Regulation of the Helicobacter pylori Cellular Receptor Decay-accelerating Factor
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m801144200
ISSN1083-351X
AutoresD. O’Brien, Judith Romero‐Gallo, Barbara Schneider, Rupesh Chaturvedi, Alberto Delgado, Elizabeth J. Harris, Uma Krishna, Seth R. Ogden, Dawn A. Israel, Keith T. Wilson, Richard M. Peek,
Tópico(s)Veterinary medicine and infectious diseases
ResumoChronic gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulceration and distal gastric cancer, and adherence of H. pylori to gastric epithelial cells is critical for induction of inflammation. One H. pylori constituent that increases disease risk is the cag pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Decay-accelerating factor (DAF) is a cellular receptor for H. pylori and a mediator of the inflammatory response to this pathogen. H. pylori induces DAF expression in human gastric epithelial cells; therefore, we sought to define the mechanism by which H. pylori up-regulates DAF and to extend these findings into a murine model of H. pylori-induced injury. Co-culture of MKN28 gastric epithelial cells with the wild-type H. pylori cag+ strain J166 induced transcriptional expression of DAF, which was attenuated by disruption of a structural component of the cag secretion system (cagE). H. pylori-induced expression of DAF was dependent upon activation of the p38 mitogen-activated protein kinase pathway but not NF-κB. Hypergastrinemic INS-GAS mice infected with wild-type H. pylori demonstrated significantly increased DAF expression in gastric epithelium versus uninfected controls or mice infected with an H. pylori cagE- isogenic mutant strain. These results indicate that H. pylori cag+ strains induce up-regulation of a cognate cellular receptor in vitro and in vivo in a cag-dependent manner, representing the first evidence of regulation of an H. pylori host receptor by the cag pathogenicity island. Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulceration and distal gastric cancer, and adherence of H. pylori to gastric epithelial cells is critical for induction of inflammation. One H. pylori constituent that increases disease risk is the cag pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Decay-accelerating factor (DAF) is a cellular receptor for H. pylori and a mediator of the inflammatory response to this pathogen. H. pylori induces DAF expression in human gastric epithelial cells; therefore, we sought to define the mechanism by which H. pylori up-regulates DAF and to extend these findings into a murine model of H. pylori-induced injury. Co-culture of MKN28 gastric epithelial cells with the wild-type H. pylori cag+ strain J166 induced transcriptional expression of DAF, which was attenuated by disruption of a structural component of the cag secretion system (cagE). H. pylori-induced expression of DAF was dependent upon activation of the p38 mitogen-activated protein kinase pathway but not NF-κB. Hypergastrinemic INS-GAS mice infected with wild-type H. pylori demonstrated significantly increased DAF expression in gastric epithelium versus uninfected controls or mice infected with an H. pylori cagE- isogenic mutant strain. These results indicate that H. pylori cag+ strains induce up-regulation of a cognate cellular receptor in vitro and in vivo in a cag-dependent manner, representing the first evidence of regulation of an H. pylori host receptor by the cag pathogenicity island. Helicobacter pylori induces an inflammatory response in the stomach that persists for decades and increases the risk not only for peptic ulceration but also for gastric adenocarcinoma and non-Hodgkin's lymphoma of the stomach (1Peek Jr., R.M. Blaser M.J. Nat. Rev. Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1445) Google Scholar, 2Moss S.F. Sood S. Curr. Opin. Infect. Dis. 2003; 16: 445-451Crossref PubMed Scopus (66) Google Scholar). Gastric adenocarcinoma is the second leading cause of cancer-related death in the world, and chronic gastritis induced by H. pylori is the strongest known risk factor for this malignancy (1Peek Jr., R.M. Blaser M.J. Nat. Rev. Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1445) Google Scholar, 3Moss S.F. Blaser M.J. Nat. Clin. Pract. Oncol. 