Proc. Natl. Acad. Sci. USA Vol. 94, pp. 7595–7599, July 1997 Microbiology Induction of host signal transduction pathways by Helicobacter pylori (phosphorylationyinterleukin 8yvasodilator-stimulated phosphoprotein) ELLYN D. SEGAL*†‡,C.LANGE§,A.COVACCI§,L.S.TOMPKINS*†¶i, AND S. FALKOW*†¶i Departments of *Microbiology and Immunology and ¶Medicine, and †Digestive Disease Center, Stanford University School of Medicine, Stanford, CA 94305; iRocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; and §Immunolbiological Research Institute, Siena, Italy Contributed by S. Falkow, May 7, 1997 ABSTRACT Adherence of Helicobacter pylori to cultured tween type I strains and the ability to induce IL-8 secretion in gastric epithelial cells is associated with several cellular gastric epithelial cell lines has been demonstrated by several events, including the tyrosine phosphorylation of a 145-kDa laboratories (10). Tummuru et al. (11) have identified a gene, host protein; the reorganization of the host cell actin and picB, with significant amino acid homology to the B. pertussis associated cellular proteins, like vasodilator-stimulated phos- toxin-secretion protein (PtlC) and have demonstrated that the phoprotein, adjacent to the attached bacterial cell; and the picB gene product is involved in inducing IL-8 in vitro. The picB subsequent release of the cytokine, interleukin 8 (IL-8). H. gene now is known to be within the H. pylori cag1 PAI. VacA, pylori isolated from patients with ulcer disease and gastric CagA, and urease are not functionally involved with the cancer contain a DNA insertion, the cag pathogenicity island induction of IL-8 from cultured gastric cells (12, 13). (PAI), that is not present in bacteria isolated from individuals We have previously shown (14) that H. pylori attachment to with asymptomatic infection. Mutations in a number of PAI gastric epithelial cells is associated within effacement of genes abolish tyrosine phosphorylation and IL-8 synthesis but microvilli, and rearrangement of the cytoskeleton occurs be- not the cytoskeletal rearrangements. Kinase inhibition stud- neath the attached bacterium (pedestal formation). These ies suggest there are two distinct pathways operative in findings are similar to that described for the enteropathogenic stimulating IL-8 release from host cells and one of these H. Escherichia coli (EPEC) which are associated with genes pylori pathways is independent of the tyrosine phosphoryla- encoded within a PAI (15). Furthermore, like EPEC, H. pylori tion step. binding to epithelial cells induces tyrosine phosphorylation of host cell proteins adjacent to the site of bacterial adherence. Helicobacter pylori, a human pathogen and class I carcinogen These effects of H. pylori attachment to cells suggests that (1), causes acute and chronic inflammation of the stomach alteration of host cell signal transduction might lead to chronic (gastritis). Progression of the disease in some, but not all, inflammation and perhaps may lead to the oncogenic trans- patients can lead to peptic ulcer disease, gastric cancer, or formation that are the hallmarks of symptomatic H. pylori lymphoma originating in the mucosal-associated lymphoid infection. tissue. Several putative H. pylori virulence factors have been To better understand the mechanism(s) involved in the identified, including urease; the vacuolating cytotoxin, VacA; induction of the host cell response to H. pylori attachment, and a cytotoxin-associated antigen, CagA. However, muta- wild-type type I and type II strains and isogenic type I H. pylori tions in the genes encoding these factors do not have a mutants were screened for their ability to attach to gastric measurable effect on the virulence of H. pylori in cell culture epithelial cells, to induce tyrosine phosphorylation of the or animal infection studies (2, 3). 145-kDa host protein, and for their capacity to generate an Most of the clinical isolates of H. pylori isolated from peptic inflammatory reaction in vitro as measured by the induction of ulcer disease or patients suffering from malignant disease IL-8. In addition, we have performed experiments to analyze express CagA. Consequently, H. pylori strains have been further the cellular pathways affected by H. pylori infection. divided into two broad categories. Type I strains are cag1 and vacA1. Type II strains are cagA2 and fail to produce a MATERIALS AND METHODS functional VacA toxin. Yet, DNA sequences homologous to the vacA gene are present in the chromosomes of type II Bacterial Strains and Cell Lines. Helicobacter pylori strain strains. Type I isolates are associated with duodenitis, duode- 87A300 was obtained from the State of California, Depart- nal ulcers, and gastric cancer (4–6). A pathogenicity island ment of Health Services, Berkeley. It is a human clinical isolate (PAI), called cag1, has been identified exclusively in H. pylori producing the vacuolating cytotoxin (vacA), urease, CagA, and type I strains. The