{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T05:41:17Z","timestamp":1792734077142,"version":"4.5.0"},"reference-count":28,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5457","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,2,25]]},"abstract":"<jats:p>\n                    The Gram-negative bacterium\n                    <jats:italic>Helicobacter pylori<\/jats:italic>\n                    is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (\n                    <jats:italic>cagA<\/jats:italic>\n                    <jats:sup>+<\/jats:sup>\n                    ) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the\n                    <jats:italic>cag<\/jats:italic>\n                    pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic\n                    <jats:italic>Helicobacter<\/jats:italic>\n                    infection with yet unknown consequences.\n                  <\/jats:p>","DOI":"10.1126\/science.287.5457.1497","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:48:21Z","timestamp":1027748901000},"page":"1497-1500","source":"Crossref","is-referenced-by-count":1092,"title":["Translocation of\n                    <i>Helicobacter pylori<\/i>\n                    CagA into Gastric Epithelial Cells by Type IV Secretion"],"prefix":"10.1126","volume":"287","author":[{"given":"Stefan","family":"Odenbreit","sequence":"first","affiliation":[{"name":"Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ju\u0308rgen","family":"Pu\u0308ls","sequence":"additional","affiliation":[{"name":"Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bettina","family":"Sedlmaier","sequence":"additional","affiliation":[{"name":"Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elke","family":"Gerland","sequence":"additional","affiliation":[{"name":"Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wolfgang","family":"Fischer","sequence":"additional","affiliation":[{"name":"Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rainer","family":"Haas","sequence":"additional","affiliation":[{"name":"Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","unstructured":"M. J. Blaser Sci. Am. 274 104 (February 1996).","DOI":"10.1038\/scientificamerican0296-104"},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","unstructured":"Z. Y. Xiang et al. Infect. Immun. 63 94 (1995).","DOI":"10.1128\/iai.63.1.94-98.1995"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1073\/pnas.93.3.1259","volume":"93","author":"Segal E. D.","year":"1996","unstructured":"Segal E. D., Falkow S., Tompkins L. S., Proc. Natl. Acad. Sci. U.S.A. 93, 1259 (1996).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","unstructured":"S. Censini et al. Proc. Natl. Acad. Sci. U.S.A. 93 14648 (1996).","DOI":"10.1073\/pnas.93.25.14648"},{"key":"e_1_3_1_6_2","doi-asserted-by":"crossref","first-page":"7595","DOI":"10.1073\/pnas.94.14.7595","volume":"94","author":"Segal E. D.","year":"1997","unstructured":"Segal E. D., Lange C., Covacci A., Tompkins L. S., Falkow S., Proc. Natl. Acad. Sci. U.S.A. 94, 7595 (1997).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.284.5418.1328"},{"key":"e_1_3_1_8_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.273.5278.1107"},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1016\/0966-842X(96)81513-7","volume":"4","author":"Winans S. C.","year":"1996","unstructured":"Winans S. C., Burns D. L., Christie P. J., Trends. Microbiol. 4, 64 (1996).","journal-title":"Trends. Microbiol."},{"key":"e_1_3_1_10_2","unstructured":"Bacterial strains P1 P2 P3 P12 have been described (22). Strains 26695 J99 ATCC43526 and NCTC11637 are reference strains obtained from the American Type Culture Collection (ATCC). Human gastric cell lines AGS (ATCC CRL 1739) KatoIII (ATCC HTB103) St 3051 or nongastric epithelial cell lines ME180 (ATCC HTB 33) HeLa (ATCC CCL 2) or Chang (ATCC CCL 20.2) were grown in RPMI 1640 medium (Gibco) containing 10% fetal calf serum and l -glutamine in a water-saturated atmosphere at 37\u00b0C and 5% CO 2 ."