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Clandestine intracellular delivery of Helicobacter pylori CagA: Guess who's coming to dinner?

2004; Elsevier BV; Volume: 127; Issue: 2 Linguagem: Inglês

10.1053/j.gastro.2004.06.030

1528-0012

Richard M. Peek,

Eosinophilic Esophagitis

See article on page 514.Virtually all persons who are colonized by Helicobacter pylori develop coexisting gastritis, a signature feature of which is the capacity to persist for decades. This is in marked contrast to inflammatory reactions induced by other Gram-negative enteric pathogens, such as Salmonella, that either resolve within days to weeks or progress to eliminate the host. One mechanism through which H. pylori may evade immune clearance is by limiting the bactericidal effects of proinflammatory molecules, such as nitric oxide.1Gobert A.P. McGee D.J. Akhtar M. Mendz G.L. Newton J.C. Cheng Y. Mobley H.L. Wilson K.T. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells a strategy for bacterial survival.Proc Natl Acad Sci U S A. 2001; 98: 13844-13849Crossref PubMed Scopus (308) Google Scholar Another level of host defense that may be circumvented by H. pylori is innate immunity. Toll-like receptors (TLRs) are an evolutionarily conserved family of eukaryotic receptors that function in innate immunity via recognition of invariant regions in bacterial molecules termed pathogen- or microbe-associated molecular patterns. It is becoming increasingly clear, however, that H. pylori has evolved strategies to avoid activation of this system. For example, TLR4 recognizes bacterial lipopolysaccharide (LPS), yet H. pylori LPS is relatively anergic compared with that of other enteric bacteria, primarily because of lipid A core modifications.2Perez-Perez G.I. Shepherd V.L. Morrow J.D. Blaser M.J. Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide.Infect Immun. 1995; 63: 1183-1187PubMed Google Scholar, 3Backhed F. Rokbi B. Torstensson E. Zhao Y. Nilsson C. Seguin D. Normark S. Buchan A.M. Richter-Dahlfors A. Gastric mucosal recognition of Helicobacter pylori is independent of toll-like receptor 4.J Infect Dis. 2003; 187: 829-836Crossref PubMed Scopus (115) Google Scholar In contrast to flagellins expressed by Gram-negative mucosal pathogens, which activate TLR5-mediated proinflammatory responses, H. pylori flagellin is noninflammatory.4Lee S.K. Stack A. Katzowitsch E. Aizawa S.I. Suerbaum S. Josenhans C. Helicobacter pylori flagellins have very low intrinsic activity to stimulate human gastric epithelial cells via TLR5.Microbes Infect. 2003; 5: 1345-1356Crossref PubMed Scopus (148) Google Scholar, 5Gewirtz A.T. Yu Y. Krishna U.S. Israel D.A. Lyons S.L. Peek R.M. Helicobacter pylori flagellin evades Toll-like receptor 5-mediated innate immunity.J Infect Dis. 2004; 189: 1914-1920Crossref PubMed Scopus (216) Google ScholarH. pylori can also facilitate persistence by varying the antigenic repertoire of surface-exposed proteins6Aras R.A. Fischer W. Perez-Perez G.I. Crosatti M.L. Ando T. Haas R. Blaser M.J. Plasticity of repetitive DNA sequences within a bacterial (type IV) secretion system component.J Exp Med. 2003; 198: 1349-1360Crossref PubMed Scopus (77) Google Scholar and by actively suppressing the host adaptive immune response.7Gebert B. Fischer W. Weiss E. Hoffmann R. Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T-lymphocyte activation.Science. 2003; 301: 1099-1102Crossref PubMed Scopus (446) Google Scholar, 8Boncristiano M. Paccani S.R. Barone S. Ulivieri C. Patrussi L. Ilver D. Amedei A. D'Elios M.M. Telford J.L. The Helicobacter pylori vacuolating cytotoxin inhibits T-cell activation by two independent mechanisms.J Exp Med. 2003; 198: 1887-1897Crossref PubMed Scopus (236) Google Scholar, 9Sundred M.S. Torres V.J. Unutmaz D. Cover T.L. Inhibition of primary human T-cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion.Proc Natl Acad Sci U S A. 2004; 101: 7727-7732Crossref PubMed Scopus (201) Google Scholar However, there is a biological cost to long-term relationships between H. pylori and humans in that chronic inflammation confers a significantly increased risk of serious disease, including noncardia gastric adenocarcinoma, and persistent colonization is the strongest identified risk factor for malignancies that arise within the stomach.10Peek Jr, R.M. Blaser M.J. Helicobacter pylori and gastrointestinal tract adenocarcinomas.Nat Rev Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1454) Google ScholarInvestigations into mechanisms that underlie H. pylori-induced gastric cancer have emphasized that disease risk involves specific and well-choreographed interactions between pathogen and host, which, in turn, are dependent on strain-specific bacterial factors and/or host characteristics. One virulence-associated H. pylori constituent is the cag pathogenicity island, a multigene locus that is present in 60%–70% of U.S. strains and carriage of cag+ strains augments the risk for distal gastric adenocarcinoma compared to that incurred by cag− strains.11Crabtree J.E. Wyatt J.I. Sobala G.M. Miller G. Tompkins D.S. Primrose J.N. Morgan A.G. Systemic and mucosal humoral responses to Helicobacter pylori in gastric cancer.Gut. 1993; 34: 1339-1343Crossref PubMed Scopus (202) Google Scholar, 12Blaser M.J. Perez-Perez G.I. Kleanthous H. Cover T.L. Peek R.M. Chyou P.H. Stemmermann G.N. Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach.Cancer Res. 1995; 55: 2111-2115PubMed Google Scholar Several cag genes encode products that bear homology to components of a type IV bacterial secretion system, which functions as a molecular syringe to export proteins, and the product of the terminal gene in the island (CagA) is translocated into host cells after bacterial attachment. After its injection into epithelial cells by the cag secretion system, CagA undergoes tyrosine phosphorylation by members of the Src family of kinases. Phospho-CagA subsequently activates a eukaryotic phosphatase (SHP-2) as well as ERK, a member of the mitogen-activated protein kinase (MAPK) family, leading to morphological changes (e.g., cell scattering) that are reminiscent of unrestrained stimulation by growth factors.13Blaser M.J. Atherton J.C. Helicobacter pylori persistence biology and disease.J Clin Invest. 2004; 113: 321-333Crossref PubMed Scopus (750) Google Scholar It is likely no coincidence that manipulation of the interleukin (IL)-6 family coreceptor gp130 leading to altered SHP-2 signaling similarly culminates in the development of intestinal-type gastric adenocarcinoma in genetically engineered mice.14Tebbutt N.C. Giraud A.S. Inglese M. Jenkins B. Waring P. Clay F.J. Malki S. Alderman B.M. Grail D. Hollande F. Heath J.K. Ernst M. Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice.Nat Med. 2002; 8: 1089-1097Crossref PubMed Scopus (409) Google Scholar A CagA-independent consequence of cag island-mediated H. pylori–epithelial cell contact is secretion of cytokines, including IL-8, an event that is dependent on activation of the transcription factor NF-κB and MAPK by certain cag genes.15Tummuru M.K. Sharma S.A. Blaser M.J. Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells.Mol Microbiol. 1995; 18: 867-876Crossref PubMed Scopus (348) Google Scholar Thus, contact between H. pylori cag+ strains and gastric epithelial cells activates multiple signaling pathways that regulate cellular responses, which may heighten the risk for transformation, particularly over prolonged periods of colonization.Although cag+ strains are disproportionately represented among hosts who develop serious sequelae of infection and genes within the cag island are necessary for induction of pathogenic epithelial responses, most persons colonized by these strains remain completely asymptomatic. This paradox has fostered the need for studies that can more clearly define mechanisms underpinning differences in the biological activity of CagA proteins that are present in different H. pylori strains. Higashi et al. demonstrated that the number of tyrosine phosphorylation motifs (TPMs) within CagA proteins isolated from persons residing in Western countries can vary substantially and that SHP-2 binding affinity and induction of cell scattering are potentiated by an increasing number of such motifs.16Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (461) Google Scholar In contrast, TPMs within CagA proteins from East Asian H. pylori strains are unique and the sequences flanking East Asian–type TPMs perfectly match the consensus binding site for SHP-2. As expected, binding of SHP-2 and cell scattering are induced more potently by CagA proteins containing East Asian–type TPMs compared with Western-type TPMs, which may explain, in part, the strikingly different rates of gastric cancer in these regions.The aforementioned cell culture studies that have focused on H. pylori: epithelial interactions have contributed to the genesis of a molecular portrait through which CagA can commandeer host signaling pathways. However, although these events may recapitulate specific steps in the development of gastric injury and ultimately carcinoma, infiltrating inflammatory cells and extracellular matrix proteins also are likely to be involved in alterations of epithelial cell responses induced by H. pylori in vivo. To better understand the clinical ramifications of mechanistic studies, it is important to extend such findings into human populations by rigorously analyzing H. pylori strains isolated from well-characterized patients in whom gastric malignancy has been unambiguously verified. Argent et al. have now taken an important step in this critical process by examining the capacity of H. pylori cagA+ strains harvested from persons with and without gastric cancer to translocate and phosphorylate CagA and induce cytoskeletal derangements, and by investigating the relationship between such highly interactive strains and disease.17Argent R.H. Kidd M. Owen R.J. Thomas R.J. Limb M.C. Atherton J.C. Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.Gastroenterology. 2004; 127: 514-523Abstract Full Text Full Text PDF PubMed Scopus (175) Google ScholarOf 44 H. pylori strains that contained the gene cagA as determined by polymerase chain reaction (PCR), only 34 (77%) strains expressed CagA protein, and the ability to translocate and phosphorylate CagA as well as induce IL-8 production was absolutely concordant with CagA protein expression. These results suggest that molecular genotyping using PCR-based techniques that target cag genes may not reliably identify strains with increased virulence potential. The 3′ terminus of CagA from a subset of strains (n = 22) was then sequenced to determine the number and type of CagA TPMs in this population. All of the potential TPM sites were “Western-type” and varied in number from 3 to 6. Using both transformed gastric epithelial cells and a novel cell-free assay, Argent et al. showed that the relationship between cagA TPM sites and phosphorylation was directly proportional in that H. pylori CagA proteins with a higher number of TPMs became more intensely phosphorylated. The functional effects of CagA phosphorylation motif load were also examined by quantifying the extent of cell scattering in relation to TPM number and, similar to phosphorylation, the sheer number of cells displaying a scatter-type morphology, as well as the actual length of the cellular protrusions that define this phenotype, were increased by H. pylori strains bearing a higher number of CagA TPM sites. These in vitro findings were then extended by defining the relationship between CagA TPMs and disease. Five of 6 H. pylori strains that had the capacity to translocate CagA and that possessed >3 CagA variable region TPMs were harvested from patients with gastric cancer, whereas only 1 of 19 strains isolated from patients without cancer possessed these properties.There are several important points that emanate from the current study. First, segregation of pathogenic H. pylori strains on the basis of cag genotype per se is suboptimal, and a phenotypic analysis that reflects the true functionality of the cag island will likely yield more clinically relevant information. Second, the use of cell-free assays such as the system used in the current study will not only permit a less cumbersome assessment of H. pylori virulence potential, but may also facilitate the development of novel techniques that can rapidly and accurately stratify H. pylori strains based on differing gastric cancer risk. Finally, the ability to translate focused observations from a model of pathogen-induced cellular dysfunction under rigorously controlled laboratory conditions to the clinical arena is critical for understanding the effects of H. pylori on gastric carcinogenesis. Although the current results linking H. pylori strains with a functional cag island and a heightened number of CagA TPMs with gastric cancer did not achieve statistical significance (P = 0.09), these findings mirror previous studies demonstrating that H. pylori strains expressing high-copy TPM CagA proteins are more closely associated with gastric atrophy and gastric cancer,18Yamaoka Y. Kodama T. Kashima K. Graham D.Y. Sepulveda A.R. Variants of the 3′ region of the cagA gene in Helicobacter pylori isolates from patients with different H. pylori–associated diseases.J Clin Microbiol. 