cag1 region contains over 22 open reading a contiguous cag1 PAI. This strain was grown as described frames (ORF) encoded in 40 kb of DNA (7). The majority of (14). Urease mutants of strain 87A300 were described (16). H. the proteins predicted to be encoded by the cag1 ORF appear pylori strains G27, 314, G50, G198, and their mutants were to be membrane-associated with features similar to bacterial provided by Antonio Covacci (Immunobiological Research secretion systems, particularly the Type IV system epitomized Institute, Siena, Italy) and are described by Censini et al. (7). by Bordetella pertussis toxin secretion (8). A summary of all strains used in this study and their respective The cytokine interleukin 8 (IL-8) is a neutrophil chemotac- genotypes is presented in Table 1. AGS cells (ATCC CRL tic factor that has been shown to be increased in H. pylori- 1739), a human gastric adenocarcinoma epithelial cell line, infected patients with active gastritis (9). A correlation be- were grown in DMEM plus 10% fetal calf serum as described (14). The publication costs of this article were defrayed in part by page charge Abbreviations: PAI, pathogenicity island; IL-8, interleukin 8; VASP, payment. This article must therefore be hereby marked ‘‘advertisement’’ in vasodilator-stimulated phosphoprotein; IF, immunofluorescence; accordance with 18 U.S.C. §1734 solely to indicate this fact. PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase © 1997 by The National Academy of Sciences 0027-8424y97y947595-5$2.00y0 G. PNAS is available online at http:yywww.pnas.org. ‡To whom reprint requests should be addressed. 7595 Downloaded by guest on October 1, 2021 7596 Microbiology: Segal et al. Proc. Natl. Acad. Sci. USA 94 (1997) Table 1. H. pylori strains used in this study Infection Assays and Determination of Tyrosine Phosphor- Strain Genotype Phenotype ylation of Host Cell Proteins. AGS cells were cultured for 24 hr in 60-mm tissue culture dishes containing DMEM as 87A300 Type 1 cag1, vac1, urease1 described (14). The cells were washed once with PBS (pH 7.4), Mutants* and 2 ml of fresh media was added to each dish. Approximately ure1 urease2 1 3 107 H. pylori from a liquid overnight culture was added to G27 Type 1 cag1, vac1, urease1 the AGS cells at a multiplicity of infection of 10:1. After Mutants† incubation in a 5% CO2 incubator for 2 hr, cell lysates were 10B4 cagE::Kan made as described (14) and stored at 280°C until needed. Mfe:Kan cagF::Kan Whole cell lysates of H. pylori were made by pelleting the 62A cagG::Kan bacteria and suspending the pellet in an equal amount of PBS 3F cagH::Kan and 23 SDS lysis buffer (250 mM Tris, pH 6.8y4% SDSy20% 5-3 cagI::Kan glyceroly0.002% bromophenol bluey10% 2-mercaptoethanol). 2B cagL::Kan SDS, PAGE, and electrotransfer was performed as described 14A-4 cagN::Kan (14). Anti-phosphotyrosine binding and detection was done H12-10 cagN::Kan using the ECL system (Amersham) according to the manu- H12-18A cagN::Kan facturer’s instructions. H12-47 cagM::Kan IL-8 Assay. Experiments were performed with the AGS cell H12-4 cagM::Kan line grown and infected with H. pylori as described above. After H12-16 cagM::Kan incubation in a 5% CO incubator for 2 hr, the medium was H12-5 cagM::Kan 2 removed from the dish, spun to remove any remaining bacteria 314 Type 1 cag1, vac1, urease1 (10,000 rpm for 10 min at 4°C), and the aliquots were frozen G50 Type 2 cag2, vac2, urease1 at 280°C. IL-8 levels of these extracts was determined by G104 Type 2 cag2, vac2, urease1 ELISA (Amersham). G198 Type 2 cag2, vac2, urease1 Kinase Inhibition Assays. The concentration of kinase *Isolates generated by ethyl methanesulfonate mutagenesis. inhibitors used was as follows: 1 mM staurosporine, 250 mM † Isolates generated by kanamycin insertion into the gene. genistein, 2 mM bisindolymaleimide I, 9.8 mM H-89 (dihydro- chloride), and 47 mM KT5823. All inhibitors were purchased Antibodies. Rabbit polyclonal anti-H. pylori antibodies were from Calbiochem. For the assay, AGS cells were seeded as produced against whole heat-killed H. pylori strain 87A300. described above. The medium was removed before the assay Working dilution of the polyclonal anti-H. pylori was 1:100. and replaced with fresh DMEM with the appropriate inhibitor Monoclonal anti-phosphotyrosine PY20 and anti-vasodilator- and incubated for 30 min. H. pylori was added as above and stimulated phosphoprotein (VASP) were purchased from incubated for an additional 3 hr. Lysates were prepared and Transduction Laboratories (Lexington, KY) and used at a analyzed for tyrosine phosphorylation as described earlier. working dilution of 1:1000. Anti-mouse IgG crystalline tetra- methylrhodamine isothiocyanate conjugate and anti-rabbit IgG fluorescein isothiocyanate conjugate was obtained from RESULTS Sigma. Anti-mouse IgG 7-amino-4-methylcoumarin-3-acetic H. pylori Attachment to Cultured Gastric Epithelial Cells acid conjugate was obtained from Vector Laboratories.