- Email: [email protected]
Increased urease activity of H. pylori contributes to impaired duodenal mucosal bicarbonate secretion in duodenal ulcer
A736 AGA ABSTRACTS
3988 IDENTIFICATION OF THE INTERACTION OF UREGIUREE AND UREFIUREH IN H. PYLORI USING A YEAST TWO-HYBRID SCREEN. Petra R. Voland, George Sachs, Vaglahs & UCLA, Los Angeles, CA. Background: Accessory proteins (Ure E, F. G. H) of the H. pylori urease gene cluster may interact with each other during urease assembly. The Yeast Two-Hybrid system is a method to characterize known protein interactions and to identify genes that encode interacting partners. Aim: Identification of H. pylori proteins interacting with the accessory proteins of the urease gene cluster. Methods: The Yeast Two-Hybrid System (Matchmaker II, Clontech) was used to screen a genomic DNA library of H. pylorifor proteins interacting with UreE, F, G and H. A high-efficiency mating procedure was applied. Diploid clones were tested for ~ galactosidase activity with a lift assay to identify clones containing interacting proteins. Plasmid-DNA was isolated from blue clones for restriction analysis, DNA-sequencing and co-transformation into yeast. After lift assay, a subset of blue clones underwent a quantitative chemiluminescent ~ga lactosidase assay. Interactions were verified by co-immuno-precipitation of 35S_Cys labeled in vitrotranscriptionltranslation products of UreE, G, F and H using antibodies against c-Myc and HA tags. Results: Using UreG as bait in a library screen we found 36 blue clones in the colony lift assay. Further analysis by restriction enzyme digest and DNA sequencing identified sequences coding for UreE. The interaction between UreGlUreE was quantified by f3-galactosidase assay with 5 independent fish -clones showing 20-55x higher activity than controls. Using UreF as bait we found 53 blue clones. Restriction analysis and DNA sequencing revealed that 17 clones contained sequences coding for UreH. Interaction of UreFlUreH was quantified by f3-galactosidase assay with 4 independent clones being 1O-250x higher than controls. We confirmed the interaction between UreF/ UreH by co-immuno-precipitation of 35S_Cyslabeled in vitrotranscriptionl translation products. The interacting proteins UreFIH could be co-precipitated with antibodies against the fusion tag (c-Myc, HA) of each partner. Conclusions: The Yeast Two-Hybrid screen shows strong interaction of UreGlUreE and UreFlUreH, confirmed by co-immuno-precipitation of in vitro translation products. These proteins thus form a pair of complexes necessary for assembly and NiH insertion into the apoenzyme.
3989 PHASE VARIATION IN A TYPE m RESTRICTION-MODIFICA· TION SYSTEM OF HEUCOBACTER PYWRI. Nicolette Vries de, Dirk Duinsbergen, Ernst J. Kuipers, Patricia Wiesenekker, Christina M. Vandenbroucke-Grauls, Johannes G. Kusters, Vrije Univ, Amsterdam, Netherlands. Objective:The on- and off-switching of the expression of virulence factors (phase variation) plays an important role in the pathogenesis of many bacterial infections. LPS phase variation in Helicobacter pylori occurs at the translational level and has been studied well. In contrast, phase variation at the transcriptional level has not been demonstrated in H. pylori. Therefore, we investigated transcriptional on- and off-switching of gene expression in H. pylori. Methods: A library with random genomic transcriptionallacZ fusions in H. pylori strain 1061 (HPI061) was screened for mutants that showed blue and white sectored colonies on X-Gal. As the X-Gal substrate is converted into a blue product when the lacZ gene is expressed into the f3-galactosidase, sectored colonies indicate transcriptional phase variation. Results: One HP1061 mutant displayed frequent onand off-switching of lacZ expression. The on-to-off switch frequency was 2.67 %, while the off-to-on frequency was 0.75 %. Sequencing revealed that the lacZ gene was fused to a putative type III methylase gene (mod). RNA spot blot hybridization demonstrated that specific lacZ and mod probes bound to mRNA from blue colonies, but not to mRNA from white colonies. This proved that mod switched on and off at the transcriptional level. An open reading frame, encoding a putative type III restriction enzyme gene (res), is located immediately upstream of mod and contains a polynucleotide C-tract. This Cvtract, which may cause phase variation of res through translational frameshifts, might indirectly act on the transcription of mod. However, sequence analysis showed that the number of cytosines in res was not related to the on- or off-status of mod. Conclusion: In H. pylori. the putative type III methylase gene, mod, displays phase variation at the transcriptional level. It is known that methylation of promoter sequences can affect the transcription of bacterial virulence factors. We propose a specific role of mod and related restriction-modification systems in H. pylori in the regulation of virulence genes.
