Determinants and Consequences of Different Levels of CagA Phosphorylation for Clinical Isolates of Helicobacter pylori

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1 GASTROENTEROLOGY 2004;127: Determinants and Consequences of Different Levels of CagA Phosphorylation for Clinical Isolates of Helicobacter pylori RICHARD H. ARGENT,* MARK KIDD, ROBERT J. OWEN, RACHAEL J. THOMAS,* MARIE C. LIMB,* and JOHN C. ATHERTON* *Institute of Infection, Immunity and Inflammation, and the Wolfson Digestive Diseases Centre, University of Nottingham, Queen s Medical Centre, Nottingham, England; School of Medicine, Yale University, New Haven, Connecticut; and Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency, London, England See editorial on page 669. Background & Aims: The Helicobacter pylori cag pathogenicity island encodes a secretory system that translocates CagA into epithelial cells, where it becomes tyrosine phosphorylated and induces cytoskeletal rearrangements. Strains with more CagA tyrosine phosphorylation motifs are most closely associated with gastric cancer. Here we assess whether clinical strains can deliver CagA, whether strains with different numbers of CagA phosphorylation motifs have CagA phosphorylated to different degrees, and whether this induces different amounts of epithelial cytoskeletal change. Methods: Forty-four H. pylori strains from South African patients, all caga gene positive, were cocultured with the gastric adenocarcinoma cell line AGS. CagA expression and phosphorylation were determined by Western blot and interleukin-8 secretion by enzyme-linked immunosorbent assay. The caga 3 variable regions of 22 strains were sequenced and shown to possess 3 6 phosphorylation motifs. These strains were used to quantify CagA phosphorylation and cytoskeletal rearrangements. Results: caga genotype and typing of cag pathogenicity island genes were poorly predictive of phenotype. Thirty-four of 44 strains expressed CagA protein that could be delivered to and phosphorylated within AGS cells. Only these 34 strains induced interleukin-8 secretion from AGS cells. Among those strains, the number of CagA tyrosine phosphorylation motifs determined the degree of CagA phosphorylation and the level of biologic activity in terms of degree and extent of AGS cell elongation. Conclusions: H. pylori strains that deliver CagA with more phosphorylation motifs induce higher levels of CagA phosphorylation in epithelial cells, induce more cytoskeletal changes, and are more likely to be associated with gastric cancer. Helicobacter pylori colonizes the stomachs of half of the world s population and is associated with the development of gastroduodenal diseases such as peptic ulcer disease and distal gastric adenocarcinoma and lymphoma. H. pylori strains possessing the caga gene have been linked with an increased risk of developing gastric cancer and peptic ulceration. 1 9 However, this association is not absolute; many strains that do not cause disease possess caga, and some strains that are associated with disease do not. Some studies fail to show any significant association between caga status and clinical outcome, especially studies from East Asia, where more than 90% of isolated H. pylori strains test positive for caga. Thus, there is considerable interest in whether there are differing levels of pathogenicity among cagapositive strains. caga is but one gene in the cag pathogenicity island (PaI), a 40-kilobase region of the H. pylori chromosome comprising genes. 19 The cag PaI encodes a type IV secretory system (TFSS), which forms a syringe-like structure that penetrates into epithelial cells and delivers CagA into the host cytosol. 19,20 Within the cytosol, CagA becomes phosphorylated on tyrosine residues by Src family kinases 23,26,27 that recognize tyrosine phosphorylation motifs (TPMs). Phosphorylated CagA interacts with the phosphatase SHP causing dephosphorylation of cortactin 31 and cytoskeletal rearrangements forming the hummingbird phenotype. 22,32,33 Phosphorylated CagA can also interact with C-terminal Src kinase, which attenuates SHP-2 signaling, decreasing hummingbird cell formation, and inactivates Src kinases (preventing further phosphorylation of CagA). 34 In addition to inducing hummingbird cell formation, CagA also promotes cell spreading or scattering similar to the scattered phenotype induced by scatter factor/hepatocyte growth factor on binding the c-met receptor. 35 Contact Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; IL, interleukin; PaI, pathogenicity island; PCR, polymerase chain reaction; TFSS, type IV secretory system; TPM, tyrosine phosphorylation motif by the American Gastroenterological Association /04/$30.00 doi: /j.gastro

