Helicobacter pylori cag-Type IV Secretion System Facilitates Corpus Colonization to Induce Precancerous Conditions in Mongolian Gerbils
2005; Elsevier BV; Volume: 128; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.02.064
ISSN1528-0012
AutoresGabriele Rieder, Juanita L. Merchant, Rainer Haas,
Tópico(s)Eosinophilic Esophagitis
ResumoBackground & Aims: Epidemiological studies suggest that atrophic corpus-dominant gastritis is an increased risk factor for gastric carcinogenesis. The role of the Helicobacter pylori type IV secretion system (T4SS) for pathogenesis in the Mongolian gerbil model was explored. Methods: Mongolian gerbils were infected for 32 weeks either with H pylori type I strain B128 or with isogenic mutant strain B128Δ cytotoxin-associated gene (cagY) or B128ΔcagA, defective in T4SS or in the production of its effector protein CagA, respectively. Quantitative H pylori reisolation was performed from the gastric antrum and corpus separately, cytokines were measured by quantitative reverse-transcription polymerase chain reaction, and gastric pH and hormones were determined. Results: B128-infected gerbils harbored high numbers of bacteria in the gastric antrum and corpus, whereas B128ΔcagY and B128ΔcagA colonized the antrum more densely than the corpus. All infected animals showed a strong antral inflammation and epithelial cell proliferation. B128-infected, rather than mutant-infected, gerbils presented a severe transmural inflammation with huge lymph aggregates, increased proliferation, significant atrophy, and mucous gland metaplasia in the corpus. Plasma gastrin levels and gastric pH values were significantly increased only in B128-infected gerbils. In all infected animals, the expression of the proinflammatory cytokines interleukin 1β, interferon γ, and growth-regulated protein was considerably increased in the antrum, but only in wild type-infected animals was an increase seen in the corpus mucosa. Conclusions: The presence of an intact T4SS allows H pylori to colonize the gastric corpus. This results in atrophic corpus-dominant gastritis, a severe precancerous condition, thus highlighting T4SS and CagA as major risk factors for gastric cancer development. Background & Aims: Epidemiological studies suggest that atrophic corpus-dominant gastritis is an increased risk factor for gastric carcinogenesis. The role of the Helicobacter pylori type IV secretion system (T4SS) for pathogenesis in the Mongolian gerbil model was explored. Methods: Mongolian gerbils were infected for 32 weeks either with H pylori type I strain B128 or with isogenic mutant strain B128Δ cytotoxin-associated gene (cagY) or B128ΔcagA, defective in T4SS or in the production of its effector protein CagA, respectively. Quantitative H pylori reisolation was performed from the gastric antrum and corpus separately, cytokines were measured by quantitative reverse-transcription polymerase chain reaction, and gastric pH and hormones were determined. Results: B128-infected gerbils harbored high numbers of bacteria in the gastric antrum and corpus, whereas B128ΔcagY and B128ΔcagA colonized the antrum more densely than the corpus. All infected animals showed a strong antral inflammation and epithelial cell proliferation. B128-infected, rather than mutant-infected, gerbils presented a severe transmural inflammation with huge lymph aggregates, increased proliferation, significant atrophy, and mucous gland metaplasia in the corpus. Plasma gastrin levels and gastric pH values were significantly increased only in B128-infected gerbils. In all infected animals, the expression of the proinflammatory cytokines interleukin 1β, interferon γ, and growth-regulated protein was considerably increased in the antrum, but only in wild type-infected animals was an increase seen in the corpus mucosa. Conclusions: The presence of an intact T4SS allows H pylori to colonize the gastric corpus. This results in atrophic corpus-dominant gastritis, a severe precancerous condition, thus highlighting T4SS and CagA as major risk factors for gastric cancer development. 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Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils.Am J Pathol. 