3990 INCREASED UREASE ACTIVITY OF H. PYLORI CONTRIBUTES TO IMPAIRED DUODENAL MUCOSAL BICARBONATE SECRETION IN DUODENAL ULCER. Akinori Yanaka, Hideo Suzuki, Akira Nakahara, Naomi Tanaka, Hiroshi Muto, Inst of Clin Med, Univ of Tsukuba, Tsukuba, Japan. It has been well known that approximately half of worldwide population is infected with H. pylori (Hp), but peptic ulcers develop only in less than 15 % of the infected subjects. Recent studies have suggested that Hps isolated from duodenal ulcer (DU) show higher urease activity and causes more severe damage in gastric mucosa than those obtained from gastritis (Gut 44: 456,'99). We have previously shown that Hp-derived vac A protein prepared from DU patients inhibits duodenal mucosal bicarbonate secre-
GASTROENTEROLOGY Vol. 118, No.4
tion (DMBS) in vitro (Gastro 114: A339, '98). Based on this background, we hypothesized that DU-derived Hps have higher urease activity and more increased acid resistance than Hps from chronic gastritis (CG), thereby contributes to development of DU. Methods: Hps were isolated from 15 DU, 15 gastric ulcer (GU), and 15 CG pts. 1. Effects of ambient pH on urease activity, pHi regulation, and viability of Hps. Urease activity was estimated from ammonia production in the presence of 5 mM urea. Regulation of pHi was evaluated by measurement of pHi at various ambient pHs (5.0 - 8.0), using a ph-sensitive fluorescein, BCECF. Viability of Hps at different ambient pHs (2.0 - 8.0) was evaluated by Bacterial Viability kits (Molecular Probe), which allows dual staining of the live and the dead Hps with SYTO-9 (Ex 470, Em 510), and with propidium iodide (Ex470, Em 620), respectively. 2. Effects of Hps on DMBS. Intact sheets of rat proximal duodenal mucosae were incubated in Ussing chambers in vitro. The serosal and the luminal sides were bathed with 25 mM HC03Ringer/95%02-5%_C02, and with 150 mM NaCI/lOO% 02, respectively. The mucosae were preincubated with live Hps in the luminal solution (pH 3.0) in the presence of 5 mM urea for Ihr . After removing the Hps from the luminal side, PGE2-stimulated DMBS was measured. Results: 1. In the presence of urea, Hps derived from DU showed higher urease activity, more excellent pHi regulation, and higher acid resistance than Hps from GU or CG, effects abolished by an urease inhibitor, acetohydroxamic acid (AHA) (0.5 mg/l), and mitigated by a PPI, rabeprazole (10 uM/l). 2. Pretreatment of the duodenal mucosa with DU derived Hps inhibited DMBS more prominently than that with Hps from GU or CG, effects abolished by the presence of AHA or rabeprazole. Conclusion: These results indicate that Hp strains isolated from DU show higher urease activity, more increased acid resistance, and more potent inhibition of DMBS than Hps from GU or CG, effects appear to be related to pathogenesis of DO.