2 August 2004 CAGA PHOSPHORYLATION AND EPITHELIAL CELL ELONGATION 515 of the TFSS with the epithelial cell, or possibly translocation of a second, unidentified factor, also leads to activation and nuclear translocation of nuclear factor B and the secretion of proinflammatory cytokines and chemokines such as interleukin (IL)-8. 19,36 38 CagA is not needed for this effect. CagA is differently sized in different strains due to the presence of repeat sequences, encoding TPMs, within the 3 variable region of caga. 27,29,33,39 42 Several studies have investigated the effects of multiple TPMs within the CagA variable region on the associations with disease. 27,29,41 43 Yamaoka et al. and Azuma et al. found that H. pylori strains possessing more that 3 TPMs in the CagA variable region were significantly associated with gastric carcinoma and atrophic gastritis in Japan. 41,42 Yamaoka et al. also showed that H. pylori strains with more than 3 CagA variable region TPMs were significantly associated with gastric mucosal atrophy and intestinal metaplasia in patients from Colombia, the United States, Italy, and South Korea. 43 Several studies have used mutagenesis to assess functionality of CagA TPMs. Stein et al. and Higashi et al. showed by mutagenesis of laboratory strains of H. pylori with multiple TPMs that reducing the number of functional TPMs reduced CagA phosphorylation and the number of cells forming the hummingbird phenotype. 27,29 In contrast, however, Aras et al. showed that although CagA phosphorylation was important for induction of the hummingbird phenotype, there was no correlation between the number of potential TPMs and the number of AGS hummingbird cells formed. 44 On this background, we have performed a study of clinical isolates of H. pylori from South African patients with and without gastric cancer. Firstly, we have assessed whether CagA is expressed and can be translocated into epithelial cells and whether this can be predicted from routine genotypic analysis of strains. Secondly, and most importantly, we have defined the numbers of TPMs in these strains and shown that this determines the level of CagA phosphorylation and the biological activity of CagA in terms of inducing epithelial cytoskeletal (hummingbird) change. Materials and Methods Materials Anti-CagA antibodies were obtained from Austral Biologicals (San Ramon, CA). Anti-phosphotyrosine monoclonal antibodies (clone PY99; Santa Cruz Biotechnology, Santa Cruz, CA) were obtained from Autogen Bioclear (Calne, Wiltshire, England). Tissue culture medium and reagents were obtained from Invitrogen (Paisley, Scotland). Blood-agar plates were obtained from Oxoid (Basingstoke, England). Unless stated, all other reagents were purchased from Sigma (Poole, England). Maintenance and Culture of H. pylori Strains H. pylori strains were originally isolated from patients living in the Western Cape region of South Africa between 1989 and ,46 Forty-four strains were used in this study, isolated from 13 patients with gastritis alone, 13 patients with duodenal ulceration, and 18 patients with gastric adenocarcinoma. Strains were grown on blood-agar plates in a microaerobic environment within a MACS-VA500 workstation (Don Whitley Scientific, Shipley, England), usually for 6 passages from frozen stocks but occasionally more to synchronize experiments. We observed no differences in genotyping or phenotyping between passage numbers. Polymerase Chain Reaction Amplification of cag Genes and Nucleotide Sequence Analysis of the 3 Region of caga H. pylori genomic DNA isolation and polymerase chain reaction (PCR) amplification of cag PaI genes were performed as previously described. 46,47 PCR amplification of the 3 variable region of the caga gene was performed using the primers cag2 and cag4. 