1999; 154: 951-960Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar By evaluating the antral and corpus mucosa separately, it was possible for the first time to dissect the local contributions of bacterial virulence factors individually, leading to the postulated sequelae of cancer development. We present data showing a crucial role for the T4SS and CagA translocation in gastric corpus colonization, modulation of gastric physiology, and disease induction related to an atrophic corpus-dominant gastritis, which is a precancerous condition. Fifty-five female Mongolian gerbils (Meriones unguiculatus; RCC Ltd, Itingen, Switzerland; outbred animals), 16–20 weeks old, were used under specific pathogen-free conditions. All experiments and procedures performed were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Regierung von Oberbayern (AZ 211-2531-43/02). The streptomycin-resistant H pylori strain B128 (type I, VacA, s1m2, CagA)25Israel D.A. Salama N. Arnold C.N. Moss S.F. Ando T. Wirth H.P. Tham K.T. Camorlinga M. Blaser M.J. Falkow S. Peek R.M. Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.J Clin Invest. 2001; 107: 611-620Crossref PubMed Scopus (307) Google Scholar, 29Fischer W. Buhrdorf R. Gerland E. Haas R. Outer membrane targeting of passenger proteins by the vacuolating cytotoxin autotransporter of Helicobacter pylori.Infect Immun. 2001; 69: 6769-6775Crossref PubMed Scopus (57) Google Scholar allowed a quantitative recovery from the animal stomach by antibiotic selection (streptomycin 250 mg/L).30Kavermann H. Burns B.P. Angermüller K. 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Püls J. Sedlmaier B. Gerland E. Fischer W. Haas R. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.Science. 2000; 287: 1497-1500Crossref PubMed Scopus (1101) Google Scholar and monoclonal anti-P-Tyr antibody (P99; Santa Cruz Biotechnology, Santa Cruz, CA). The capability of wild-type (WT) or mutant strains to translocate CagA or to induce IL-8 in AGS (human gastric adenocarcinoma) epithelial cells was determined as previously described.14Fischer W. Püls J. Buhrdorf R. Gebert B. Odenbreit S. Haas R. Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island essential genes for CagA translocation in host cells and induction of interleukin-8.Mol Microbiol. 2001; 42: 1337-1348Crossref PubMed Scopus (438) Google Scholar Motility was tested by plating the bacteria on soft agar (0.4%). Animals were challenged orogastrically 3 times on 5 consecutive days with approximately 109 viable H pylori or brucella broth and were killed 32 weeks after inoculation. The stomach was opened along the greater curvature, and antral and corpus tissues were macroscopically distinguished and harvested separately. For RNA isolation, the tissue was immediately frozen in liquid nitrogen. Sections for histological analysis were fixed in 10% neutral buffered formalin. Quantitative reisolation of H pylori of antral and corpus tissue sections was performed as described previously.30Kavermann H. Burns B.P. Angermüller K. Odenbreit S. Fischer W. Melchers K. Haas R. Identification and characterization of Helicobacter pylori genes essential for gastric colonization.J Exp Med. 2003; 197: 813-822Crossref PubMed Scopus (231) Google Scholar From each gerbil, we reisolated 4 colonies from antral and corpus tissue, respectively. All of those colonies were tested by Western blot for urease, oxidase, and catalase activity, for CagY and CagA protein, and for tyrosine phosphorylation of CagA. Furthermore, IL-8 induction of AGS epithelial cells was tested. The pH of the gastric mucosa was measured by using color-fixed indicator test sticks (pH-Fix 0.0-6.0; Macherey-Nagel, Dueren, Germany). The gastrin radioimmunoassay was performed as described previously.35Zavros Y. Rathinavelu S. Kao J.Y. Todisco A. Del V.J. Weinstock J.V. Low M.J. Merchant J.L. Treatment of Helicobacter gastritis with IL-4 requires somatostatin.Proc Natl Acad Sci U S A. 2003; 100: 12944-12949Crossref PubMed Scopus (90) Google Scholar Briefly, plasma samples were incubated in duplicate at 4°C with 125I-Met human gastrin amide (G-17) label and antiserum 5135 (Center for Ulcer Research and Education, University of Michigan, Ann Arbor, MI; UCLA). The median infective dose was 1 fmol/mL, and interassay and intra-assay coefficients of variation were <2% and 11%, respectively. H&E-stained longitudinal paraffin sections of antrum and corpus were graded on the intensity of inflammation, metaplasia, and ulcer development (scale 0–5), by a pathologist blinded to the treatment, according to Garhart et al.36Garhart C.A. Redline R.W. Nedrud J.G. Czinn S.J. Clearance of Helicobacter pylori infection and resolution of postimmunization gastritis in a kinetic study of prophylactically immunized mice.Infect Immun. 