High prevalence of functional type IV secretion system for CagA protein in Japanese clinical Helicobacter pylori

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Diversity of the cag7 and cag2 Genes in H. Pylori Ouanjiang Dung, Adelaide & Meath Hosp. Trinity Coil, Dublin Ireland; Sinead Byme, Colm A. O'Morain, Martin Buckley, Adelaide & Meath Hosp, Dublin Ireland

Helicehaofer Pylori Vacenlating Cylotoxin (VacA) Inhibits Tubulovenicle Fusion Possible Mechanism For The INibition Of Acid Secretion in Isolated Gastric Glands Semen Karvar, Joseph G. Duman, Xuebiao Yao, Rihong Zhou, Univ OF CA AT Berkeley, Berkeley, CA; Timothy L. Cover, DEPTOF MEDICINE,VANDERBILTUniv Sch OF MEDICINE, Nashville,TN; John G. Forte, Univ OF CA AT Berkeley,Berkeley,CA

Background:The can pathogenecityisland of He/icobacterpy/oriisassociatedwith the severer mucosal damageandthe developmentof duodenalulcer and gastric cancer. It shows significant struture diversity and microvariability in single genes. Aim: To investigatethe relationship of the diversity of the cog7 and can2 genes to the H. pylori associated diseases, Methods: A tntal of 80 ca~;Npositive isolates, including 34 isolates from gastritis, 44 from peptic ulcer and 2 from gastric cancer were enrolled in the study. H. pyioristrain NCTC11638 was used as the reference. The diversity of the can7 and can2 genes was investigated using PCR, southern blot and sequencing. Results: (1) The can7 genotypes: The first repeat region of the cagTgenewas detected using PCR. The absenceof the cagTgene was demonstrated by PCR and southern blot. The size of the the first repeat region was variable. Five types were identified according to the size and the sequenceanalysis (table). Type A and U were more prevalent in gastritis isolates than in ulcer isolates (p
repeat regions

gastritis

peptic ulcer

9astxl¢cancer

A B C D U

>3 2 1 1 Absent

9 14 1 1 5

2" 33 4 4 1*

0 2 0 0 0

BACKGROUND:Colonizationof the human gastric epithelium by Helicobacterpylori (H. pylori) is strongly associatedwith peptic ulceration, MALT-lymphoma,and adenocarcinoma.H. pylori strains cenying the rag pathogenicity island and expressingan active VacA toxin have been associatedwith an increasedrisk for peptic ulcer diseasecomparedto strains that lack these determinants. Here we studied the effect of purified VacA toxin on acid secretion in isolated rabbit gastric glands. METHODS:Gastricglands were isolatedfrom rabbit stomach and treated with acid-activated VacA toxin (1-5p,g/ml) for 4 hr in the absence of ammonium chloride. After that the glands were stimulated with histamine and IBMX for 30 min. Acid secretion was measured by 14C Aminopyrine accumulation. To test possible effect of vacA toxin on intracellnlar membrane fusion, parietal cell tubulovesicles and apical membranes were collected.Fusion betweentubulovesiclesand tubulovesicleswith apical membraneswas measured using an in vitro octedecylrhodamine(R18) dequenchingassay. The cells were stained for H,K-ATPase and F-aetin. RESULTS:VacA toxin had a small but significant dose-dependent inhibitory effect on histamine stimulated acid secretion (1/,,,gVacA toxin 20.5_+3.42%, 5pO VacA toxin 31.1_+4.5% inhibition There was no effect of VacA on acid secretion unstimulated glands. In parallel, we detected about 20-30% inhibition of tubulovesicle fusion using the R18 degnenching assay. Addition of hafilOmydn A1 (a known inhibitory of VacA-induced vacuole formation in intact cells), did not relieve the inhibition. No changes were detected based on H,K-ATPaseand F-Actin staining. CONCLUSION:Clinical and experimental studies havedernor~batedthat acid secredonis inhibited by acute H. pylori infection. The mechanisms of these phenomena are still unknown. Our results suggest that VacA toxin has a negative effect on tubulovesiclefusion. It is possible that reducedacid secretion observed in vivo, and here in vitro, may be relatedto some effect on membranefusion. The fusion inhibitory effect of VacA toxin was not dependenton V-ATPaseand may be mediated by altered function of docldng~sien proteins (such as Synta,dn-3 or VAMP-2), or regulatory proteins (such as rablla or rob25). The presentobservationssuggest that VacA toxin inhibits membranefusion and thus may decreasethe acid output in parietal cells.