2005; 2: 90-97Crossref PubMed Scopus (228) Google Scholar, 4Correa P. Gut. 2004; 53: 1217-1219Crossref PubMed Scopus (84) Google Scholar, 5Blanchard T.G. Drakes M.L. Czinn S.J. Curr. Opin. 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BabA is an outer-membrane protein (OMP) 2The abbreviations used are:OMPouter-membrane proteinDAFdecay-accelerating factorNF-κBnuclear factor-κBNODnucleotide binding oligomerization domainMAPKmitogen-activated protein (MAP) kinaseERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinasem.o.i.multiplicity of infectionMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseRTreverse transcriptionqRTquantitative real-timeIHCimmunohistochemicalGAPDHglyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are:OMPouter-membrane proteinDAFdecay-accelerating factorNF-κBnuclear factor-κBNODnucleotide binding oligomerization domainMAPKmitogen-activated protein (MAP) kinaseERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinasem.o.i.multiplicity of infectionMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseRTreverse transcriptionqRTquantitative real-timeIHCimmunohistochemicalGAPDHglyceraldehyde-3-phosphate dehydrogenase. encoded by the strain-specific gene babA2, which binds the Lewisb (Leb) histo-blood-group antigen on gastric epithelial cells (9Gerhard M. 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Angstrom J. Larsson T. Teneberg S. Karlsson K.A. Altraja S. Wadstrom T. Kersulyte D. Berg D.E. Dubois A. Petersson C. Magnusson K.E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarstrom L. Boren T. Science. 2002; 297: 573-578Crossref PubMed Scopus (696) Google Scholar). Gastric inflammation induced by H. pylori up-regulates the expression of sLex on epithelial cells, which amplifies interactions between this molecule and SabA. outer-membrane protein decay-accelerating factor nuclear factor-κB nucleotide binding oligomerization domain mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase c-Jun N-terminal kinase multiplicity of infection mitogen-activated protein kinase/extracellular signal-regulated kinase kinase reverse transcription quantitative real-time immunohistochemical glyceraldehyde-3-phosphate dehydrogenase. outer-membrane protein decay-accelerating factor nuclear factor-κB nucleotide binding oligomerization domain mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase c-Jun N-terminal kinase multiplicity of infection mitogen-activated protein kinase/extracellular signal-regulated kinase kinase reverse transcription quantitative real-time immunohistochemical glyceraldehyde-3-phosphate dehydrogenase. We recently identified another H. pylori receptor, Decay-accelerating factor (DAF), that is up-regulated after bacterial contact (13O'Brien D.P. Israel D.A. Krishna U. Romero-Gallo J. Nedrud J. Medof M.E. Lin F. Redline R. Lublin D.M. Nowicki B.J. Franco A.T. Ogden S. Williams A.D. Polk D.B. Peek Jr., R.M. J. Biol. Chem. 2006; 281: 13317-13323Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). DAF is an intrinsic regulator of complement that is attached to the outer leaflet of the cell membrane by a glycophosphatidylinositol anchor (14Servin A.L. Clin. Microbiol. Rev. 2005; 18: 264-292Crossref PubMed Scopus (163) Google Scholar). DAF protects cells from complement activation on their surfaces by dissociating membrane-bound C3 convertases that are required for cleaving C3 and further propagating the complement cascade. 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Expression of DAF is increased within H. pylori-infected human gastric tissue compared with uninfected mucosa, and the intensity of expression is directly related to the density of H. pylori colonization and severity of inflammation (19Berstad A.E. Brandtzaeg P. Gut. 1998; 42: 522-529Crossref PubMed Scopus (59) Google Scholar, 20Rautemaa R. Rautelin H. Puolakkainen P. Kokkola A. Karkkainen P. Meri S. Gastroenterology. 