3991 VITAMIN C AT CONTRATIONS COMPARABLE TO THOSE IN GASTRIC JUICE INHIBITS THE GROWTH OF GASTRIC CANCER CELLS, BUT NOT H.PYLORI. Zun-Wu Zhang, Mohamed Abdullahi, Michael 1. Farthing, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Vitamin C may be protective against the development of gastric cancer, but intra-gastric concentrations are significantly reduced by H pylori infection. However, the precise interactions between vitamin C, gastric epithelial cells and H. pylori are poorly understood. Methods: Gastric cancer cell lines AGS and MKN45, and H. pylori strains 11637, SSI and ND5 (a phospholipase mutant) were used. The effects of vitamin C on gastric cancer cell growth and DNA synthesis were determined using a trypan blue exclusion analysis and a BrdU incorporation ELISA. To assess the effect of vitamin C on H. pylori growth and adhesion to gastric cells, the bacterium was grown in Brain-heart infusion broth containing a range of concentrations of L-ascorbic acid (Sigma, UK). H. pylori growth was determined using viability count and spectrophotometry. The results are expressed as cfu/ml. H. pylori adhesion to gastric epithelial cells was analysed using immunofluorescence staining with fluorescence microscopy and flow cytometry. Results: Vitamin C induced a significant dose-dependent inhibition of cell growth and DNA synthesis in gastric cancer cell lines AGS and MKN45. At concentrations similar to those found in gastric juice of healthy subjects (2: 100 fLM), vitamin C was markedly cytotoxic to gastric cancer cells, but this effect was significantly reduced at levels similar to those in gastric juice of H pylori infected patients « 50 fLM). At levels comparable to those in gastric juice, it had no obvious effect on the growth of H. pylori strains, SSI and ND5. Although vitamin C had no effect on H. pylori adhesion to gastric AGS cells compared to untreated controls, it significantly enhanced H. pylori associated apoptosis and induced cell cycle arrest in these cells. Conclusion: Vitamin C inhibits the growth of gastric cancer cells at concentrations comparable to those in normal gastric juice, but had no effect on H. pylori growth or adhesion to gastric epithelial cells. However, the inhibitory effect on gastric cancer cells was lost at vitamin C concentrations similar to those found in gastric juice of H pylori infected patients.
3992 EFFECT OF H.PYLORl VIRULENCE FACTORS ON THE INDUC· TION OF APOPTOSIS AND CELL CYCLE PHASE PROGRES· SION OF GASTRIC AGS CELLS. Zun-Wu Zhang, Nick Dorrell, Brendan W. Wren, Michael J. Farthing, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom; London Sch of Hygiene & Tropical Medicine, London, United Kingdom. Abrogation of gastric epithelial cell homeostasis, a balance between cell proliferation and apoptosis, is a prominent feature of H. pylori associated gastroduodenal disease and may be responsible for the diversity of disease expression. Using an in vitro model of H. pylori infection, we examined the role of some bacterial virulence factors in the induction of gastric AGS cell apoptosis and cell cycle phase progression. Methods: Gastric cell lines AGS and H. pylori strains 11637 (cytotoxic), SSI and four isogenic mutants (ureB, pldA, cagE and jliQ) were used. Sub-confluent AGS cells were incubated with or without H pylori for 48h. Camptothecin served as an inducer of apoptosis and cell cycle arrest (positive control). Cell cycle phase distribution was analysed using flow cytometry with propidium
3988 IDENTIFICATION OF THE INTERACTION OF UREGIUREE AND UREFIUREH IN H. PYLORI USING A YEAST TWO-HYBRID SCREEN. Petra R. Voland, George Sachs, Vaglahs & UCLA, Los Angeles, CA. Background: Accessory proteins (Ure E, F. G. H) of the H. pylori urease gene cluster may interact with each other during urease assembly. The Yeast Two-Hybrid system is a method to characterize known protein interactions and to identify genes that encode interacting partners. Aim: Identification of H. pylori proteins interacting with the accessory proteins of the urease gene cluster. Methods: The Yeast Two-Hybrid System (Matchmaker II, Clontech) was used to screen a genomic DNA library of H. pylorifor proteins interacting with UreE, F, G and H. A high-efficiency mating procedure was applied. Diploid clones were tested for ~ galactosidase activity with a lift assay to identify clones containing interacting proteins. Plasmid-DNA was isolated from blue clones for restriction analysis, DNA-sequencing and co-transformation into yeast. After lift assay, a subset of blue clones underwent a quantitative chemiluminescent ~ga lactosidase assay. Interactions were verified by co-immuno-precipitation of 35S_Cys labeled in vitrotranscriptionltranslation products of UreE, G, F and H using antibodies against c-Myc and HA tags. Results: Using UreG as bait in a library screen we found 36 blue clones in the colony lift assay. Further analysis by restriction enzyme digest and DNA sequencing identified sequences coding for UreE. The interaction between UreGlUreE was quantified by f3-galactosidase assay with 5 independent fish -clones showing 20-55x higher activity than controls. Using UreF as bait we found 53 blue clones. Restriction analysis and DNA sequencing revealed that 17 clones contained sequences coding for UreH. Interaction of UreFlUreH was quantified by f3-galactosidase assay with 4 independent clones being 1O-250x higher than controls. We confirmed the interaction between UreF/ UreH by co-immuno-precipitation of 35S_Cyslabeled in vitrotranscriptionl translation products. The interacting proteins UreFIH could be co-precipitated with antibodies against the fusion tag (c-Myc, HA) of each partner. Conclusions: The Yeast Two-Hybrid screen shows strong interaction of UreGlUreE and UreFlUreH, confirmed by co-immuno-precipitation of in vitro translation products. These proteins thus form a pair of complexes necessary for assembly and NiH insertion into the apoenzyme.