},{"key":"e_1_3_1_11_2","unstructured":"For infection experiments cells were grown in a preconfluent monolayer (5 \u00d7 10 5 cells) and infected with bacteria at a multiplicity of infection (MOI) of 100 for 4 hours at 37\u00b0C and 5% CO 2 . Nonadherent bacteria were removed by washing five times with PBS* [phosphate-buffered saline (PBS) 1 mM CaCl 2 0.5 mM MgCl 2 ]. Cells were suspended in 1 ml of ice-cold PBS* (PBS 1 mM EDTA 1 mM orthovanadate 1 mM phenylmethylsulfonyl fluoride (PMSF) 1 \u03bcM leupeptin 1 \u03bcM pepstatin) (cell scraper) collected by centrifugation and resuspended in 30 \u03bcl PBS*. For kinase inhibition studies the cell culture medium was supplemented with 250 \u03bcM genistein or 1 \u03bcM staurosporine 1 hour before and during infection. For dephosphorylation cells were collected in 30 \u03bcl of PBS 0.1% Triton X-100 and incubated with 50 U of YopH phosphatase (NEB) for 1 hour at 30\u00b0C. For SDS\u2013polyacrylamide gel electrophoresis 40 \u03bcl of a twofold sample solution were added before boiling (5 min). Proteins were separated on a 6% polyacrylamide gel blotted on a polyvinylidene difluoride membrane and examined for CagA (AK257 protein A\/AP) or tyrosine phosphorylation (PY99 Santa Cruz Biotechnology; anti-mouse immunoglobulin G (IgG)\u2013horseradish peroxidase (HRP). The HRP-conjugated antibody was visualized by enhanced chemiluminescence (Renaissance NEN). For immunofluorescence studies AGS cells were grown to a preconfluent state and infected with Hp at a MOI of 100 for 3 hours. After removal of nonadherent bacteria cells were fixed in 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100. Hp was detected with AK175 (13) and fluorescein isothiocyanate\u2013conjugated anti-rabbit IgG. The FLAG-tagged CagA was visualized with the FLAG-specific mAb M2 and TRITC-coupled anti-mouse IgG."},{"key":"e_1_3_1_12_2","unstructured":"Plasmid pWS30 (22) was used for inactivation of cag A. For construction of a cag E \u2212 insertion mutant the Hp 26695 chromosomal region (position 574639 to 579587) was amplified by polymerase chain reaction (PCR). The PCR fragment was cloned in pMin1 mutated by a Tn Max transposon derivative as described ["},{"key":"e_1_3_1_12_3","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/0378-1119(95)00671-0","volume":"167","author":"Kahrs A. F.","year":"1995","unstructured":"Kahrs A. F., et al., Gene 167, 53 (1995);","journal-title":"Gene"},{"key":"e_1_3_1_12_4","unstructured":"] and used for transformation of strain P12 resulting in the cag E \u2212 mutant. For construction of P12\u0394 cag A region 579021 \u2013to 579905 ( cag A-upstream) and 583483 to 584934 ( cag A-downstream) of the Hp 26695 genome were amplified by PCR with 18-nucleotide flanking sequences as primers. Fragments were cloned into pBluescript Xho I\u2013Bam HI sites separated by an aph A-3 gene cassette. For complementation the cag A gene of 26695 was amplified by PCR and cloned into the pHel2 vector (Xho I\u2013Bam HI) (15). The FLAG-tag (Sigma) sequence was fused to the cag A-downstream primer (5\u2032-CGGGATCCTTACTTGTCATCGTCGTCCTTGTAGTCAGATTTTTGGAAACCACCTT-3). The 18-nucleotide cag A upstream primer starts at position 579701 (19). Transformation of Hp strains was performed as described ["},{"key":"e_1_3_1_12_5","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1111\/j.1365-2958.1993.tb01618.x","volume":"8","author":"Haas R.","year":"1993","unstructured":"Haas R., et al., Mol. Microbiol. 8, 753 (1993)].","journal-title":"Mol. Microbiol."},{"key":"e_1_3_1_13_2","unstructured":"AGS cells (1 \u00d7 10 7 ) were infected with Hp P12 incubated for 4 hours (37\u00b0C 5% CO 2 ) washed 5\u00d7 with PBS* and lysed in 500 \u03bcl of modified RIPA buffer [50 mM tris-HCl (pH 7.4) 150 mM NaCl 1 mM EDTA 1% NP-40 0.25% Na-deoxycholate 1 mM PMSF 1 mM orthovanadate 1 \u03bcM leupeptin 1 \u03bcM pepstatin] (1 hour 4\u00b0C gentle shaking). The lysate was cleared (15 000 rpm 4\u00b0C 10 min) and the protein content adjusted to 3 mg\/ml with PBS*. AK257 (1.7 \u03bcg\/ml) was added to a total volume of 900 \u03bcl; after 1 hour at 4\u00b0C 40 \u03bcl of Protein G\u2013agarose (Roche) was added and the mixture incubated for a further 2 hours at 4\u00b0C. After a short centrifugation step (10 s 6.000 g ) the supernatant was discarded and beads were washed four times with 1 ml of PBS* and suspended in 40 \u03bcl of sample solution."