2001; 120: 470-479Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We recently demonstrated that DAF influences the inflammatory response to H. pylori as infected DAF-deficient mice developed significantly less severe inflammation compared with infected wild-type mice, suggesting that the interaction between H. pylori and DAF is important for pathogenesis (13O'Brien D.P. Israel D.A. Krishna U. Romero-Gallo J. Nedrud J. Medof M.E. Lin F. Redline R. Lublin D.M. Nowicki B.J. Franco A.T. Ogden S. Williams A.D. Polk D.B. Peek Jr., R.M. J. Biol. 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Gastroenterology. 2007; 132: 1309-1319Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). H. pylori peptidoglycan components delivered by the cag secretion system are recognized by the intracellular pattern recognition receptor NOD1, which initiates cell-signaling events including activation of NF-κB (24Viala J. Chaput C. Boneca I.G. Cardona A. Girardin S.E. Moran A.P. Athman R. Memet S. Huerre M.R. Coyle A.J. DiStefano P.S. Sansonetti P.J. Labigne A. Bertin J. Philpott D.J. Ferrero R.L. Nat. Immunol. 2004; 5: 1166-1174Crossref PubMed Scopus (977) Google Scholar). In vivo, the presence of the cag island also influences the topography of colonization, as H. pylori cag- strains predominate within the mucus gel layer, whereas cag+ strains are found immediately adjacent to epithelial cells (27Camorlinga-Ponce M. Romo C. Gonzalez-Valencia G. Munoz O. Torres J. J. Clin. Pathol. 2004; 57: 822-828Crossref PubMed Scopus (44) Google Scholar). Compared with cag- strains, H. pylori cag+ strains augment the risk for severe pathologic outcomes, such as peptic ulceration and gastric cancer (1Peek Jr., R.M. Blaser M.J. Nat. Rev. Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1445) Google Scholar). Because adherence likely plays a critical role in pathogenesis, we sought to delineate the host and bacterial factors that mediate H. pylori induction of DAF. We demonstrate that H. pylori cag+ strains up-regulate DAF expression in gastric epithelial cells in vitro and in vivo in a cag-dependent manner and that this induction is mediated by p38 MAP kinase activation. Reagents and Constructs—Actinomycin D, cycloheximide, the p38 inhibitor SB203580, and the JNK1/2/3 inhibitor JNK inhibitor II were obtained from Calbiochem, and the MEK1/2 inhibitor PD98059 was obtained from Cayman Chemical. Anti-DAF IA10 (BD Pharmingen), anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-phospho-MAPKAPK-2 (Thr-334), and anti-phospho-c-Jun (Ser-73) antibodies (Cell Signaling) were used for Western analysis. The anti-DAF antibody MCA1614 (AbD Serotec) was used for immunohistochemistry. The pNF-κB luciferase vector (Clontech) and pRL Renilla luciferase vector (Promega) were used for NF-κB luciferase studies. Dominant-negative mutant IκBα S32A/S36A and dominant-negative IκB kinase β K44A constructs were used for NF-κB inhibition studies (generous gifts of Dr. Andrew Neish, Emory University School of Medicine) (28Zeng H. Wu H. Sloane V. Jones R. Yu Y. Lin P. Gewirtz A.T. Neish A.S. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 290: 96-108Crossref PubMed Scopus (104) Google Scholar). Cell Culture—MKN28 human gastric epithelial cells (kindly provided by Dr. Robert Coffey, Vanderbilt University) were grown in RPMI 1640 (Invitrogen) with 10% heat-inactivated fetal bovine serum and 20 μg/ml gentamicin in an atmosphere of 5% CO2 at 37 °C. Bacterial Strains—Experiments were performed with the H. pylori cag+ strains J166 and 7.13 (13O'Brien D.P. Israel D.A. Krishna U. Romero-Gallo J. Nedrud J. Medof M.E. Lin F. Redline R. Lublin D.M. Nowicki B.J. Franco A.T. Ogden S. Williams A.D. Polk D.B. Peek Jr., R.M. J. Biol. Chem. 2006; 281: 13317-13323Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 29Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O'Brien D P. Romero-Gallo J. Peek Jr., R.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10646-10651Crossref PubMed Scopus (391) Google Scholar). Isogenic cagA, cagE, and cagM null mutants were constructed by insertional mutagenesis using aphA (conferring kanamycin resistance) as previously described (30Peek Jr., R.M. Blaser M.J. Mays D.J. Forsyth M.H. Cover T.L. Song S.Y. Krishna U. Pietenpol J.A. Cancer Res. 1999; 59: 6124-6131PubMed Google Scholar, 31Crawford H.C. Krishna U.S. Israel D.A. Matrisian L.M. Washington M.K. Peek Jr., R.M. Gastroenterology. 