1998; 36: 2258-2263PubMed Google Scholar, 19Azuma T. Yamakawa A. Yamazaki S. Fukuta K. Ohtani M. Ito Y. Dojo M. Yamazaki Y. Kuriyama M. Correlation between variation of the 3′ region of the cagA gene in Helicobacter pylori and disease outcome in Japan.J Infect Dis. 2002; 186: 1621-1630Crossref PubMed Scopus (117) Google Scholar and therefore bolster the validity of this relationship.Colonization of humans by pathogenic bacteria is relatively common, but the development of diseases such as cancer follows in only a fraction of colonized persons. Consideration must be given to strain-specific differences that exist among isolates of an individual species, such as H. pylori, as well as the functions associated with such differences. However, it is likely that CagA phosphorylation is not a penultimate event in gastric adenocarcinogenesis. In addition to the effects of phospho-CagA on signaling pathways that alter cellular morphology, CagA phosphorylation also results in activation of C-terminal Src kinase, which inhibits the activity of Src, leading to a reciprocal decrease in the level of phosphorylated CagA. Although this negative feedback loop likely contributes to the long-term equilibrium between H. pylori and its host, emerging data indicate that unphosphorylated CagA can exert effects within the cell that contribute to pathogenesis. For example, unmodified CagA binds to growth factor receptor bound 2 (Grb2), which results in activation of the Ras/MEK/ERK MAPK pathway.20Mimuro H. Suzuki T. Tanaka J. Asahi M. Haas R. Sasakawa C. Grb2 is a key mediator of Helicobacter pylori CagA protein activities.Mol Cell. 2002; 10: 745-755Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Translocation, but not phosphorylation, of CagA leads to disruption of apical-junctional complexes in polarized epithelial cells and a loss of cellular polarity, alterations that have previously been shown to play a role in carcinogenesis.21Amieva M.R. Vogelmann R. Covacci A. Tompkins L.S. Nelson W.J. Falkow S. Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA.Science. 2003; 300: 1430-1434Crossref PubMed Scopus (612) Google ScholarH. pylori loci exogenous to the cag island also influence the progression to gastric cancer. Approximately 50% of H. pylori isolates express a toxin with vacuolating activity, and these strains are more common among patients with distal gastric cancer than among patients with gastritis alone.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (522) Google Scholar BabA, encoded by the strain-specific gene babA2, is an adhesin that binds the Lewisb histo-blood-group antigen, and H. pylori babA2+ strains are associated with an increased risk for gastric adenocarcinoma compared with strains that lack babA2.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (522) Google Scholar Another H. pylori adhesin, SabA, has recently been shown to bind the sialyl-Lewis a antigen, an established tumor antigen and marker of gastric dysplasia that is up-regulated by gastric inflammation.23Mahdavi J. Sonden B. Hurtig M. Olfat F.O. Forsberg L. Roche N. Angstrom J. Larsson T. Teneberg S. Karlsson K.A. Altraja S. Wadstrom T. Kersulyte D. Berg D.E. Dubois A. Petersson C. Magnusson K.E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarstrom L. Boren T. Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation.Science. 2002; 297: 573-578Crossref PubMed Scopus (701) Google Scholar In addition to characterizing microbial constituents that influence disease outcome, host responses to such factors must also be examined to completely ascertain mechanisms that lead to human disease. Polymorphisms within immune response genes, such as IL-1β, TNF-α, and IL-10, which are associated with increased proinflammatory gene expression, heighten the risk for atrophic gastritis and distal gastric adenocarcinoma among H. pylori–infected persons, and a synergistic effect on gastric cancer risk is present when virulence determinants of infecting H. pylori isolates are examined in conjunction with the polymorphism status of their cognate hosts.24El-Omar E.M. Rabkin C.S. Gammon M.D. Vaughan T.L. Risch H.A. Schoenberg J.B. Stanford J.L. Mayne S.T. Goedert J. Blot W.J. Fraumeni Jr, J.F. Chow W.H. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms.Gastroenterology. 2003; 124: 1193-1201Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar, 25Figueiredo C. Machado J.C. Pharoah P. Seruca R. Sousa S. Carvalho R. Capelinha A.F. Quint W. Caldas C. van Doorn L.J. Carneiro F. Sobrinho-Simoes M. Helicobacter pylori and interleukin 1 genotyping an opportunity to identify high-risk individuals for gastric carcinoma.J Natl Cancer Inst. 2002; 94: 1680-1687Crossref PubMed Scopus (553) Google ScholarStudies such as the one from Argent et al. are extremely valuable, primarily because of the significance of H. pylori as a human pathogen. Gastric cancer is a highly lethal disease when diagnosed in the United States, and establishment of H. pylori as a risk factor for this malignancy permits an approach to identify persons at increased risk; however, infection with this organism is extremely common and most colonized persons never develop cancer. Thus, techniques to identify high-risk subpopulations must use other biological markers. Analytical tools now exist, including