48 To sequence this region, PCR-amplified products were excised from a 1.2% (wt/vol) agarose gel and purified using a QIAEX II (Qiagen, Crawley, England) gene extraction kit. Sequencing was performed by GRI Genomics (Braintree, England) using both primers. Phosphorylation of CagA by Coculture and IL-8 Enzyme-Linked Immunosorbent Assay AGS human gastric epithelial cells were seeded into 25-cm 2 flasks ( cells/flask) in nutrient mixture F12 Ham and grown at 37 C ina5% CO 2 air-humidified atmosphere until almost confluent. H. pylori strains were swabbed from 24- to 48-hour growth plates into medium, the optical density at 550 nm was determined, and the strains were diluted to OD before addition to AGS cells (5 ml/flask; multiplicity of infection 100). AGS cells were cocultured with H. pylori for 6 hours at 37 C ina5%co 2 air-humidified atmosphere before the medium was saved to measure the secreted IL-8, and the cells were washed twice with phosphatebuffered saline (PBS) containing 1 mmol/l calcium chloride and 0.5 mmol/l magnesium chloride. Cells were scraped from the flasks in 5 ml PBS containing 1 mmol/l sodium vanadate, harvested by centrifugation at 1000g for 10 minutes, and then resuspended in 100 L PBS/sodium vanadate and 50 L 4 sample loading buffer (0.2 mol/l Tris-HCl, ph 6.8, 0.4 mol/l dithiothreitol, 8% [wt/vol] sodium dodecyl sulfate, 40% [vol/ vol] glycerol, 0.4% [wt/vol] bromophenol blue). The samples were boiled for 5 minutes and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting using anti-caga and anti-phosphotyrosine monoclonal antibodies. Blots were developed using the Amersham (Little

3 516 ARGENT ET AL. GASTROENTEROLOGY Vol. 127, No. 2 Chalfont, England) ECL detection reagents. Quantification was by densitometry using a Bio-Rad (Hemel Hempstead, England) GS-800 calibrated densitometer. The IL-8 containing medium from the AGS cells was centrifuged at 15,000g for 10 minutes, and the supernatant was stored at 80 C. IL-8 measurements were performed using an R&D Systems (Abingdon, England) DuoSet human IL-8 enzyme-linked immunosorbent assay (ELISA), and results were normalized between experiments by comparing IL-8 secretion induced by the H. pylori strains with the IL-8 induced by a reference strain (ATCC 49503). Phosphorylation of CagA Using Cell-Free Extracts An AGS cell-free extract was prepared as follows. Cells were harvested by centrifugation at 500g for 5 minutes, washed with ice-cold PBS, and then resuspended in 500 L20 mmol/l Tris-HCl, ph 7.4, 150 mmol/l sodium chloride, 1 mmol/l EDTA, 1 mmol/l ethylene glycol-bis(2-aminoethylether)tetraacetic acid, 0.1% (vol/vol) Triton X-100, 1 mmol/l -glycerophosphate, 1 mmol/l sodium vanadate, 0.1 mmol/l leupeptin, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l 1,10-phenanthroline, 5 mol/l trans-epoxysuccinyl-leucylamido(4-guanidino)butane, and 1 mol/l pepstatin A. The cells were held on ice for 30 minutes and then sheared through a 21-gauge needle 10 times and a 25-gauge needle twice before centrifugation at 15,000g for 10 minutes at 4 C. The supernatant was removed and stored at 4 C. H. pylori water extracts were prepared by resuspending the bacteria from 24- to 48- hour growth plates in 1 ml of water, followed by mixing for 30 seconds, standing at room temperature for 1 hour, mixing again, and removing intact bacteria and membranes by centrifugation at 15,000g for 10 minutes. The supernatant was passed through a 0.2- m filter and stored at 4 C. To determine phosphorylation of CagA, H. pylori water extracts were incubated with AGS cell-free extract in the presence of 1 mmol/l adenosine triphosphate and kinase buffer (25 mmol/l Tris-HCl, ph 7.4, 10 mmol/l magnesium chloride, 5 mmol/l -glycerophosphate, 2 mmol/l dithiothreitol, 0.1 mmol/l sodium vanadate) at 37 C for 2 minutes before the reaction was terminated by the addition of boiling 4 sample loading buffer and boiling the sample for 5 minutes. Samples were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting as previously described. Cytoskeletal Rearrangements Induced by CagA AGS cells were seeded into 6-well plates at a density of cells/well and allowed to adhere at 37 C ina5%co 2 air-humidified atmosphere overnight. Coculture with H. pylori strains was performed as previously described except that cells were incubated for up to 2 days. Cells were examined for the hummingbird phenotype by microscopy of randomly chosen fields, and measurements of the lengths of hummingbird cells formed after