2003; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and were selected on Brucella agar with kanamycin (25 μg/ml). Heat-killed H. pylori were generated by heating the bacteria to 80 °C for 10 min. H. pylori lysates were generated by sonication as previously described (32Gewirtz A.T. Yu Y. Krishna U.S. Israel D.A. Lyons S.L. Peek Jr., R.M. J. Infect. Dis. 2004; 189: 1914-1920Crossref PubMed Scopus (213) Google Scholar). Lysates were then sterilized using a 0.2-μm pore size filter. Western Analysis—MKN28 gastric epithelial cells were grown to confluence, then cultured in serum-free medium for 24 h and then co-cultured with H. pylori for specified times at a multiplicity of infection (m.o.i.) of 100. H. pylori-infected and uninfected MKN28 cells were lysed in radioimmune precipitation assay buffer (50 mm Tris, pH 7.2, 150 mm NaCl, 1% Triton X-100, 0.1% SDS), and protein concentrations were quantified by the BCA assay (Pierce) (31Crawford H.C. Krishna U.S. Israel D.A. Matrisian L.M. Washington M.K. Peek Jr., R.M. Gastroenterology. 2003; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Proteins (30 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pall Corp., Ann Arbor, MI). DAF levels were examined in gastric cells by Western blotting using an anti-DAF (1:1000, IA10) antibody. Levels of the phosphorylated MAPK targets ERK1/2, MAPKAPK-2, and c-Jun were detected using the respective antibodies described above (1:1000). Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and visualized by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) according to the manufacturer's instructions. Western blots were imaged, and band intensities were quantified using the ChemiGenius Gel Bio Imaging System (Syngene). Real-time Quantitative RT-PCR—MKN28 gastric epithelial cells were grown to confluence, cultured in serum-free medium for 24 h, and then co-cultured with H. pylori for specified times (m.o.i. = 100). RNA was prepared from H. pylori:gastric cell co-cultures using the RNeasy RNA purification kit (Qiagen) following the manufacturer's instructions. Reverse transcriptase PCR was performed using TaqMan reverse transcription reagents (Applied Biosystems), which was followed by real-time quantitative PCR using the TaqMan Gene Expression Assay and a 7300 real-time PCR system (Applied Biosystems). Daf and gapdh cDNA were quantified using a TaqMan® Gene Expressions primer set purchased from Applied Biosystems. -Fold induction of daf mRNA was determined from the threshold cycle values normalized for gapdh mRNA expression, and this ratio was then normalized to the value derived from cells cultured with medium alone. Transfections and Luciferase Assay—MKN28 cells were transiently transfected using FuGENE 6 reagent (Roche Applied Science) per the manufacturer's instructions. Cells were allowed to incubate with the transfection mixture for 24 h, cultured in serum-free medium for an additional 24 h, and then co-cultured with H. pylori strain J166 (m.o.i. = 100). Samples were assayed for luciferase activity on a TD-20/20 Luminometer (Turner Designs) using the Dual Luciferase® reporter kit (Promega) according to the manufacturer's instructions. Experimental Animal Infections—All procedures were approved by the Institutional Animal Care Committee of Vanderbilt University. Male INS-GAS transgenic mice on the FVB/N background 6–8 weeks of age were challenged with either sterile Brucella broth, wild-type H. pylori strain 7.13, or a 7.13 cagE- mutant by oral gavage as previously described (33Fox J.G. Wang T.C. Rogers A.B. Poutahidis T. Ge Z. Taylor N. Dangler C.A. Israel D.A. Krishna U. Gaus K. Peek Jr., R.M. Gastroenterology. 2003; 124: 1879-1890Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Mice were euthanized at 4, 12, and 24 weeks post-challenge. At necropsy, linear strips extending from the squamocolumnar junction through proximal duodenum were fixed in 10% neutral-buffered formalin, paraffin-embedded, and cut at 5 μm increments. Sections were then deparaffinized, and DAF immunohistochemical (IHC) staining was carried out as previously described (29Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O'Brien D P. Romero-Gallo J. Peek Jr., R.