2002; 70: 3529-3538Crossref PubMed Scopus (122) Google Scholar The presence of metaplasia was confirmed by a periodic acid-Schiff/alcian blue stain (pH 2.5). Longitudinal paraffin sections of antrum and corpus were deparaffinized, treated with citrate buffer (Vector Laboratories, Burlingame, CA), and permeabilized in 3% H2O2. Slides were preincubated with 20% goat serum/phosphate-buffered saline and 0.1% Triton X-100 and were then incubated with a mouse anti-Ki-67 antibody (Novocastra Laboratories Ltd, Newcastle, UK), followed by a secondary biotinylated anti-mouse immunoglobulin G antibody (Vector Laboratories). They were then visualized with avidin-biotin complexes and diaminobenzidine (DAB) (Vectastain Elite ABC Kit; Vector Laboratories) as substrate for horseradish peroxidase. Negative controls were performed by omitting the primary antibody. Morphometric analysis of the proliferating cells in antrum and corpus were analyzed with MetaMorph (Visitron, Puchheim, Germany) software to count 2 independent fields at a magnification of 100×. To identify Mongolian gerbil gene sequences, known messenger RNA (mRNA) sequences of human, mouse, and rat were aligned, and conserved regions were used to design appropriate oligonucleotide primers. The PCR primers used for the amplification of up to 500 base pairs are listed (see online supplemental material at http://www.mvp.uni-muenchen.de/Gastroenterology.425.0.html). The cross-species reverse-transcription PCR (RT-PCR) reaction (10 μL) was performed in a LightCycler (version 3.5; Roche, Mannheim, Germany) by using gerbil complementary DNA (cDNA) and a Fast Start DNA Master SYBR-Green Kit (Roche) for amplification. The DNA sequences of the amplification products were applied to a Blast analysis, and appropriate gene sequences were verified by cross-species homology. Sequences were submitted to European Molecular Biology Laboratory/Genbank/DNA Data Bank of Japan databases (18S ribosomal RNA, AJ877917; H+K+ [ATPase], AJ877918; IL-1β, AJ877919; IFN-γ, AJ877920; KC, AJ877921; Sst, AJ877922). Shock-frozen tissue specimens from antrum and corpus were stored at −80°C until use. Samples were then homogenized with a polytron PT2100 homogenizer (Kinematica, Luzern, Switzerland), and total RNA was extracted by using RNeasy Mini Kit spin columns (Qiagen, Hilden, Germany). RNA was treated on a column with ribonuclease-free deoxyribonuclease (Qiagen). Total RNA (1 μg) was used for cDNA synthesis by applying random hexamer oligonucleotide primers and a TaqMan Reverse Transcriptase Kit (Roche). Aliquots of cDNA were used as a template for real-time RT-PCR with oligonucleotides primers and probes specific for H+K+-ATPase, IL-1β, IFN-γ, KC, IL-12p40, Sst, and the housekeeping gene 18S ribosomal RNA (see online supplemental material). The real-time RT-PCR was run for 45 cycles under the standard condition of the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) by amplifying a product between 80 and 100 base pairs (duplicated samples). RNA samples were used as negative controls to confirm that DNA was absent. AM0 cells are AGS cells stably transfected with a plasmid containing 240 base pairs of the human gastrin promoter expressing the luciferase reporter (240 GasLuc).37Ford M.G. Valle J.D. Soroka C.J. Merchant J.L. EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures.J Clin Invest. 1997; 99: 2762-2771Crossref PubMed Scopus (44) Google Scholar AM4 cells express the 240 GasLuc construct with a 4–base pair mutation within the GC-rich element conferring epidermal growth factor (EGF) responsiveness (gERE) to the gastrin promoter.38Shiotani A. Merchant J.L. cAMP regulates gastrin gene expression.Am J Physiol. 