},{"key":"e_1_3_1_14_2","unstructured":"The COOH-terminal part of CagA was produced as a fusion protein with MS2-polymerase and an NH 2 -terminal His 6 -tag by using the Escherichia coli expression vector pEV40 [J. Pohlner et al. Gene 130 121 (1993)]. The fusion protein was expressed and purified by Ni 2+ \u2013nitrilotriacetic acid affinity chromatography by standard procedures and used to immunize a rabbit to obtain antiserum AK257. AK175 was generated by immunization of a rabbit with whole heat-killed Hp bacteria. Immunoblotting was performed and alkaline phosphatase (AP)\u2013coupled protein A was used to visualize the antibody bound by decomposition of nitro blue tetrazolium."},{"key":"e_1_3_1_15_2","unstructured":"After standard infection of AGS cells with Hp strains (10) cells were washed three times with PBS + harvested with a cell scraper and treated with PBS* containing 0.05% (w\/v) saponin for 10 min at room temperature. Cellular debris and bacteria were separated from the soluble material by centrifugation (5 min 6000 g ) and 0.2-\u03bcm pore diameter filtration of the supernatant. Pellets were resuspended in 30 \u03bcl of sample solution and supernatants were precipitated with chloroform-methanol as described ["},{"key":"e_1_3_1_15_3","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/0003-2697(84)90782-6","volume":"138","author":"Wessel D.","year":"1984","unstructured":"Wessel D., Fl\u00fcgge U. I., Anal. Biochem. 138, 141 (1984);","journal-title":"Anal. Biochem."},{"key":"e_1_3_1_15_4","unstructured":"] and resuspended in 30 \u03bcl of sample solution. Equal amounts (8 \u03bcl) of probes were loaded on 6 or 10% polyacrylamide gels."},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1007\/s004380050677","volume":"257","author":"Heuermann D.","year":"1998","unstructured":"Heuermann D., Haas R., Mol. Gen. Genet. 257, 519 (1998).","journal-title":"Mol. Gen. Genet."},{"key":"e_1_3_1_17_2","unstructured":"Available at www.motif.genome.ad.jp\/."},{"key":"e_1_3_1_18_2","unstructured":"The sequence of the P12 cagA gene was obtained by PCR amplification of the complete gene fragment from corresponding chromosomal DNA and sequencing of the cloned genes by primer walking. ATCC43526 CagA (GenBank accession number 2073135) CagA sequences of J99 (18) and 26695 (19)."},{"key":"e_1_3_1_19_2","unstructured":"R. A. Alm et al. Nature 397 176 (1999)."},{"key":"e_1_3_1_20_2","unstructured":"J.-F. Tomb et al. Nature 388 539 (1997)."},{"key":"e_1_3_1_21_2","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1038\/361730a0","volume":"361","author":"Galyov E. E.","year":"1993","unstructured":"Galyov E. E., H\u00e5kansson S., Forsberg A., Wolf-Watz H., Nature 361, 730 (1993).","journal-title":"Nature"},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","unstructured":"O. Ilan et al. EMBO J. 18 3241 (1999).","DOI":"10.1093\/emboj\/18.12.3241"},{"key":"e_1_3_1_23_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1111\/j.1365-2958.1994.tb01019.x","volume":"12","author":"Schmitt W.","year":"1994","unstructured":"Schmitt W., Haas R., Mol. Microbiol. 12, 307 (1994).","journal-title":"Mol. Microbiol."},{"key":"e_1_3_1_24_2","unstructured":"We thank W.-D. Hardt M. Aepfelbacher and J. Heesemann for constructive comments on the manuscript A. Covacci for the gift of Hp strain G27 and K. Melchers for Hp strain ATCC43526. Supported by grants from the Deutsche Forschungsgemeinschaft to R.H. (HA 2697\/2-1)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.287.5457.1497","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:17:08Z","timestamp":1705123028000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.287.5457.1497"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,2,25]]},"references-count":28,"journal-issue":{"issue":"5457","published-print":{"date-parts":[[2000,2,25]]}},"alternative-id":["10.1126\/science.287.5457.1497"],"URL":"https:\/\/doi.org\/10.1126\/science.287.5457.1497","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,2,25]]}}}