H. pylori coculture were performed on 100 randomly selected hummingbird cells. We define a hummingbird cell as a cellular needle-like protrusion 2 m in length. Statistics were performed using a 2-tailed Student t test. Results PCR Assessment of the cag PaI All 44 strains tested positive for caga by PCR using 2 sets of primers: one specific for the 5 region and one for the 3 region. Forty (90.9%) tested positive for cage, 41 (93.2%) for cagm, 39 (88.6%) for cagt, and 33 (75%) for cag6 7. Overall, 31 strains (70.4%) tested positive for all 5 cag PaI PCRs and so on the basis of this limited testing might be predicted to have an intact cag PaI. Expression of CagA, Phosphorylation of CagA by AGS Cells, and IL-8 Secretion Because all strains possessed the caga gene, we next determined whether they expressed the CagA protein by Western blotting H. pylori lysates with anti- CagA antibodies. Only 34 strains (77.3%) expressed the CagA protein (Figure 1A and Tables 1 and 2). We next assessed whether CagA could be translocated into and phosphorylated within the adenocarcinoma cell line AGS; all strains expressing CagA were able to translocate the protein into AGS cells, and in every case it became phosphorylated. As expected, CagA and phosphorylated CagA were not shown in epithelial cells cocultured with strains shown previously not to express CagA (Figure 1A and Tables 1 and 2). Because our PCR tests had predicted that many strains would not have the complete cag-encoded TFSS (which stimulates epithelial cells to express IL-8 independently of CagA 19,36 38 ), we next assessed whether our strains stimulated IL-8 secretion by AGS cells. We took the medium from the AGS cells after coculture in the CagA translocation experiments and determined IL-8 secretion from the cells by ELISA. This showed that PCR prediction of cag PaI intactness was a poor test of the presence of a TFSS capable of inducing IL-8 expression of AGS cells (Figure 1B and Tables 1 and 2). The 34 strains that induced IL-8 secretion were the same 34 strains that had CagA translocated into and phosphorylated within AGS cells. None of the 10 strains that did not express CagA induced significant IL-8 secretion, indicating that they lacked a functional TFSS. This shows that, in this clinical strain population, CagA expression, CagA translocation, CagA phosphorylation, and TFSS-induced IL-8 secretion are concordant; the TFSS in these strains is either functional for both CagA translocation and IL-8 stimulation or neither. The data also show that prediction of PaI intactness by PCR amplification of cag genes is poor

4 August 2004 CAGA PHOSPHORYLATION AND EPITHELIAL CELL ELONGATION 517 Figure 1. CagA expression, translocation, and phosphorylation and IL-8 secretion by H. pylori strains. (A) Western blots showing CagA expression by H. pylori strains and translocation of CagA into and phosphorylation within AGS cells following coculture for 6 hours. Samples were probed with anti-caga monoclonal antibodies (H. pylori alone and after AGS cell coculture) or anti-phosphotyrosine monoclonal antibodies (AGS cell coculture). This shows that of the H. pylori strains shown here, strains GC90, HP186, HP501, HP465, HP467, and HP471 do not express CagA. (B) IL-8 secretion from AGS cells following coculture with H. pylori strains for 6 hours, expressed as a percentage of the reference strain ATCC The strains have been separated into 2 populations based on their ability to induce high levels of IL-8 (P , 2-tailed Student t test). with this H. pylori strain population because there were 6 false-negative and 3 false-positive results (Tables 1 and 2). This implies that simple genotypic testing by limited PCR analysis is not an adequate guide to cag phenotype. Sequencing the 3 Variable Region of caga Shows Variation in the Number of TPMs As in other studies, the CagA proteins from our clinical strains showed size variation by Western blot (Figure 1), and PCR amplification of the 3 variable region of caga produced fragments of different length from 550 to 800 base pairs (data not shown). This has been shown to be due to variation in the number of repeat sequences containing TPMs. 27,29,33,39 42 We next set out to define the number of TPMs in each strain. From those strains expressing CagA, we chose all those with 3 caga PCR fragments 600 base pairs and a random selection of strains giving smaller PCR fragments (22 strains in total: 7 gastritis alone, 5 duodenal ulceration, and 10 gastric adenocarcinoma). We performed nucleotide sequence analysis of the caga 3 variable region of these strains. The sequencing data (Table 3) showed that the variable region of caga possessed from 3 to 6 EPIYA TPMs. All of the sequenced caga variable regions were of the western type, because they encoded the polypeptide sequence QAASGLGGVGQAGFPLKRHDK- VDDLSKV but not the sequence EATSAINRKID- RINKIASAGKGVGGFSGA that has been found in CagA in East Asian strains. 