3989 PHASE VARIATION IN A TYPE m RESTRICTION-MODIFICA· TION SYSTEM OF HEUCOBACTER PYWRI. Nicolette Vries de, Dirk Duinsbergen, Ernst J. Kuipers, Patricia Wiesenekker, Christina M. Vandenbroucke-Grauls, Johannes G. Kusters, Vrije Univ, Amsterdam, Netherlands. Objective:The on- and off-switching of the expression of virulence factors (phase variation) plays an important role in the pathogenesis of many bacterial infections. LPS phase variation in Helicobacter pylori occurs at the translational level and has been studied well. In contrast, phase variation at the transcriptional level has not been demonstrated in H. pylori. Therefore, we investigated transcriptional on- and off-switching of gene expression in H. pylori. Methods: A library with random genomic transcriptionallacZ fusions in H. pylori strain 1061 (HPI061) was screened for mutants that showed blue and white sectored colonies on X-Gal. As the X-Gal substrate is converted into a blue product when the lacZ gene is expressed into the f3-galactosidase, sectored colonies indicate transcriptional phase variation. Results: One HP1061 mutant displayed frequent onand off-switching of lacZ expression. The on-to-off switch frequency was 2.67 %, while the off-to-on frequency was 0.75 %. Sequencing revealed that the lacZ gene was fused to a putative type III methylase gene (mod). RNA spot blot hybridization demonstrated that specific lacZ and mod probes bound to mRNA from blue colonies, but not to mRNA from white colonies. This proved that mod switched on and off at the transcriptional level. An open reading frame, encoding a putative type III restriction enzyme gene (res), is located immediately upstream of mod and contains a polynucleotide C-tract. This Cvtract, which may cause phase variation of res through translational frameshifts, might indirectly act on the transcription of mod. However, sequence analysis showed that the number of cytosines in res was not related to the on- or off-status of mod. Conclusion: In H. pylori. the putative type III methylase gene, mod, displays phase variation at the transcriptional level. It is known that methylation of promoter sequences can affect the transcription of bacterial virulence factors. We propose a specific role of mod and related restriction-modification systems in H. pylori in the regulation of virulence genes.
3990 INCREASED UREASE ACTIVITY OF H. PYLORI CONTRIBUTES TO IMPAIRED DUODENAL MUCOSAL BICARBONATE SECRETION IN DUODENAL ULCER. Akinori Yanaka, Hideo Suzuki, Akira Nakahara, Naomi Tanaka, Hiroshi Muto, Inst of Clin Med, Univ of Tsukuba, Tsukuba, Japan. It has been well known that approximately half of worldwide population is infected with H. pylori (Hp), but peptic ulcers develop only in less than 15 % of the infected subjects. Recent studies have suggested that Hps isolated from duodenal ulcer (DU) show higher urease activity and causes more severe damage in gastric mucosa than those obtained from gastritis (Gut 44: 456,'99). We have previously shown that Hp-derived vac A protein prepared from DU patients inhibits duodenal mucosal bicarbonate secre-
GASTROENTEROLOGY Vol. 118, No.4
tion (DMBS) in vitro (Gastro 114: A339, '98). Based on this background, we hypothesized that DU-derived Hps have higher urease activity and more increased acid resistance than Hps from chronic gastritis (CG), thereby contributes to development of DU. Methods: Hps were isolated from 15 DU, 15 gastric ulcer (GU), and 15 CG pts. 1. Effects of ambient pH on urease activity, pHi regulation, and viability of Hps. Urease activity was estimated from ammonia production in the presence of 5 mM urea. Regulation of pHi was evaluated by measurement of pHi at various ambient pHs (5.0 - 8.0), using a ph-sensitive fluorescein, BCECF. Viability of Hps at different ambient pHs (2.0 - 8.0) was evaluated by Bacterial Viability kits (Molecular Probe), which allows dual staining of the live and the dead Hps with SYTO-9 (Ex 470, Em 510), and with propidium iodide (Ex470, Em 620), respectively. 2. Effects of Hps on DMBS. Intact sheets of rat proximal duodenal mucosae were incubated in Ussing chambers in vitro. The serosal and the luminal sides were bathed with 25 mM HC03Ringer/95%02-5%_C02, and with 150 mM NaCI/lOO% 02, respectively. The mucosae were preincubated with live Hps in the luminal solution (pH 3.0) in the presence of 5 mM urea for Ihr . After removing the Hps from the luminal side, PGE2-stimulated DMBS was measured. Results: 1. In the presence of urea, Hps derived from DU showed higher urease activity, more excellent pHi regulation, and higher acid resistance than Hps from GU or CG, effects abolished by an urease inhibitor, acetohydroxamic acid (AHA) (0.5 mg/l), and mitigated by a PPI, rabeprazole (10 uM/l). 2. Pretreatment of the duodenal mucosa with DU derived Hps inhibited DMBS more prominently than that with Hps from GU or CG, effects abolished by the presence of AHA or rabeprazole. Conclusion: These results indicate that Hp strains isolated from DU show higher urease activity, more increased acid resistance, and more potent inhibition of DMBS than Hps from GU or CG, effects appear to be related to pathogenesis of DO.