* ThepercentageoftypeAandtypeUwashigherinthegastfitisisolatesthanin pep~culcer isolates H. pylod Stralen whiz the Novel Gime MboX Are Conelated with Gastric Cancer in ACiN I ~ Takofumi Acdo, New York Univ Sch of Medicine, New York, NY; Richard M PeekJr., Vaoderbilt Univ Sch of Medicine, Nashville, TN; Kazoo Kusugami, Nagoya Univ Sch of Medicine, NagoyaJapan; Lean-JanVan Doom, Delft Diagnostic Lab, Delft The Netherlands; Tn~y Wassenasr, Fdn for Bacteriology,Arlington, VA; Sung-Kook Kim, Vanderbilt Univ Sch of Medicine, Nashville, TN; Martin J. Blaser, New York Univ Sch of Medicine, New York, NY

3533 Test of the Hypothesisthat Triple Positive I~llcobacter pylm'l (ca~4, Tara sl and babA2 Gene) Has Predictive Value with Relation to Clinical Preenntatlen. Yoshio Yamaoka, Julia Souchek, VAMC and Beylor Coil of Medicine, Houston, TX; Tadashi Kudama, Kyoto Prefectural Univ of Medicine, Kyntn Japan; Rainer Haas, Max Von Pettenkofer-lnstltut, Munchen Germany; Kei Kashima, Kynto Prefectural Univ of Medicine, Kyoto Japan; Michael S. Osato, VAMC and 8aylor Coil of Medicine, Houston, TX; Oscar Gutierrez, Univ Nacionai de Colombia, Bogota Colombia; Jong G, Kim, Guru Hosp and Korea Univ Coil of Medicine, Seoul South Korea; David Y. Graham, VAMC and Baylor Coil of Medicine, Houston, TX Background:Theability to identity/-/, py/ori virulence factors that predicted an increased risk for symptomatic clinical outcomes would be useful. Basedon a small sample,it was suggested that strains triple positive for cagA, vacAsl and babA2wereassociatedwith duodenal ulcer. We examinedthe cagA, vacAand babAgenotypes of H. py/orifrom 4 countries with respect to the disease presentation. Methods: PCR was used to examine caO pathogenicity island (PAl), vacA,and babAstatus of 1,114 H. py/oriisolatesfrom patients with peptic ulcer, gastric cancer, and gastritis (Colombia 196, Houston 282, Korea 275, Japan 361). PCR sensitivity and specificity of the can PAl status (combination of 5 sets of primers including ca~l, cagE and cagG) was 98% and 100%, respectively, using Southern hybridization and Western blotting analysis using antibodies raised against recombinantCaoAprotein as standard (n = 220). PCR sensitivity and specificity of the babA2status (combination of 2 sets of primers) was 97% and 98%, respectively, using Western blotting analysis using antibodies raised against recombinant BabA2 protein as standard (n = 260). Results: Triple positive isolates for can PAl, vacA sl and babA2were the most common strains in every country. Although triple positive isolates were less common in gastritis cases compared with peptic ulcer, the differences were not great and were only statistically significant for the US cases (Korea: 86% vs. 93%, Japan: 88% vs. 94%, Colombia: 62% vs. 70% and US: 62% vs. 82%, p = 0.002). vacA genotype, can PAl and babA2 positivlty alone were also independent of the clinical presentation except for the US cases (US: vacA s type: 81% vs. 94%, p = 0.002, can PAl: 83% vs. 96%, p = 0.001, bahai. 72% vs. 86%, p = 0.01). The cagA gene was closely associatedwith the presenceof the babA2or vacAsl types in the U.S. and Colombia (p< 0.0001) but not in Japan or Korea (e.g. all 636 isolates from Japan and Korea were vacA sl). Conclusion: Triple positive status gives no additional information compared with the can PAl status, vacAsl and the babA2genotypesare surrogates for the presenceof the can PAl. Although can PAl positivity was statistically higher in peptic ulcer than in gastritis cases in the US, the overall high can PAl posltivlty in gastritis cases (63%) suggests that neither can PAl nor any combination of the genes are useful in predicting the clinical outcome of an H. pylori infection.

