Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging

Science

Volumn 279, Issue 5349, 1998, Pages 373-377

  Ilver, Dag ^a   Arnqvist, Anna ^a   Ögren, Johan ^a   Frick, Inga Maria ^b   Kersulyte, Dangeruta ^c   Incecik, Engin T ^c,f   Berg, Douglas E ^c   Covacci, Antonello ^d   Engstrand, Lars ^e,g   Borén, Thomas ^a  

^a UMEÅ UNIVERSITY   (Sweden)

^b LUND UNIVERSITY   (Sweden)

^c WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^d Instituto Ricerche Immunobiol Siena   (Italy)

^e UPPSALA UNIVERSITY HOSPITAL   (Sweden)

^f UNIVERSITY OF MUNICH   (Germany)

^g SWEDISH INSTITUTE FOR INFECTIOUS DISEASE CONTROL   (Sweden)

Author keywords

[No Author keywords available]

Indexed keywords

ADHESIN;

ANTIGEN BINDING; ARTICLE; BACTERIUM ADHERENCE; BLOOD GROUP LEWIS SYSTEM; HELICOBACTER PYLORI; PATHOGENICITY; PHENOTYPE; PRIORITY JOURNAL; RECEPTOR BINDING; VIRULENCE;

ADHESINS, BACTERIAL; AMINO ACID SEQUENCE; ANTIGENS, BACTERIAL; BACTERIAL ADHESION; BACTERIAL PROTEINS; BASE COMPOSITION; BASE SEQUENCE; BIOTINYLATION; CELL MEMBRANE; CLONING, MOLECULAR; CODON, INITIATOR; FUCOSE; GASTRIC MUCOSA; GENES, BACTERIAL; GLYCOCONJUGATES; HELICOBACTER PYLORI; HUMANS; LEWIS BLOOD-GROUP SYSTEM; LIGANDS; MOLECULAR SEQUENCE DATA; VIRULENCE;

EID: 15444357984     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5349.373     Document Type: Article

References (41)