41 Additionally, none of the sequences here possessed the consensus SHP-2 binding motif py-(s/t/a/v/i)-x-(v/i/l)-x-(w/f), 49 which is also present in CagA of East Asian strains and leads to greater activation of the phosphatase. 29 CagA Phosphorylation Increases With Increasing Numbers of TPMs Because the sequenced caga alleles showed a range in the number of predicted variable region TPMs, we next determined whether these were functional in terms of tyrosine phosphorylation and whether one or more TPMs could become phosphorylated. To do this, we cocultured AGS cells with H. pylori strains as previously described. Because of the potential inaccuracies associated with coculture of bacterial and eukaryotic cells, we also developed a second cell-free method that we used in parallel. Here, we used a cell-free extract from AGS cells to measure the level of phosphorylation of CagA in water extracts prepared from the various H. pylori strains. Strains possessing caga with higher numbers of TPMs expressed CagA of higher molecular weight that became more phosphorylated on coculture with AGS cells (Fig- Table 1. Association for 44 Clinical H. pylori Isolates Between Genotypic (PCR) Testing for the cag PaI and Phenotypic Testing by Stimulation of IL-8 Secretion by AGS Cells During Coculture IL-8 secretion Total Intact PaI Incomplete PaI NOTE. PaI determined by PCR amplification of caga, cage, cagm, cagt, and cag6 7.

5 518 ARGENT ET AL. GASTROENTEROLOGY Vol. 127, No. 2 Table 2. Association for 44 Clinical H. pylori Isolates Between IL-8 Secretion and CagA Delivery and Phosphorylation in AGS Cells During Coculture CagA expression and phosphorylation IL IL ure 2A) and when incubated with the cell-free extract (Figure 2B); this indicated that CagA, from the H. pylori strains used in this study, can become phosphorylated at multiple TPMs. This result also shows that the cell-free extract method can be used as a simple rapid assay that produces clear-cut results. Level of Induction of IL-8 Secretion Is Independent of the Degree of CagA Phosphorylation Although bacterial mutant studies have shown that IL-8 secretion is dependent on an intact TFSS but that CagA is not needed for its induction, 19,36 38 the finding that IL-8 secretion was entirely concordant with CagA expression and phosphorylation in our strains (Tables 1 and 2) prompted us to look at whether highly phosphorylated CagA could increase IL-8 induction. We found, in agreement with other recent work using a different approach, 50 that the degree of CagA phosphorylation had no significant association with the amount of IL-8 secreted following AGS cell coculture (Figure 3). Increasing CagA Phosphorylation Increases the Number of Hummingbird AGS Cells Having shown that CagA can become highly phosphorylated in a cell-free system and within AGS cells by coculture (Figure 2), we next aimed to assess the effects of the different forms of CagA on the AGS cytoskeletal structure. Higashi et al. showed, using mutant forms of caga from the laboratory-adapted strain NCTC11637 in which the 5 CagA variable region TPMs have been mutated into nonfunctional motifs, that AGS cells transfected with caga constructs with higher numbers of functional TPMs induce greater numbers of cells to transform to the hummingbird phenotype. 29 Similarly, Stein et al. showed that decreasing the number of CagA TPMs of laboratory-adapted strain G27 reduced AGS cell hummingbird formation following coculture. 