Backgrouod: H. pylori exhibits substantial genomic diversity that at least in part influences clinical outcome. Type II r e - - m o d i f i c a t i o n (R-M) systems are highly diverse between strains, a characteristic unique for H. pylori. Hpylll, a restriction enzymefound in H. pylori, is highly homologous to Mpol which recognizesGATC.We sought to determinethe variability of hwIII R-M genotyposin H. pylori strains and whether differences are linked to geographic origin or clinical features, Method: We examined 228 strains (US 50, South America 12, Europe 15, Asia 147, Oceania4), by assessing digestion of chromosomal DNA by Mbol, and by performing PCR for hpyllL We assessed the correlation between hpylll status, other genotypes, and clinical features among 182 strains (50 US, 85 Japan, 28 China, and 19 Korea). cagA, vacA, and iceA status were determined by PCR and LIPA. Results: Eleven(5%) of 228 strains tested were Mpol-sensitive, and by PCR, 10 (91%) of these 11 did not have hpylllR. In these 10 strains, we identified a novel gone (that we termed MPOX)in the hpylll position, followed by the cognate methylase, hpylllM. MPOXhas substantial homology with C. jejuni Cj1602, but no function is known for either. Strain 88-29 which is Mbol-sensitive, possesseshpyltl, hut the upstream promotor is truncated. We next examinedthe hpylli locus in 217 Mbol-resistant strains, and found that 148 (68%) have hpylllR, and 69 (32%) have MboX. In Western countries, MboX is more prevalent (55%) than in Asia (25%) (p
High Prevalence of Feneffoenl Type IV Secretion System for CagA Protein in Japanese Clinical Hellcobacter pylori Yoshihiro Hirats, Shin Maeda,Yuzo Mltuno, Masao Akanuma, Takao Kawabe,Haruhiko Yoshida, Yasushi Shiratori, Mason Omata, Univ of Tokyo, Tokyo Japan Background/Aim:CagAprotein of He/icobacterpyloriis reportedlysecreted into epithelial cells via type IV secretion system encoded by can PAl. We have shown this system is crucial for the pathogenesisin Mongolian gerbils. The aim of this study is to elucidate the functionality of type IV secredonsystem. The size of CagAprotein, reportedlyto be associatedwith gastric diseases, was also examined. Methods: H. py/ori strains isolated from 50 Japanesepatients (25 from gastric cancer patients and 25 from control) were co-cultured with AGS cells for 10 hrs. Functionality of type IV secretion system was evaluated by immunoprecipitation of tyrosin-phosphorylated CegA protein. TN2 strain was used as a positive control, and cagE knocked out strain was used as a negative control. Molecular weight of CagA protein was

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also analyzedby immunoblot analysis. Results:Tyrosin phosphorylationof CagAwas induced by 96% of Japaneseclinical isolates (100% in strains from gastric cancer and 92% in strains from other diseases).Two strains negativefor tyrosin-phosphorylationof CagAdid not produce CagA protein. Most strains (46 strains) had low molecular size CagA protein (128-135kDa), and only 2 strains had high molecular weight CagA protein (138kDa-). Conclusions: Almost all strains in Japan have functional type IV secretion system. This is compatible with our previous report that cag PAl genes are preservedin 97% of Japaneseclinical isolates. High prevalenceof functional type IV secretion system may reflect high prevalenceof gastric cancer in Japan. The size of CagA protein seems to have little association with gastric cancer.