1. J. R. Warren, Lancet i, 1273 (1983); B. Marshall, ibid., p. 1273.
2. J. R. Warren, Lancet i, 1273 (1983); B. Marshall, ibid., p. 1273.
3. A. Dubois, Emerg. Infect. Dis. 1, 79 (1995).
4. M. J. Blaser, Sci. Am. 274, 92 (February 1996).
5. R. P. Logan, Lancet 344, 1078 (1994).
6. M. J. Blaser, Trends Microbiol. 1, 255 (1993); D. E. Kirschner and M. J. Blaser, J. Theor. Biol. 176, 281 (1995).
7. M. J. Blaser, Trends Microbiol. 1, 255 (1993); D. E. Kirschner and M. J. Blaser, J. Theor. Biol. 176, 281 (1995).
8. T. Borén, P. Falk, K. A. Roth, G. Larson, S. Normark, Science 262, 1892 (1993); P. Falk et al., Proc. Natl. Acad. Sci. U.S.A. 90, 2035 (1993); P. Falk, T. Borén, S. Normark, Methods Enzymol. 236, 353 (1994).
9. T. Borén, P. Falk, K. A. Roth, G. Larson, S. Normark, Science 262, 1892 (1993); P. Falk et al., Proc. Natl. Acad. Sci. U.S.A. 90, 2035 (1993); P. Falk, T. Borén, S. Normark, Methods Enzymol. 236, 353 (1994).
10. T. Borén, P. Falk, K. A. Roth, G. Larson, S. Normark, Science 262, 1892 (1993); P. Falk et al., Proc. Natl. Acad. Sci. U.S.A. 90, 2035 (1993); P. Falk, T. Borén, S. Normark, Methods Enzymol. 236, 353 (1994).
11. b antigen-binding activity.
12. 125I-labeled conjugate (that is, an excess of receptor substrate) for 30 min in phosphate-buffered saline containing 0.5% albumin and 0.05% Tween-20. After centrifugation, the radioactivity bound to the bacterial pellet was measured with a gamma counter. Binding experiments were reproducible and performed in triplicate. In addition, the extent of BAB activity of each strain was stable.
13. 125I-labeled conjugate (that is, an excess of receptor substrate) for 30 min in phosphate-buffered saline containing 0.5% albumin and 0.05% Tween-20. After centrifugation, the radioactivity bound to the bacterial pellet was measured with a gamma counter. Binding experiments were reproducible and performed in triplicate. In addition, the extent of BAB activity of each strain was stable.
14. A. Covacci et al., Proc. Natl. Acad. Sci. U.S.A. 90, 5791 (1993); M. Marchetti et al., Science 267, 1655 (1995).
15. A. Covacci et al., Proc. Natl. Acad. Sci. U.S.A. 90, 5791 (1993); M. Marchetti et al., Science 267, 1655 (1995).
16. Z. Xiang et al., Infect. Immun. 63, 94 (1995).
17. Strains were analyzed for the presence of cagA as described [M. K. Tummuru, T. L. Cover, M. J. Blaser, ibid. 61, 1799 (1993)].
18. S. Censini et al., Proc. Natl. Acad. Sci. U.S.A. 93, 14648 (1996).
19. For the total cag PAI deletion, polymerase chain reaction (PCR) products from both ends of the cag PAI [generated with primers 2F and 4R (0.4 kb), and 24F and 25R (0.6 Kb)] were cloned in pBluescript SK (Stratagene, La Jolla, CA). The camR gene [Y. Wang and D. E. Taylor, Gene 94, 23 (1990)] was ligated between the fragments, and the product was used to transform H. pylori strain P119 (37). The cag PAI deletion status of the transformants was verified by their failure either to hybridize with any of several cag region probes or to yield PCR products with primers specific for internal regions of the cag PAI. The sequences of primers 2F and 4R, derived from cosmid 36 (24), and of 24F and 25R (9) are as follows: 2F, 5′-ACATTTTGGCTAAATAAACGCTG; 4R, 5′-TCTCCATGTTGCCATTATGCT; 24F, 5′-GGAATTATCACACCTTATAATGCCC; and 25R, 5′-TCATGCGAGCGGCGATGTG.
20. Confocal microscopy was performed with a Nikon/ Multiprobe 2001 instrument (Molecular Dynamics, Sunnyvale, CA). Electron microscopy was performed with a JEOL 100 CX instrument.
21. A. G. Scatchard, Ann. N.Y. Acad. Sci. 51, 600 (1949).
22. O. Mol and B. Oudega, FEMS Microbiol. Rev. 19, 25 (1996).
23. Cell extracts were solubilized in SDS sample buffer (without mercaptoethanol) at 37°C for 10 min and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated overnight with biotinylated Leb glycoconjugate (1 μg/ml) or biotinylated albumin (negative control), prepared as previously described (6). After washing, the biotinylated structures bound by the BabA protein band were visualized with horseradish peroxidase-streptavidin and ECL reagents (Amersham).
24. J.-F. Tomb et al., Nature 388, 539 (1997).
25. J. Brunner, Trends Cell Biol. 6, 154 (1996); J. D. Bleil and P. M. Wassarman, Proc. Natl. Acad. Sci. U.S.A. 87, 5563 (1990).
26. J. Brunner, Trends Cell Biol. 6, 154 (1996); J. D. Bleil and P. M. Wassarman, Proc. Natl. Acad. Sci. U.S.A. 87, 5563 (1990).