27 We now aimed to assess for clinical strains whether the number of TPMs affected the extent of hummingbird change. We cocultured AGS cells with H. pylori strains and showed that the number of hummingbird cells was increased when incubated with strains possessing CagA with higher numbers of TPMs (Figure 4A and D). Strains with 3 CagA TPMs induced hummingbird cells in only 15% of AGS cells, whereas strains with 4 or more CagA TPMs induced hummingbird cells in 43% (P ). Additionally, H. pylori strains possessing CagA with progressively higher numbers of TPMs progressively and significantly increased the overall length of hummingbird cells formed (Figure 4B and C). This is further emphasized by the finding that few hummingbird cells 6 m in length are induced by H. pylori possessing CagA with 3 TPMs, whereas strains with 4 or more (and especially 5 or 6) TPMs induce the formation of many more long hummingbird protrusions (Figure 4C). These findings show that increased tyrosine phosphorylation of CagA leads to increased biological activity in terms of inducing these cytoskeletal changes. Increased CagA Phosphorylation May Be Associated With the Development of Gastric Adenocarcinoma We have shown that increasing the number of CagA TPMs increases phosphorylation of the protein, increases the number of AGS cells undergoing cytoskeletal alterations, and increases the degree of cellular rearrangement in terms of the length of hummingbird cell formed. Because these cytoskeletal rearrangements are Table 3. CagA Variable Region TPMs and Repeat Patterns Strain Pathology CagA variable region pattern a No. of EPIYA motifs HP175 Gastritis R1R2R1R3R1R4 3 HP184 Gastritis R1R2R1R3R1R4 3 HP190 Gastritis R1R2R1R3R1R4 3 HP196 Gastritis R1R2R1R3R1R4 3 HP199 Gastritis R1R2R1R3R1R4 3 HP504 Gastritis R1R2R1R3R1R4 3 HP508 Gastritis R1R2R1R3R1R4 3 HP14 DU R1R2R1R3R1R4 3 HP192 DU R1R2R1R3R1R4 3 HP458 DU R1R2R1R3R1R4 3 HP460 DU R1R2R1R3R1R4 3 HP464 DU R1R2R1R3R1R4R1R4 4 GC16 GC R1R2R1R3R1R4 3 GC17 GC R1R2R1R3R1R4 3 GC59 GC R1R2R1R3R1R4R1R4 4 GC77 GC R1R2R1R3R1R4R1R4R1R4 5 GC78 GC R1R2R1R3R1R4R1R2R1R3R1R4 6 GC80 GC R1R2R1R3R1R4 3 GC86 GC R1R2R1R3R1R4 3 GC94 GC R1R2R1R3R1R4 3 GC102 GC R1R2R1R3R1R4R1R4 4 GC1507 GC R1R2R1R3R1R4R1R3 4 DU, duodenal ulceration; GC, gastric cancer. a R1 EPIYA; R2 KVNKKKTGQVASPE; R3 QVAKKVNAKIDRLN- QAASGLGGVGQA(A)GFPLKRHDKVDDLSKV; R4 TIDDLGGPFPLKRHD- KVDDLSKVG. 42

6 August 2004 CAGA PHOSPHORYLATION AND EPITHELIAL CELL ELONGATION 519 considered to promote scattering and spreading of cells, 35 they may be important in carcinogenesis. Analysis of the disease state of 34 patients from whom our strains were obtained shows that 5 of 6 strains able to translocate CagA with more than 3 CagA variable region TPMs were isolated from patients with gastric cancer, whereas only one of 19 infected patients without gastric cancer had such an isolate (P 0.09; Fisher exact test). This is consistent with other published studies showing an association between the number of CagA TPMs and gastric cancer. 41,42 Discussion People infected with H. pylori are more likely to develop gastroduodenal diseases, including gastric cancer, than uninfected patients. Among those infected, the risk of disease is further increased if the strains possess caga, 1 9 although the association is not absolute and many studies have failed to confirm it One reason for difficulty in showing such an association could be that PCR tests for caga do not predict CagA expression. We have shown that this is the case in this South African strain population, in that 10 of 44 strains we examined were PCR positive for caga (by 2 separate reactions) but did not express the CagA protein. We have not investigated the reason for this in our population, but one possibility is that it may be due to deletions in the caga promoter, or truncations within the caga gene, as has been found for some Japanese strains. 51 We have also shown that PCR detection of selected cag PaI genes (at least with the primers used for this study) is a far from accurate method for predicting functionality of cag PaI encoded proteins (Table 1); given the very high nucleotide sequence variability in H. pylori, it is unlikely that any PCR-based method will be totally accurate. There- Figure 2. The degree of CagA phosphorylation increases with increasing numbers of EPIYA motifs. (A) AGS cells were cocultured with H. pylori strains for 6 hours before the cells were lysed and the samples subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting with anti-phosphotyrosine monoclonal antibodies (upper panel). The blots were