genome sequences (H. pylori and human), measurable phenotypes (CagA phosphorylation), and practical animal models, to discern the fundamental biological basis of H. pylori–associated neoplasia, which should have direct clinical applications. It is important to gain more insight into the pathogenesis of H. pylori–induced gastric adenocarcinoma, not only to develop more effective treatments for this common cancer, but also because it might serve as a paradigm for the role of chronic inflammation in the genesis of other malignancies that arise within the gastrointestinal tract. See article on page 514. See article on page 514. See article on page 514. Virtually all persons who are colonized by Helicobacter pylori develop coexisting gastritis, a signature feature of which is the capacity to persist for decades. This is in marked contrast to inflammatory reactions induced by other Gram-negative enteric pathogens, such as Salmonella, that either resolve within days to weeks or progress to eliminate the host. One mechanism through which H. pylori may evade immune clearance is by limiting the bactericidal effects of proinflammatory molecules, such as nitric oxide.1Gobert A.P. McGee D.J. Akhtar M. Mendz G.L. Newton J.C. Cheng Y. Mobley H.L. Wilson K.T. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells a strategy for bacterial survival.Proc Natl Acad Sci U S A. 2001; 98: 13844-13849Crossref PubMed Scopus (308) Google Scholar Another level of host defense that may be circumvented by H. pylori is innate immunity. Toll-like receptors (TLRs) are an evolutionarily conserved family of eukaryotic receptors that function in innate immunity via recognition of invariant regions in bacterial molecules termed pathogen- or microbe-associated molecular patterns. It is becoming increasingly clear, however, that H. pylori has evolved strategies to avoid activation of this system. For example, TLR4 recognizes bacterial lipopolysaccharide (LPS), yet H. pylori LPS is relatively anergic compared with that of other enteric bacteria, primarily because of lipid A core modifications.2Perez-Perez G.I. Shepherd V.L. Morrow J.D. Blaser M.J. Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide.Infect Immun. 1995; 63: 1183-1187PubMed Google Scholar, 3Backhed F. Rokbi B. Torstensson E. Zhao Y. Nilsson C. Seguin D. Normark S. Buchan A.M. Richter-Dahlfors A. Gastric mucosal recognition of Helicobacter pylori is independent of toll-like receptor 4.J Infect Dis. 2003; 187: 829-836Crossref PubMed Scopus (115) Google Scholar In contrast to flagellins expressed by Gram-negative mucosal pathogens, which activate TLR5-mediated proinflammatory responses, H. pylori flagellin is noninflammatory.4Lee S.K. Stack A. Katzowitsch E. Aizawa S.I. Suerbaum S. Josenhans C. Helicobacter pylori flagellins have very low intrinsic activity to stimulate human gastric epithelial cells via TLR5.Microbes Infect. 2003; 5: 1345-1356Crossref PubMed Scopus (148) Google Scholar, 5Gewirtz A.T. Yu Y. Krishna U.S. Israel D.A. Lyons S.L. Peek R.M. Helicobacter pylori flagellin evades Toll-like receptor 5-mediated innate immunity.J Infect Dis. 2004; 189: 1914-1920Crossref PubMed Scopus (216) Google ScholarH. pylori can also facilitate persistence by varying the antigenic repertoire of surface-exposed proteins6Aras R.A. Fischer W. Perez-Perez G.I. Crosatti M.L. Ando T. Haas R. Blaser M.J. Plasticity of repetitive DNA sequences within a bacterial (type IV) secretion system component.J Exp Med. 2003; 198: 1349-1360Crossref PubMed Scopus (77) Google Scholar and by actively suppressing the host adaptive immune response.7Gebert B. Fischer W. Weiss E. Hoffmann R. Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T-lymphocyte activation.Science. 2003; 301: 1099-1102Crossref PubMed Scopus (446) Google Scholar, 8Boncristiano M. Paccani S.R. Barone S. Ulivieri C. Patrussi L. Ilver D. Amedei A. D'Elios M.M. Telford J.L. The Helicobacter pylori vacuolating cytotoxin inhibits T-cell activation by two independent mechanisms.J Exp Med. 2003; 198: 1887-1897Crossref PubMed Scopus (236) Google Scholar, 9Sundred M.S. Torres V.J. Unutmaz D. Cover T.L. Inhibition of primary human T-cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion.Proc Natl Acad Sci U S A. 2004; 101: 7727-7732Crossref PubMed Scopus (201) Google Scholar However, there is a biological cost to long-term relationships between H. pylori and humans in that chronic inflammation confers a significantly increased risk of serious disease, including noncardia gastric adenocarcinoma, and persistent colonization is the strongest identified risk factor for malignancies that arise within the stomach.10Peek Jr, R.M. Blaser