated from patients with peptic ulcers and from patients with chronic gastritis only . J . Clin . Microbiol . 27:225-226.
9. Gibson, J . R., E . Slater, J . Xerry, D . S . Tompkins, and R . J . Owen. 1998 . Use of an amplified-fragment length polymorphism technique to fingerprint and differentiate isolates of Helicobacter pylori . J . Clin . Microbiol . 36:2580-2585 .
10. Gonzales-Valencia, G., Atherton, J . C., Munoz, O., Dehasa, M., Madrana de la Garza, A., and J . Torres. 2000 . Helicobacter pylori vacA and cagA genotypes in Mexican adults and children . J . Infect . Dis . 182:1450-1454.
11. Israel, D . A., N . Salama, U . Krishna, U . M . Rieger, J . C . Atherton, S . Falkow, and R . M . Peek, Jr. 2001 . Helicobacter pylori genetic diversity within the gastric niche of a single human host . Proc . Natl . Acad . Sci . USA 98:14625-14630 .
12. Jorgensen, M., G . Daskalopoulos, V . Warburton, H . M . Mitchell, and S . L . Hazell. 1996 . Multiple strain colonization and metronidazole resistance in Helicobacter pylori-infected patients: identification from sequential and multiple biopsy specimens . J . Infect . Dis. 174:631-635.
13. Kersulyte, D., H . Chalkauskas, and D . E . Berg. 1999 . Emergence of recombinant strains of Helicobacter pylori during human infection . Mol . Microbiol . 31:31-43.
14. Kidd, M., A . J . Lastovica, J . C . Atherton, and J . A . Louw. 1999 . Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa? Gut 45:499-502 .
15. Kuipers, E . J., D . A . Israel, J . G . Kusters, M . M . Gerrits, J . Weel, A . van Der Ende, R . W . van Der Hulst, H . P . Wirth, J . Hook-Nikanne, S . A . Thompson, and M . J . Blaser. 2000 . Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host . J . Infect . Dis . 181:273-282.
16. Letley, D . P., and J . C . Atherton. 2000 . Natural diversity in the N terminus of the mature vacuolating cytotoxin of Helicobacter pylori determines cytotoxin activity . J . Bacteriol . 182:3278-3280 .
17. Letley, D . P., A . Lastovica, J . A . Louw, C . J . Hawkey, and J . C . Atherton. 1999 . Allelic diversity of the Helicobacter pylori vacuolating cytotoxin gene in South Africa: rarity of the vacA s1a genotype and natural occurrence of an s2/m1 allele . J . Clin . Microbiol . 37:1203-1205 .
18. Letley, D . P., J . L . Rhead, R . J . Twells, B . Dove, and J . C . Atherton. 2003 . Determinants of non-toxicity in the gastric pathogen Helicobacter pylori . J . Biol . Chem . 278:26734-26741 .
19. Leunk, R . D., P . T . Johnson, B . C . David, W . G . Kraft, and D . R . Morgan. 1988 . Cytotoxic activity in broth-culture filtrates of Campylobacter pylori . J . Med . Microbiol . 26:93-99.
20. Marchetti, M., M . Rossi, V . Giannelli, M . M . Giuliani, M . Pizza, S . Censini, A . Covacci, P . Massari, C . Pagliaccia, R . Manetti, J . L . Telford, G . Douce, G . Dougan, R . Rappuoli, and P . Ghiara. 1998 . Protection against Helicobacter pylori infection in mice by intragastric vaccination with H . pylori antigens is achieved using a non-toxic mutant of E . coli heat-labile enterotoxin (LT) as adjuvant . Vaccine 16:33-37.
21. Miehlke, S., C . Kirsch, K . Agha-Amiri, T . Gunther, N . Lehn, P . Malfertheiner, M . Stolte, G . Ehninger, and E . Bayerdorffer. 2000 . The Helicobacter pylori vacA s1, m1 genotype and cagA is associated with gastric carcinoma in Germany . Int . J . Cancer 87:322-327.
22. Morales-Espinosa, R., G . Castillo-Rojas, G . Gonzalez-Valencia, S . Ponce de León, A . Cravioto, J . C . Atherton, and Y . López-Vidal. 1999 . Colonization of Mexican patients by multiple Helicobacter pylori strains with different vacA and cagA genotypes . J . Clin . Microbiol . 37:3001-3004 .
23. Pagliaccia, C., M . de Bernard, P . Lupetti, X . Ji, D . Burroni, T . L . Cover, E . Papini, R . Rappuoli, J . L . Telford, and J . M . Reyrat. 1998 . The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity . Proc . Natl . Acad . Sci . USA 95:10212-10217 .
24. Prewett, E . J., J . Bickley, R . J . Owen, and R . E . Pounder. 1992 . DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum . Gastroenterology 102:829-833.
25. Suerbaum, S., J . M . Smith, K . Bapumia, G . Morelli, N . H . Smith, E . Kunstmann, I . Dyrek, and M . Achtman. 1998 . Free recombination within Helicobacter pylori . Proc . Natl . Acad . Sci . USA 95:12619-12624 .
26. Tomb, J . F., O . White, A . R . Kerlavage, R . A . Clayton, G . G . Sutton, R . D . Fleischmann, K . A . Ketchum, H . P . Klenk, S . Gill, B . A . Dougherty, K . Nelson, J . Quackenbush, L . Zhou, E . F . Kirkness, S . Peterson, B . Loftus, D . Richardson, R . Dodson, H . G . Khalak, A . Glodek, K . McKenney, L . M . Fitzegerald, N . Lee, M . D . Adams, J . C . Venter, et al. 1997 . The complete genome sequence of the gastric pathogen Helicobacter pylori . Nature 388:539-547.

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