then stripped and reprobed with anti-caga polyclonal antibodies (lower panel). The densities of the CagA and phosphorylated CagA bands were determined and plotted as a ratio. This figure shows results from 5 representative strains, each with different numbers of CagA EPIYA motifs. (B) An AGS cell-free extract (CFE) was incubated with H. pylori water extracts for 2 minutes at 37 C before the samples were analyzed as described in A. This figure shows 7 representative strains with different numbers of CagA EPIYA motifs. These experiments were repeated 3 times each, with representative blots shown. Figure 3. The level of IL-8 secretion is independent of the degree of CagA phosphorylation. H. pylori strains expressing CagA with 3 6 EPIYA motifs, or a strain lacking the PaI, were cocultured with AGS cells for 6 hours in quadruplicate, along with control cells not cultured with H. pylori, before the concentration of IL-8 secreted from the cells was determined by ELISA.

7 520 ARGENT ET AL. GASTROENTEROLOGY Vol. 127, No. 2 Figure 4. CagA possessing greater numbers of TPMs increases the number of hummingbird AGS cells and increases the length of cellular protrusions. H. pylori strains expressing CagA with greater numbers of variable region TPMs increase (A) the number of AGS cells displaying the hummingbird phenotype, (B) the mean length of hummingbird protrusion, and (C) the number of hummingbird cells 6 m long. AGS cells were cocultured with 3 representative strains expressing CagA with 3 EPIYA motifs, 2 representative strains expressing CagA with 4 EPIYA motifs, and one strain each expressing CagA with 5 or 6 EPIYA motifs that were grouped together for statistical analysis (A and B). Values presented are means SE. *P 0.001, **P , ***P (D) Hummingbird cell formation by representative H. pylori strains expressing CagA with 3 6 TPMs or a strain lacking the cag PaI. fore, we argue that phenotypic detection of CagA expression and phosphorylation is more relevant than genotyping H. pylori strains for the presence of PaI genes. We find that, in our South African H. pylori strain population, CagA expression, CagA phosphorylation, and the induction of IL-8 secretion, consistent with a functional TFSS, are entirely concordant. Should this concordance hold true for all H. pylori strains, testing for one of these phenotypes would be sufficient to predict them all and rapid phenotypic cag testing of strains would be more feasible. In support of our findings, Maeda et al. showed that Japanese isolates also displayed this concordance. 51 However, in contrast, Backert et al. showed that, although most German isolates were concordant, several clinical strains expressed CagA but lacked a functional TFSS or induced IL-8 secretion without expression of CagA. 52 Further work is needed on this important point using different strain populations. The size variation in CagA between strains is due to the number of repeats within the 3 variable region. 27,29,33,39 42 Many of the strains used in this study have CagA with 3 EPIYA motifs, but 6 strains have 4 or more of these motifs, with 5 of these occurring in strains isolated from patients with gastric carcinoma. We find that strains with greater numbers of EPIYA motifs become more phosphorylated in vitro, although we do not know which motifs become phosphorylated. Several studies have now shown that CagA can become tyrosine phosphorylated at all EPIYA motifs. 27,29,34,53,54 However, it is unclear whether CagA from all strains of H. pylori become phosphorylated at all TPMs because Aras et al. showed, using an identical pair of naturally occurring H. pylori strains but with deletions/insertions in caga, that CagA with the first 2 EPIYA motifs alone was not phosphorylated and did not induce hummingbird cell formation, whereas CagA with 3 EPIYA motifs was