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10646-10651Crossref PubMed Scopus (391) Google Scholar) using the anti-DAF antibody MCA1614 (Serotec). A single pathologist (E. Harris), experienced in murine pathology and blinded to treatment groups, scored DAF IHC staining on an ordinal scale from 0 to 4 by as previously described (34Shattuck-Brandt R.L. Lamps L.W. Heppner Goss K.J. DuBois R.N. Matrisian L.M. Mol. Carcinog. 1999; 24: 177-187Crossref PubMed Scopus (81) Google Scholar). To assess colonization, gastric tissue was homogenized, plated, and incubated under microaerobic conditions at 37 °C for 5–6 days as previously described (33Fox J.G. Wang T.C. Rogers A.B. Poutahidis T. Ge Z. Taylor N. Dangler C.A. Israel D.A. Krishna U. Gaus K. Peek Jr., R.M. Gastroenterology. 2003; 124: 1879-1890Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Colonies were verified as H. pylori by Gram's stain, urease, catalase, and oxidase reactions as described (33Fox J.G. Wang T.C. Rogers A.B. Poutahidis T. Ge Z. Taylor N. Dangler C.A. Israel D.A. Krishna U. Gaus K. Peek Jr., R.M. Gastroenterology. 2003; 124: 1879-1890Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Successful colonization was confirmed by IHC staining using an anti-H. pylori antibody. Gastric Epithelial Cell Isolation and Detection of DAF Protein by Flow Cytometry—Gastric epithelial cells were isolated from frozen stomach samples using a dissociation and dispersion technique as previously described (35Whitehead R.H. VanEeden P.E. Noble M.D. Ataliotis P. Jat P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 587-591Crossref PubMed Scopus (333) Google Scholar). Briefly, epithelial cells were treated with 10 mm dithiothreitol at room temperature for 30 min and then with 1.0 mm EDTA for 60 min at 4 °C. Dispersed cells were filtered through a 0.4 μm filter to isolate single cells. Cell surface DAF protein was stained using hamster anti-mouse DAF antibody conjugated with phycoerythrin (BD Pharmingen) (1:50 dilution) at 4 °C for 45 min. After washing, cells were fixed and permeabilized with 0.1% paraformaldehyde and ice-cold methanol. To confirm epithelial lineage, cells were also stained with a mouse anti-pan cytokeratin antibody conjugated with fluorescein isothiocyanate (Abcam, Cambridge, MA) (1:50 dilution) for 30 min at 4 °C. Cells were acquired using a LSR II flow cytometer (BD Biosciences) and analyzed by Flowjo (Star tree, Ashland, OR). Extraction of Total RNA from Murine Gastric Mucosa—Serial sectioning of frozen gastric tissue was performed using a cryostat. The thickness of the serial sections was 7 μm. Hematoxylin and eosin stains were performed on the first and last section in the series to determine cellular composition of the tissue and serial sections were selected in which the majority cell type was epithelial. RNA was isolated using the RNeasy RNA extraction kit according to the manufacturer's instructions. daf mRNA expression was measured using real-time RT-PCR as described above and normalized to levels of gapdh mRNA. Taqman® probes were used to detect daf and gapdh expression; Mm00438377_m1 and Rodent GAPDH control reagents, respectively (Applied Biosystems). Statistical Analysis—An analysis of variance one-way analysis of variance and the Tukey-Kramer post test were used for analysis of in vitro data. The Mann-Whitney U test of intergroup comparisons was used for analysis of in vivo IHC and real-time PCR data. The Newman-Keuls test was used for analysis of flow cytometric data. Significance was defined as p ≤ 0.05. All calculations were performed with the GraphPad Prism 4 statistical analysis software package (GraphPad Software, Inc.). H. pylori Induction of DAF Is Regulated at the Transcriptional Level—We previously demonstrated that H. pylori up-regulates DAF in vitro (13O'Brien D.P. Israel D.A. Krishna U. Romero-Gallo J. Nedrud J. Medof M.E. Lin F. Redline R. Lublin D.M. Nowicki B.J. Franco A.T. Ogden S. Williams A.D. Polk D.B. Peek Jr., R.M. J. Biol. Chem. 2006; 281: 13317-13323Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To determine whether DAF induction was transcriptionally or post-transcriptionally mediated, the H. pylori cag+ strain J166 was co-cultured with MKN28 gastric epithelial cells that