3991 VITAMIN C AT CONTRATIONS COMPARABLE TO THOSE IN GASTRIC JUICE INHIBITS THE GROWTH OF GASTRIC CANCER CELLS, BUT NOT H.PYLORI. Zun-Wu Zhang, Mohamed Abdullahi, Michael 1. Farthing, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Vitamin C may be protective against the development of gastric cancer, but intra-gastric concentrations are significantly reduced by H pylori infection. However, the precise interactions between vitamin C, gastric epithelial cells and H. pylori are poorly understood. Methods: Gastric cancer cell lines AGS and MKN45, and H. pylori strains 11637, SSI and ND5 (a phospholipase mutant) were used. The effects of vitamin C on gastric cancer cell growth and DNA synthesis were determined using a trypan blue exclusion analysis and a BrdU incorporation ELISA. To assess the effect of vitamin C on H. pylori growth and adhesion to gastric cells, the bacterium was grown in Brain-heart infusion broth containing a range of concentrations of L-ascorbic acid (Sigma, UK). H. pylori growth was determined using viability count and spectrophotometry. The results are expressed as cfu/ml. H. pylori adhesion to gastric epithelial cells was analysed using immunofluorescence staining with fluorescence microscopy and flow cytometry. Results: Vitamin C induced a significant dose-dependent inhibition of cell growth and DNA synthesis in gastric cancer cell lines AGS and MKN45. At concentrations similar to those found in gastric juice of healthy subjects (2: 100 fLM), vitamin C was markedly cytotoxic to gastric cancer cells, but this effect was significantly reduced at levels similar to those in gastric juice of H pylori infected patients « 50 fLM). At levels comparable to those in gastric juice, it had no obvious effect on the growth of H. pylori strains, SSI and ND5. Although vitamin C had no effect on H. pylori adhesion to gastric AGS cells compared to untreated controls, it significantly enhanced H. pylori associated apoptosis and induced cell cycle arrest in these cells. Conclusion: Vitamin C inhibits the growth of gastric cancer cells at concentrations comparable to those in normal gastric juice, but had no effect on H. pylori growth or adhesion to gastric epithelial cells. However, the inhibitory effect on gastric cancer cells was lost at vitamin C concentrations similar to those found in gastric juice of H pylori infected patients.
3992 EFFECT OF H.PYLORl VIRULENCE FACTORS ON THE INDUC· TION OF APOPTOSIS AND CELL CYCLE PHASE PROGRES· SION OF GASTRIC AGS CELLS. Zun-Wu Zhang, Nick Dorrell, Brendan W. Wren, Michael J. Farthing, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom; London Sch of Hygiene & Tropical Medicine, London, United Kingdom. Abrogation of gastric epithelial cell homeostasis, a balance between cell proliferation and apoptosis, is a prominent feature of H. pylori associated gastroduodenal disease and may be responsible for the diversity of disease expression. Using an in vitro model of H. pylori infection, we examined the role of some bacterial virulence factors in the induction of gastric AGS cell apoptosis and cell cycle phase progression. Methods: Gastric cell lines AGS and H. pylori strains 11637 (cytotoxic), SSI and four isogenic mutants (ureB, pldA, cagE and jliQ) were used. Sub-confluent AGS cells were incubated with or without H pylori for 48h. Camptothecin served as an inducer of apoptosis and cell cycle arrest (positive control). Cell cycle phase distribution was analysed using flow cytometry with propidium