8 August 2004 CAGA PHOSPHORYLATION AND EPITHELIAL CELL ELONGATION 521 phosphorylated and did induce hummingbird cell formation. 44 Also, CagA from H. pylori strain J99 naturally possesses only 2 EPIYA motifs and is not phosphorylated on delivery into AGS cells, 33 although these motifs can be phosphorylated in vitro as determined by mass spectrometry. 53 Should the first 2 EPIYA motifs prove not to become phosphorylated, then, for the strains used in this study, the CagA variable regions would possess 1 3 phosphorylated TPMs. Either way, our results are consistent with the degree of CagA phosphorylation increasing with the number of (functional) TPMs. The degree of CagA phosphorylation in our strains correlates well with the level of cytoskeletal rearrangement occurring during coculture of the H. pylori clinical isolates with AGS cells. Strains expressing CagA with 3 variable region TPMs induce less cells to undergo transformation into the hummingbird phenotype than strains expressing CagA with 4 or more TPMs. Additionally, strains expressing CagA with greater numbers of TPMs induce AGS cells to produce longer hummingbird protrusions. This latter point is important because it suggests that delivery of more highly phosphorylated CagA to a cell results in more profound biological effects on that cell. Because the hummingbird cytoskeletal changes are secondary to activation by phosphorylated CagA of SHP-2 phosphatase, our study also suggests that strains with western CagA can activate SHP-2 phosphatase despite not possessing the SHP-2 binding consensus sequence described in East Asian strains. The alternative, that these strains induce the same hummingbird changes through a different mechanism, seems unlikely. Studies are now indicated to see which other effects of CagA are enhanced by increased CagA phosphorylation and subsequently to explore which of these effects are important in gastric carcinogenesis. It has previously been shown that East Asian H. pylori strains expressing CagA with higher numbers of TPMs are more closely associated with gastric atrophy and cancer. 41,42 Yamaoka et al. showed that, in 155 Japanese isolates, those possessing CagA with 4 variable region TPMs were significantly associated with gastric cancer (6 of 7 strains), although the 2 strains with 5 CagA TPMs were isolated from patients with chronic gastritis only. 41 Azuma et al. found that, in 90 isolates from Fukui, Japan, those possessing CagA with 5 or more TPMs were significantly associated with atrophic gastritis, and 6 of 15 strains possessing 4 or more TPMs were isolated from patients with gastric cancer. 42 Lai et al., however, found no association between the number of CagA TPMs and disease outcome in 58 Taiwanese isolates. 55 In this study, we show a weak association (P 0.09) between delivery of CagA with 4 or more phosphorylation sites and a gastric cancer origin of the strain. Although this is reassuring, it is not a robust finding. Our population is small and nonrandom; strains were selected because they were obtained from patients with specific disease states. Large surveys are now needed to assess the association between the level of CagA phosphorylation and risk of disease. The cell-free system we describe here for assessing the level of CagA phosphorylation could be modified to provide a rapid and simple quantitative test of phenotype for such studies. Although CagA is an important H. pylori virulence factor and the presence of this protein has been associated with disease outcome, 6 9 our data, in agreement with others, 15,29,41,42,55 suggest that the degree of CagA phosphorylation may be more important than the expression of CagA per se. Here we show that cag genotype, in terms of PCR-determined presence of caga and other genes in the cag PaI, is a poor predictor of phenotype in our population and thus probably in others. However, we show that, in the strains we study, the number of CagA TPMs as determined by sequence analysis is a good predictor of the level of CagA phosphorylation, which in turn determines the level of effect CagA induces in cells and is associated with cancer risk. Sequence analysis of caga is not practical in larger studies, and there may be examples in which it does not predict phenotype clearly (e.g., if there were single amino acid substitutions in TPMs). We speculate that measuring phenotype, perhaps in terms of level of CagA phosphorylation, is likely to be simpler, cheaper, and more accurate than genotypic testing. The degree of CagA phosphorylation may prove to be an important and pathogenically relevant marker of a subgroup of H. pylori strains that predispose strongly to gastric cancer. References 1. van Doorn LJ, Figueiredo C, Rossau R, Jannes G, van Asbroeck M, Sousa JC, Carneiro F, Quint WGV. 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