had been pretreated with either actinomycin D (inhibitor of transcription) or cycloheximide (inhibitor of translation). DAF protein expression was assessed after 24 h of co-culture (Fig. 1). Actinomycin D completely blocked DAF induction in response to H. pylori (p < 0.001), and inhibition of translation by cycloheximide blocked DAF induction in a dose-dependent manner. Vehicle-treated, H. pylori-infected cells expressed significantly more DAF than H. pylori-infected cells that had been pretreated with cycloheximide (p < 0.01), and DAF expression in cycloheximide-treated, infected cells was not significantly higher than vehicle-treated, uninfected control cells. These results indicate that up-regulation of DAF by H. pylori in human gastric epithelial cells is mediated at a transcriptional level. H. pylori Induction of daf Requires Viable Bacteria—Bacteria can activate epithelial signaling pathways via multiple mechanisms. To determine whether live H. pylori are necessary for daf induction or if inert bacterial components are sufficient, we incubated MKN28 cells with viable bacteria or with H. pylori that had either been heat-killed or lysed by sonication and assessed daf mRNA expression using real-time quantitative RT-PCR (Fig. 2). Co-culture of cells with live H. pylori, as expected, significantly induced daf mRNA. However, incubation with either heat-killed or sonicated H. pylori failed to induce expression of daf. These results indicate that induction of daf in gastric epithelial cells is dependent upon an active interplay with viable bacteria. DAF Induction Is Mediated by a Functional Type IV Secretion System—The requirement for viable H. pylori to induce daf raised the possibility that bacterial components intimately involved in epithelial contact may mediate daf expression. The cag pathogenicity island encodes a bacterial type IV secretion system that translocates effector molecules such as peptidoglycan and CagA into host cells after binding, thus affecting cell function. Therefore, we determined if H. pylori-mediated up-regulation of DAF is cag pathogenicity island-dependent. MKN28 cells were co-cultured with either wild-type H. pylori or isogenic cagA or cagE null mutant derivatives. Real-time qRT-PCR analysis demonstrated that co-culture with the cagA- mutant induced daf expression to levels similar to those induced by the wild-type strain (Fig. 3A). However, co-culture with the cagE- mutant failed to induce daf, and expression levels were no different from levels in uninfected cells. Western blot analysis confirmed that inactivation of cagE significantly attenuates the ability of H. pylori to induce DAF (Fig. 3B). Experiments were also performed with an independent H. pylori cag+ strain, 7.13, which readily infects animals and has been shown to induce gastric cancer in Mongolian gerbils and hypergastrinemic mice (29Franco A.T. Israel D.A. Washington M.K. Krishna U. Fox J.G. Rogers A.B. Neish A.S. Collier-Hyams L. Perez-Perez G.I. Hatakeyama M. Whitehead R. Gaus K. O'Brien D P. Romero-Gallo J. Peek Jr., R.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10646-10651Crossref PubMed Scopus (391) Google Scholar, 33Fox J.G. Wang T.C. Rogers A.B. Poutahidis T. Ge Z. Taylor N. Dangler C.A. Israel D.A. Krishna U. Gaus K. Peek Jr., R.M. Gastroenterology. 2003; 124: 1879-1890Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 36Romero-Gallo J. Harris E.J. Krishna U. Washington M.K. Perez-Perez G.I. Peek Jr., R.M. Lab. Investig. 2008; 88: 328-336Crossref PubMed Scopus (56) Google Scholar, 37Franco A.T. Johnston E. Krishna U. Yamaoka Y. Israel D.A. Nagy T.A. Wroblewski L.E. Piazuelo M.B. Correa P. Peek Jr., R.M. Cancer Res. 2008; 68: 379-387Crossref PubMed Scopus (223) Google Scholar). Similar to results obtained with strain J166, real-time qRT-PCR results showed that daf induction was dependent upon cagE but not cagA. The importance of the cag secretion system was more rigorously confirmed by demonstrating that inactivation of another cag gene encoding a structural component of the type IV secretion system (cagM) similarly attenuated daf expression (data not shown). These results in
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