medium 3-fold higher than that of gastric cells exposed to medium alone or gastric cells exposed to HP. The addition of ethanol to culture medium containing HP increased release of LDH into the culture medium 14% (range 5% to 21%) above that of cells cultured in medium alone, indicating that HP protectedgastric cells from ethanol inducedtoxicity. Soluble bacterial protein (obtainedfrom sonicating bacterial strains) was pre-exposedto gastric calls for one hour followed by exposureto 2 mM ethanol for one hour. LDH releaseinto the culture medium increased only 27% (on average) above that of gastric cells cultured in medium alone. The ADH activity of these HP strains varied from 0.8 to 3.9 U/ug protein. AOt-Iprotein content in these bacterial strains was identified by western blot and quantitated by scanning densitometry. ADH activity varied between HP strains with respectto ADH protein levels.AGS cells were also found to possessADH activity. In separateexperimentsAGScells were exposed overnight to cag-pos liP strains. Theseexperimentsresulted in an inversecorrelation between ADH activity and LDH release.HP ADH does not appearto be associatedwith directly toxicity to gastric epithelial cells and there was a trend toward less cytotoxicity with increasingADH activity of strains. Live bacteriaand bacterialproteins can protect againstacute ethanol induced cytotoxicity to gastric epithelial cells. One possible reason for this protection is that HP has an enzymewith activity similar to aldehydedebydrognnasewhich can metabolizeacetaldehyde.

3537 Refinement and Development of a "Dry" Novel 14C-Urea Breath Test (HeliprobeTM) for Detection of Helicobacter pylori Olga Hegedus,Ann-Sofie Rehnberg, Medicine, KarotioskaHosp, Stockholm Sweden; Carina Bengtsson, Clin Microbiology, Karolinska Hosp, Stockholm Sweden; Ragnar Befrits, Medicine, Karolinska Hosp, Stockholm Sweden; Marta GranstrSm, Clin Microbiology, Karolinska Hosp, Stockholm Sweden; Per M. HellstrOm, Medicine, Karolinska liosp, Stockholm Sweden

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Background: linlicobacter pytori (Hp) is recognizedas etiological factor for chronic gastritis, peptic ulcer and gastric malignancies.For detection of Hp, urea breathtest (UBT) is recognized as the most accurate non-invasivediagnostic test. UBT is cumbersome becauseof sampling techniques, and expensive and inaccessible analysis equipment. We have developed and validateda novel "dry" ~4C-UBT(HeliprobeTM) that simplifies the test procedureand eliminates the need for expensiveanalysis equipment. Description of Helipmbe test: After ingestion of ~4C-labeledurea,the patient exhalesinto a BreathCardTMwhich traps the labelled~4CO2.Analysis is done in the doctor's office via a cheap dnsktop-sizedcustomized analyserbasedon GeigerMdller counters. The result is thereby accessible within 5 min of test completion. Methods: After intake of ~4C-urea,regular UBT was done by saturatedtrapping of ~4C02in bnnzethoniumhydroxide/ethanol.Serology by ELISA, enhanced by western immunoblot analysis, was used as referencefor confirmation of presence or absence of Hp in antibiotic-na 3538 Full-Length Sequence Analysis of the cagE Gene of Helicobacterpylori isolates Obtained from Two Areas of Japan with Different Prevalence of Gastric Cancer. Kanako Fukuta, Toyama Medical & Pharm Univ, Toyama Japan; Takeshi Azuma, Yoshiyuki Ito, Masaru Kuriyama, Fukui Medical Univ, Fukui Japan; Hiroyuki Wakabayashi,Akiharu Watanabe, Toyama Medical & Pharm Univ, Toyama Japan Background:The cag pathogenicityisland (cag PAl) is a 40-kb fragment inherited by horizontal transfer from an unknown microorganism. Six of the cag genes are thought to be encoded by a putative type IV secretion system that is able to inject toxic proteins into enkaryutic cells. Recently,we reported that CagA is delivered from attached H. pylori into the epithelial cells by the cagtype IV secretion system where it is phosphorylatedon tyrosine residue and wired to eukaryotic signal transduction pathways, which is likely to play a major role in H. pylorkhost cell interactions and pathogenesis.The cagE gene is located upstream of cagA and considered to be one of the constructural genes for the type IV secretion system. Aim: To analyzethe full-length sequencesof cagEfrom H. pyloristrains obtained from two different areas of Japan (Fukui and Okinawa), in which the prevalenceof atrophic gastritis and the gastric cancer risk were quite different to clarity strain diversity associatedwith pathogenesis, Methods: 81 H. pyloriisolatns (42 Fukui strains; 18 gastritis strains, 12 duodenalulcer strains, and 12 gastric ulcer strains, 39 Okinawastrains; 18 gastritis strains, 11 duodenalulcer strains, and 10 gastric ulcer strains) were evaluated for cagE status by PCR. All 81 isolates were previously checked the cagA status and the genotype of vacA. We selected 18 isolates randomly, sequencedand analyzedthe full-length cagEgene. We also examinedthe phylognnntic relationships between 18 clinical isolates and 4 previously published strains from the West. Results: In this study, 80 of 81(98.8%) isolates were positive for cagE. There were no significant differences in the prevalence rate of cagE gene among disease groups in both areas. Phylogenetic analysis showed the difference of cagE sequences between Fukui and Okimawa strains. 3 of 50kinawa strains formed the same cluster as the West. 2 of 30kinawa strains in the same cluster as the West were m2 genotypeof vacA middle region. In contrast, most Fukui strains formed a different cluster from the West. The pattern of phylogenetictree was different between cagE and cagA, although these two genes were located in can PAl cassettethat was inherited by horizontaltransfer. There were no sequencediversities in cage gene associatedwith clinical outcome. Conclusions:There were no diseasespecific diversities in cagE gene in Japan. Free recombination may occur even in rag PAl.