1995; 269: G458-G464PubMed Google Scholar Both cell lines were cultured in Dulbecco's modified Eagle medium (GIBCO, Karlsruhe, Germany) under standard conditions. Cells at 80% confluence and starved for 24 hours in Nutrient Mixture F12 (HAM; GIBCO) without supplements were stimulated with 50 nmol/L EGF or bacterial suspensions of WT and mutant strains at a multiplicity of infection of 10:1 (bacteria per cell) for 5 hours. Cells were harvested, and luciferase activity was measured (MicroLumat Plus LB 96 V; Berthold Technologies, Bad Wildbad, Germany) and normalized to total cell protein. All data were statistically analyzed by using the Mann-Whitney U test for unpaired groups. The in vitro luciferase data are presented as mean ± SEM. Helicobacter pylori B128 is a gerbil-colonizing type I strain competent to deliver CagA into AGS gastric epithelial cells and to induce IL-8. B128 was transformed to streptomycin resistance to allow its quantitative reisolation from the stomach of infected animals.30Kavermann H. Burns B.P. Angermüller K. Odenbreit S. Fischer W. Melchers K. Haas R. Identification and characterization of Helicobacter pylori genes essential for gastric colonization.J Exp Med. 2003; 197: 813-822Crossref PubMed Scopus (231) Google Scholar Isogenic mutant B128ΔcagA carries an intact T4SS but does not produce or deliver CagA. B128ΔcagY produces CagA protein but is unable to deliver it into gastric epithelial cells or to induce IL-8. Such mutant strains are functionally similar (with respect to the cag-PAI) to naturally occurring type II H pylori patient isolates. A total of 55 Mongolian gerbils were infected with WT H pylori strain B128 (n = 19), B128ΔcagY (n = 13), B128ΔcagA (n = 14), or brucella broth alone (n = 9). After 32 weeks, animals were killed, their stomachs were separated into antral and corpus regions, and H pylori were quantitatively reisolated from each region independently. The reisolation rate for infected animals was 100% for B128ΔcagY, 89% for B128 WT, and 71% for B128ΔcagA (Table 1). From the antrum, both WT and mutant strains could be isolated in high numbers, and the mutants showed a slightly higher median (B128ΔcagY, P = .006; B128ΔcagA, P = .356) compared with the WT strain (Figure 1, open symbols). In WT-infected animals, the bacterial load was high, but it was approximately equal in the antrum and corpus (median of 5 × 104 and 8 × 104 colony-forming units per gram, respectively), whereas the colonization density of strains B128ΔcagY and B128ΔcagA was higher in the antrum than the corpus (1–1.5 log stages: P = .598 and P = .066, respectively; Figure 1, compare open and filled symbols). These data suggest that a functional T4SS, as present in H pylori type I, but not type II, strains, allows these bacteria to colonize the gastric corpus and to induce corpus-dominant gastritis.Table 1Macroscopic and Histopathologic Findings of Helicobacter pylori-Infected and Noninfected Mongolian GerbilsVariableNoninfected (n/n) %B128 (n/n) %ΔcagY (n/n) %ΔcagA (n/n) %No. animals9191314Reisolation vs infected animalsNA(17/19) 89(13/13) 100(10/14) 71Lymphaggregate(0/9) 0(17/17) 100(9/13) 69(7/10) 70Erosion and ulcer(0/9) 0(12/17) 71(1/13) 8(4/10) 40Metaplastic changes(0/9) 0(16/17) 94(6/13) 46(3/10) 30Enlarged stomach(0/9) 0(16/17) 94(5/13) 38(3/10) 30Increased carcinoma riskaA study in humans has shown an increased carcinoma risk if inflammation in corpus is greater than in antrum and metaplastic changes are present.49(0/9) 0(12/17) 71(1/13) 8(1/10) 10NA, not applicable.a A study in humans has shown an increased carcinoma risk if inflammation in corpus is greater than in antrum and metaplastic changes are present.49Meining A. Bayerdorffer E. Muller P. Miehlke S. Lehn N. Holzel D. Hatz R. Stolte M. Gastric carcinoma risk index in patients infected with Helicobacter pylori.Virchows Arch. 1998; 432: 311-314Crossref PubMed Scopus (110) Google Scholar Open table in a new tab NA, not applicable. After opening of the gerbil stomach at the greater curvature, the macroscopic inspection showed significant expansion of the stomach width in WT-infected gerbils as compared with the noninfected animals (Figure 2). The distinct macroscopic border between the antrum and corpus was lost, probably because of the severe inflammation. The antral mucosa was significantly enlarged (40%; P < .05) compared with the negative control group (Table 1 and Figure 2B). This was in contrast to stomachs infected with B128ΔcagY or B128ΔcagA, in which a distinct macroscopic separation of antrum
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