27. Bacteria were incubated with Leb glycoconjugate to which the Sulfo-SBED cross-linker (Pierce, Rockville, IL) had been attached by N-hydroxysuccinimide ester (NHS). The photoreactive aryl azide cross-linker group was activated by ultraviolet irradiation. Bacteria were then washed with phosphate-buffered saline (pH 7.6) containing 0.05% Tween-20, protease inhibitors (EDTA and benzamidine), and 50 mM dithiothreitol (DTT). Bacterial proteins were separated by SDS-PAGE and transferred to a PVDF membrane, after which the biotin-tagged BabA protein was detected with peroxidase-streptavidin and ECL reagents (Fig. 2E).
28. 2-terminal sequencing (41 amino acids) with a Precise 494 instrument (Applied Biosystems, Foster City, CA). The biotin-tagged BabA adhesin was purified >3000-fold from the cell extract, and the yield was ∼20%. The results of the Scatchard analysis (Fig. 1D) indicate that the level of BabA would be about five times higher compared to the resulting biotin-tagged BabA adhesin. The Retagging efficiency is dependent on the combined affinity and specificity in the receptor-ligand interaction and, in addition, the steric availability of the cross-linker structure. However, in our hands, a nonsaturated efficiency results in low background of unspecific biotin-tagging (Fig. 2E).
29. 2-terminal sequence of BabA was used to design degenerate oligonucleotides for PCR amplification of a 59-bp fragment from the CCUG17875 chromosome. This 59-bp fragment was used to probe a low-copy number plasmid (pACYC184) library of CCUG17875 chromosomal DNA partially digested with Sau 3A.
30. The integrity and identity of the COOH-terminal domain was verified by COOH-terminal peptide sequencing. The BabA band (85 pmol) (27) was excised, and the last five amino acid residues were determined to be YV(F)A-, compared with YVFAY predicted from the gene sequence, with a Procise 494C instrument (Applied Biosystems). [(F), weak signal; -, amino acid not identified, probably because of derivatization during analysis]
31. b glycoconjugate, and the location of the camR gene was analyzed by PCR with the upstream primers F2 (babA2) or F44 (babA1) in combination with primer R11. The sequences of the primers are as follows: F2, 5′-CTTAAATATCTCCCTATCCC; R39, 5′-CAAATACACGCTATAGAGCC; R41, 5′-GCGAGCCTAAAGTTAATGA; F38, 5′-ACGTGGCGAACTTCCAATTC; F44, 5′-CAGTCAAGCCCAAAGCTATGC; and R11, 5′-CGATTTGATAGCCTACGCTTGTG.
32. T. Borén et al., in preparation.
33. Pulsed-field gel mapping was performed as described [Q. Jiang, K. Hiratsuka, D. E. Taylor, Mol. Microbiol. 20, 833 (1996)]. The locations of the babA and babB genes were analyzed with PCR-generated probes. PCR was performed with the forward primers F43 (babA) or F15 (babB) in combination with primers R29 and R28, respectively. The sequences of the primers are as follows: F43, 5′-CTTGAGCAAACTTTTGACCCGAT; F15, 5′-TGGGCCTATATCCACTGCAA; R29, 5′-AGTGCCAGCTGTTGATTTGTTGTTGCATGTGG; and R28, 5′-TACGCTCACCCCCTTGCTCTTCATAACACA.
34. P. Hagblom, E. Segal, E. Billyard, M. So, Nature 315, 156 (1985); R. Haas and T. F. Meyer, Cell 44, 107 (1986).
35. P. Hagblom, E. Segal, E. Billyard, M. So, Nature 315, 156 (1985); R. Haas and T. F. Meyer, Cell 44, 107 (1986).
36. S. Langermann et al., Science 276, 607 (1997); discussed in T. Borén and P. Falk, Sci. Am. Sci. Med. 1, 28 (April 1994); L. S. Tompkins and S. Falkow, Science 267, 1621 (1995).
37. S. Langermann et al., Science 276, 607 (1997); discussed in T. Borén and P. Falk, Sci. Am. Sci. Med. 1, 28 (April 1994); L. S. Tompkins and S. Falkow, Science 267, 1621 (1995).
38. S. Langermann et al., Science 276, 607 (1997); discussed in T. Borén and P. Falk, Sci. Am. Sci. Med. 1, 28 (April 1994); L. S. Tompkins and S. Falkow, Science 267, 1621 (1995).
39. M. J. Blaser, Lancet 349, 1020 (1997).
40. H. Clausen and S. Hakomi, Vox Sang., 56, 1 (1989).
41. 2-terminal sequencing; J. Van Beeumen and B. Samyn for COOH-terminal sequencing; R. Rosqvist for assistance with confocal microscopy; L. Johansson for assistance with electron microscopy; M. Block for image processing; R. Rappual for suggestions; Z. Xiang and S. Guidotti for strains; and D. L. Milton, J. Carlsson, B.-E. Uhlin, and P. Falk for critical reading of the manuscript. Supported by the Swedish Society of Medicine, Lion's Cancer Research Foundation, Umeå University, the Magnus Bergvall Foundation (T.B.), the Swedish Medical Research Council [grants 11218 (T.B.), 10848 (L.E.), and 7480 (L. Björck)], the Swedish Society for Medical Research (T.B. and D.I.), the Royal Swedish Academy of Sciences, the J. C. Kempe Memorial Foundation (D.I.), the Umeå University-Washington University Scientific Exchange Program (T.B. and J.Ö.), and grants from the NIH and American Cancer Society (D.E.B.) and from Chiron Co. (A.C.).