M.J. Helicobacter pylori and gastrointestinal tract adenocarcinomas.Nat Rev Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1454) Google Scholar Investigations into mechanisms that underlie H. pylori-induced gastric cancer have emphasized that disease risk involves specific and well-choreographed interactions between pathogen and host, which, in turn, are dependent on strain-specific bacterial factors and/or host characteristics. One virulence-associated H. pylori constituent is the cag pathogenicity island, a multigene locus that is present in 60%–70% of U.S. strains and carriage of cag+ strains augments the risk for distal gastric adenocarcinoma compared to that incurred by cag− strains.11Crabtree J.E. Wyatt J.I. Sobala G.M. Miller G. Tompkins D.S. Primrose J.N. Morgan A.G. Systemic and mucosal humoral responses to Helicobacter pylori in gastric cancer.Gut. 1993; 34: 1339-1343Crossref PubMed Scopus (202) Google Scholar, 12Blaser M.J. Perez-Perez G.I. Kleanthous H. Cover T.L. Peek R.M. Chyou P.H. Stemmermann G.N. Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach.Cancer Res. 1995; 55: 2111-2115PubMed Google Scholar Several cag genes encode products that bear homology to components of a type IV bacterial secretion system, which functions as a molecular syringe to export proteins, and the product of the terminal gene in the island (CagA) is translocated into host cells after bacterial attachment. After its injection into epithelial cells by the cag secretion system, CagA undergoes tyrosine phosphorylation by members of the Src family of kinases. Phospho-CagA subsequently activates a eukaryotic phosphatase (SHP-2) as well as ERK, a member of the mitogen-activated protein kinase (MAPK) family, leading to morphological changes (e.g., cell scattering) that are reminiscent of unrestrained stimulation by growth factors.13Blaser M.J. Atherton J.C. Helicobacter pylori persistence biology and disease.J Clin Invest. 2004; 113: 321-333Crossref PubMed Scopus (750) Google Scholar It is likely no coincidence that manipulation of the interleukin (IL)-6 family coreceptor gp130 leading to altered SHP-2 signaling similarly culminates in the development of intestinal-type gastric adenocarcinoma in genetically engineered mice.14Tebbutt N.C. Giraud A.S. Inglese M. Jenkins B. Waring P. Clay F.J. Malki S. Alderman B.M. Grail D. Hollande F. Heath J.K. Ernst M. Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice.Nat Med. 2002; 8: 1089-1097Crossref PubMed Scopus (409) Google Scholar A CagA-independent consequence of cag island-mediated H. pylori–epithelial cell contact is secretion of cytokines, including IL-8, an event that is dependent on activation of the transcription factor NF-κB and MAPK by certain cag genes.15Tummuru M.K. Sharma S.A. Blaser M.J. Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells.Mol Microbiol. 1995; 18: 867-876Crossref PubMed Scopus (348) Google Scholar Thus, contact between H. pylori cag+ strains and gastric epithelial cells activates multiple signaling pathways that regulate cellular responses, which may heighten the risk for transformation, particularly over prolonged periods of colonization. Although cag+ strains are disproportionately represented among hosts who develop serious sequelae of infection and genes within the cag island are necessary for induction of pathogenic epithelial responses, most persons colonized by these strains remain completely asymptomatic. This paradox has fostered the need for studies that can more clearly define mechanisms underpinning differences in the biological activity of CagA proteins that are present in different H. pylori strains. Higashi et al. demonstrated that the number of tyrosine phosphorylation motifs (TPMs) within CagA proteins isolated from persons residing in Western countries can vary substantially and that SHP-2 binding affinity and induction of cell scattering are potentiated by an increasing number of such motifs.16Higashi H. Tsutsumi R. Fujita A. Yamazaki S. Asaka M. Azuma T. Hatakeyama M. Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites.Proc Natl Acad Sci U S A. 2002; 99: 14428-14433Crossref PubMed Scopus (461) Google Scholar In contrast, TPMs within CagA proteins from East Asian H. pylori strains are unique and the sequences flanking East Asian–type TPMs perfectly match the consensus binding site for SHP-2. As expected, binding of SHP-2 and cell scattering are induced more potently by CagA proteins containing East Asian–type TPMs compared with Western-type TPMs, which may explain, in part, the strikingly different rates of gastric cancer in these regions. The aforementioned cell culture studies that have focused on H. pylori: epithelial interactions have contributed to the genesis of a molecular portrait through which CagA can commandeer host signaling pathways. However, although these events may recapitulate specific steps in the development of gastric injury and ultimately carcinoma, infiltrating inflammatory cells and extracellular matrix proteins also are likely to be involved in alterations of epithelial cell responses induced by H. pylori in vivo. To better understand the clinical ramifications of mechanistic studies, it is important to extend such findings into human populations by rigorously analyzing H. pylori strains isolated from well-characterized patients in whom gastric malignancy has been unambiguously verified. Argent et al. have now taken an important step in this critical process by examining the capacity of H. pylori cagA+ strains harvested from persons with and without gastric cancer to translocate and phosphorylate CagA and induce cytoskeletal derangements, and by investigating the relationship between such highly interactive strains and disease.17Argent R.H. Kidd M. Owen R.J. Thomas R.J. Limb M.C. Atherton J.C. Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.Gastroenterology. 2004; 127: 514-523Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar Of 44 H. pylori strains that contained the gene cagA as determined by polymerase chain reaction (PCR), only 34 (77%) strains expressed CagA protein, and the ability to translocate and phosphorylate CagA as well as induce IL-8 production was absolutely concordant with CagA protein expression. These results suggest that molecular genotyping using PCR-based techniques that target cag genes may not reliably identify strains with increased virulence potential. The 3′ terminus of CagA from a subset of strains (n = 22) was then sequenced to determine the number and type of CagA TPMs in this population. All of the potential TPM sites were “Western-type” and varied in number from 3 to 6. Using both transformed gastric epithelial cells and a novel cell-free assay, Argent et al. showed that the relationship between cagA TPM sites and phosphorylation was directly proportional in that H. pylori CagA proteins with a higher number of TPMs became more intensely phosphorylated. The functional effects of CagA phosphorylation motif load were also examined by quantifying the extent of cell scattering in relation to TPM number and, similar to phosphorylation, the sheer number of cells displaying a scatter-type morphology, as well as the actual length of the cellular protrusions that define this phenotype, were increased by H. pylori strains bearing a higher number of CagA TPM sites. These in vitro findings were then extended by defining the relationship between CagA TPMs and disease. Five of 6 H. pylori strains that had the capacity to translocate CagA and that possessed >3 CagA variable region TPMs were harvested from patients with gastric cancer, whereas only 1 of 19 strains isolated from patients without cancer possessed these properties. There are several important points that emanate from the current study. First, segregation of pathogenic H. pylori strains on the basis of cag genotype per se is suboptimal, and a phenotypic analysis that reflects the true functionality of the cag island will likely yield more clinically relevant information. Second, the use of cell-free assays such as the system used in the current study will not only permit a less cumbersome assessment of H. pylori virulence potential, but may also facilitate the development of novel techniques that can rapidly and accurately stratify H. pylori strains based on differing gastric cancer risk. Finally, the ability to translate focused observations from a model of pathogen-induced cellular dysfunction under rigorously controlled laboratory conditions to the clinical arena is critical for understanding the effects of H. pylori on gastric carcinogenesis. Although the current results linking H. pylori strains with a functional cag island and a heightened number of CagA TPMs with gastric cancer did not achieve statistical significance (P = 0.09), these findings mirror previous studies demonstrating that H. pylori strains expressing high-copy TPM CagA proteins are more closely associated with gastric atrophy and gastric cancer,18Yamaoka Y. Kodama T. Kashima K. Graham D.Y. Sepulveda A.R. Variants of the 3′ region of the cagA gene in Helicobacter pylori isolates from patients with different H. pylori–associated diseases.J Clin Microbiol. 