The DNA SUmulatory Effect of Halicobacterpylori Lipopolysaccharide Is Related to the Clinical Virulence Fingerprint of Gastric Cancer Isolates Mark S. Kidd, Albert J. Lastovica, Univ of Cape Town, CapeTown South Africa; Guillermo I. Perez-Perez,New York Univ, New York, NY; Laura H. Tang, In/in M. Modlin, Yale Univ, New Haven, CT; Japie A. Louw, Univ of Gape Town, Gape Town South Africa Background/Aims: Infection with H. pylori strains with differences in virulence genes is associated with gastroduodenal disease in African populations. An intact rag pathogenicity island (PAl) and vacuolating vacA subtype el, but not cagA per se appear relevant. The mechanisms by which H. pyloriaffect cell proliferation are still unclear, but we havepreviously demmonstrated a role for lipopolysaccharidn in vitro. We postulated that LPS activity may be related to the virulence of the infecting strain and may provide a mechanism to alter cell function. Methods: LPS was isolatedfrom twelve genotypicallycharacterizedstrains (4 = nonulcer dyspeptics [NUO], 4 = peptic ulcer disease [PUD], 4 = gastric cancer [GC]). Synchronized gastric adenocarcinomaAGS cells were incubated with LPS (0.1mg/ml) for 4 hrs and DNA synthesis (measured by 3H-thymidineuptake) and apoptosis (Sob'Ga)evaluatedby flow cytometry. Control LPS was from H. pylori 84-183, C. jejuni and E. colt (055:5B). Conclusion: H. pylori LPS specifically mediates AGS cell DNA synthesis but not apoptosis compared to non-gastric organisms. Although H.pylori isolates from patients with PUD or GC shared an intact cag PAl and a vacA subtype sl, the increased DNA synthesis noted in GC isolates was associated with alterations in the cagA gene (increased 3'-fragment length). These findings suggest that LPS from GC patients may differentially alter gastrointestinal cell cycle events which may be of clinical relevance. Isolate

