Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion

Science

Volumn 287, Issue 5457, 2000, Pages 1497-1500

  Odenbreit, Stefan ^a   Püls, Jürgen ^a   Sedlmaier, Bettina ^a   Gerland, Elke ^a   Fischer, Wolfgang ^a   Haas, Rainer ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN;

ARTICLE; EPITHELIUM CELL; GASTRITIS; HELICOBACTER PYLORI; HOST CELL; MUCOSA ASSOCIATED LYMPHOID TISSUE LYMPHOMA; NONHUMAN; PEPTIC ULCER; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; PROTEIN SECRETION; PROTEIN TRANSPORT; STOMACH ADENOCARCINOMA; STOMACH EPITHELIUM;

AMINO ACID MOTIFS; ANTIGENS, BACTERIAL; BACTERIAL PROTEINS; BIOLOGICAL TRANSPORT; ENZYME INHIBITORS; EPITHELIAL CELLS; FLUORESCENT ANTIBODY TECHNIQUE; GASTRIC MUCOSA; GENES, BACTERIAL; GENETIC COMPLEMENTATION TEST; GENISTEIN; HELICOBACTER PYLORI; HUMANS; MUTATION; PHOSPHORYLATION; PHOSPHOTYROSINE; PROTEIN-TYROSINE KINASES; STAUROSPORINE; TUMOR CELLS, CULTURED; VIRULENCE;

BACTERIA (MICROORGANISMS); HELICOBACTER; HELICOBACTER PYLORI; NEGIBACTERIA;

EID: 17344379177     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5457.1497     Document Type: Article

References (24)

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10. 2]. Cells were suspended in 1 ml of ice-cold PBS* (PBS, 1 mM EDTA, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM leupeptin, 1 μM pepstatin) (cell scraper), collected by centrifugation, and resuspended in 30 μl PBS*. For kinase inhibition studies, the cell culture medium was supplemented with 250 μM genistein or 1 μM staurosporine 1 hour before and during infection. For dephosphorylation, cells were collected in 30 μl of PBS, 0.1% Triton X-100 and incubated with 50 U of YopH phosphatase (NEB) for 1 hour at 30°C. For SDS-polyacrylamide gel electrophoresis, 40 μl 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)-horseradish 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-conjugated anti-rabbit IgC. The FLAG-tagged CagA was visualized with the FLAG-specific mAb M2 and TRITC-coupled anti-mouse IgG.
11. - mutant For construction of P12ΔcagA, region 579021 -to 579905 (cagA-upstream) and 583483 to 584934 (cagA-downstream) of the Hp 26695 genome were amplified by PCR with 18-nucleotide flanking sequences as primers. Fragments were cloned into pBluescript Xho I-Bam HI sites, separated by an apnA-3 gene cassette. For complementation, the cagA gene of 26695 was amplified by PCR and cloned into the pHel2 vector (Xho I-Bam HI) (15). The FLAG-tag (Sigma) sequence was fused to the cagA-downstream primer (5′-CGGGATCCTTACTTGTCATCGTCGTCCTTGTAGTCAGATTTTTGGAAACCACCTT-3). The 18-nucleotide cagA upstream primer starts at position 579701 (19). Transformation of Hp strains was performed as described [R. Haas et al., Mol. Microbiol. 8, 753 (1993)].
12. - mutant For construction of P12ΔcagA, region 579021 -to 579905 (cagA-upstream) and 583483 to 584934 (cagA-downstream) of the Hp 26695 genome were amplified by PCR with 18-nucleotide flanking sequences as primers. Fragments were cloned into pBluescript Xho I-Bam HI sites, separated by an apnA-3 gene cassette. For complementation, the cagA gene of 26695 was amplified by PCR and cloned into the pHel2 vector (Xho I-Bam HI) (15). The FLAG-tag (Sigma) sequence was fused to the cagA-downstream primer (5′-CGGGATCCTTACTTGTCATCGTCGTCCTTGTAGTCAGATTTTTGGAAACCACCTT-3). The 18-nucleotide cagA upstream primer starts at position 579701 (19). Transformation of Hp strains was performed as described [R. Haas et al., Mol. Microbiol. 8, 753 (1993)].
13. 2), washed 5X with PBS*. and lysed in 500 μl 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 μM leupeptin,1 μM pepstatin] (1 hour, 4°C, gentle shaking). The lysate was cleared (15,000 rpm, 4°C, 10 min) and the protein content adjusted to 3 mg/ml with PBS*. AK257 (1.7 μg/ml) was added to a total volume of 900 μl; after 1 hour at 4°C, 40 μl of Protein G-agarose (Roche) was added and the mixture incubated for a further 2 hours at 4°C. After a short centrifugation step (10 s, 6.000g) the supernatant was discarded, and beads were washed four times with 1 ml of PBS* and suspended in 40 μl of sample solution.
14. 2+-nitrilotriacetic 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)-coupled protein A was used to visualize the antibody bound by decomposition of nitro blue tetrazolium.
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24. 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).