DNAsynthesis

Apoptosis

H. pylori84-183 E. colt 055:5B C. jejuni NUD PUD GC

5,2~1.t* 1.3±0.3 1.2_+0.3 1.2._+0.3 1,5..~0.1 4.8±1.6"+

0.9~0.2 1-z0.1 1.3±0.3 1.3±0.1 1.2±0.1 1~.1

*p<0.05vs. C. jejuni and E. colt +p
Helicobacter pylori Enhances an Expression of Inducible Nitric Oxide Synthase (iNOS) in Human Gastric Epithelium Qonsheng Song, Marilyn Owens, Emory Univ Sch of Medicine, Atlanta, GA; OuaneT. Smoot, HassanAshktorab, Howard Univ Sch of Medicine, Washington, DC; Benjamin D. Gold, Emory Univ Sch of Medicine, Atlanta, DC BACKGROUND:H. pyloriis a major etiologicalfactor of chronic gastric diseases.The pathobiolcoy of gastric lesions in H. py/ori infection remains poorly characterized. Previous studies showed that H. pyioriincreased expressionof inducible nitric oxide synthase(iNOS) in mouse gastric cell line. Studies on human gastric biopsies suggested an increased expression of iNOS on H. pylori-infected gastric mucosa. However,there is little data evaluatingthe role of H. py/ori and nitric oxide (NO) production in vitro, which quantitatively assess iNOS gone, enzyme and final levels of NO. AIM: To investigate iNOS gone expression of human gastric epithelium and its association with H. pylori infection using cell lines and primary gastric epithelial cells. METHODS:Gastric epithelial cells (three gastric epithelial cancer cell lines, ATCC; and two primary gastric epithelial cell lines) were grown in cell-specific culture media, with or without coincubationof human monocytnsfor 24 hours, followed by H. pyloriinfection. After incubation in C02 incubator at 37°C for 1,7, and 24 hours, media supernatants were assessedfor nitric oxide (NO2-)production. Cellsattachedin flasks were washedand collected for RNA isolation. RT-PCR followed by southern hybridization using P3Z-labeledprobe was used to analyze iNOS mRNA expression./y--actin mRNA was used as an internal control. RESULTS:RT-PCRshowedthat all gastric epithelial cells constitutively expressediNOSmRNA at baseline. Gastric epithelial cancer cell lines expressed greater iNOS mRNA than primary gastric epithelial cells. When gastric epithelial cells were infected with H. pylori without monocyte co-culture, iNOS mRNA and supernatant nitrites had no significant change or slightly decreased. However, after coincubation with monocyte and H. pylori, iNOS mRNA expression was significantly increased in primary gastric epithelial cells, but not changed significantly in the gastric epithelial cancercells. All these cells showeda concurrent increased nitrite production in the supernatant following incubation with monocytas and H. py/ori. CONCLUSION:All gastric epithelial cells, tissue culture (cancer) and primary, tested in our study, constitutively express iNOS mRNA; gastric epithelial cancer cell lines >primary gastric

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H. pylori May Protect Gastric Epithelial Cells from Acute Ethanol Cytotoxicity Tahmoures Dehesh, Gregory Winston, Anm M. Rahman, Amel Ahmed, Cornnll R. Allen, Hassan Ashktorab, Howard Univ, Washington, DC; Byron Cryer, Univ of Texas Southwestern Medical Ctr, Dallas,TX; Man Go, Veterans Admin Hosp, Salt Lake City, UT; Duane T. Smoot, Howard Univ, Washington, DC A long history of alcoholism is associatedwith a reducedprevalenceof H. pylori(HP) infection. It is not clear how H. pylorigastritis is affected by ethanol ingestion.Acute alcohol cytotoxicity is felt to be due to metabolism of ethanol to acetaldehydeby alcohol dehydrognnase(ADH). HP possessesan ADH, but not aldehydedebydrogenasewhich metabolizesacetaldehyde.The following experimentswere conductedto determinewhether or not ethanoltoxicity is additive to HP indoced toxicity to gastric epithelial cells. A human gastric carcinoma cell line (AGS) was pre-exposedto HP strains (8 cag-pos strains were used) for one to three hours then exposed to 2 mM ethanol for one hour. Gastric cells were then analyzedfor cytotoxicity by measuring releaseof LDH into the culture medium. Exposureof gastric cells to HP for 1 to 3 hours did not increase the release of LDH into the culture medium above that of cells cultured in medium alone. The addition of ethanol increased release of LDH into culture

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