1998; 36: 2258-2263PubMed Google Scholar, 19Azuma T. Yamakawa A. Yamazaki S. Fukuta K. Ohtani M. Ito Y. Dojo M. Yamazaki Y. Kuriyama M. Correlation between variation of the 3′ region of the cagA gene in Helicobacter pylori and disease outcome in Japan.J Infect Dis. 2002; 186: 1621-1630Crossref PubMed Scopus (117) Google Scholar and therefore bolster the validity of this relationship. Colonization of humans by pathogenic bacteria is relatively common, but the development of diseases such as cancer follows in only a fraction of colonized persons. Consideration must be given to strain-specific differences that exist among isolates of an individual species, such as H. pylori, as well as the functions associated with such differences. However, it is likely that CagA phosphorylation is not a penultimate event in gastric adenocarcinogenesis. In addition to the effects of phospho-CagA on signaling pathways that alter cellular morphology, CagA phosphorylation also results in activation of C-terminal Src kinase, which inhibits the activity of Src, leading to a reciprocal decrease in the level of phosphorylated CagA. Although this negative feedback loop likely contributes to the long-term equilibrium between H. pylori and its host, emerging data indicate that unphosphorylated CagA can exert effects within the cell that contribute to pathogenesis. For example, unmodified CagA binds to growth factor receptor bound 2 (Grb2), which results in activation of the Ras/MEK/ERK MAPK pathway.20Mimuro H. Suzuki T. Tanaka J. Asahi M. Haas R. Sasakawa C. Grb2 is a key mediator of Helicobacter pylori CagA protein activities.Mol Cell. 2002; 10: 745-755Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Translocation, but not phosphorylation, of CagA leads to disruption of apical-junctional complexes in polarized epithelial cells and a loss of cellular polarity, alterations that have previously been shown to play a role in carcinogenesis.21Amieva M.R. Vogelmann R. Covacci A. Tompkins L.S. Nelson W.J. Falkow S. Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA.Science. 2003; 300: 1430-1434Crossref PubMed Scopus (612) Google Scholar H. pylori loci exogenous to the cag island also influence the progression to gastric cancer. Approximately 50% of H. pylori isolates express a toxin with vacuolating activity, and these strains are more common among patients with distal gastric cancer than among patients with gastritis alone.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (522) Google Scholar BabA, encoded by the strain-specific gene babA2, is an adhesin that binds the Lewisb histo-blood-group antigen, and H. pylori babA2+ strains are associated with an increased risk for gastric adenocarcinoma compared with strains that lack babA2.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (522) Google Scholar Another H. pylori adhesin, SabA, has recently been shown to bind the sialyl-Lewis a antigen, an established tumor antigen and marker of gastric dysplasia that is up-regulated by gastric inflammation.23Mahdavi J. Sonden B. Hurtig M. Olfat F.O. Forsberg L. Roche N. Angstrom J. Larsson T. Teneberg S. Karlsson K.A. Altraja S. Wadstrom T. Kersulyte D. Berg D.E. Dubois A. Petersson C. Magnusson K.E. Norberg T. Lindh F. Lundskog B.B. Arnqvist A. Hammarstrom L. Boren T. Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation.Science. 2002; 297: 573-578Crossref PubMed Scopus (701) Google Scholar In addition to characterizing microbial constituents that influence disease outcome, host responses to such factors must also be examined to completely ascertain mechanisms that lead to human disease. Polymorphisms within immune response genes, such as IL-1β, TNF-α, and IL-10, which are associated with increased proinflammatory gene expression, heighten the risk for atrophic gastritis and distal gastric adenocarcinoma among H. pylori–infected persons, and a synergistic effect on gastric cancer risk is present when virulence determinants of infecting H. pylori isolates are examined in conjunction with the polymorphism status of their cognate hosts.24El-Omar E.M. Rabkin C.S. Gammon M.D. Vaughan T.L. Risch H.A. Schoenberg J.B. Stanford J.L. Mayne S.T. Goedert J. Blot W.J. Fraumeni Jr, J.F. Chow W.H. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms.Gastroenterology. 2003; 124: 1193-1201Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar, 25Figueiredo C. Machado J.C. Pharoah P. Seruca R. Sousa S. Carvalho R. Capelinha A.F. Quint W. Caldas C. van Doorn L.J. Carneiro F. Sobrinho-Simoes M. Helicobacter pylori and interleukin 1 genotyping an opportunity to identify high-risk individuals for gastric carcinoma.J Natl Cancer Inst. 2002; 94: 1680-1687Crossref PubMed Scopus (553) Google Scholar Studies such as the one from Argent et al. are extremely valuable, primarily because of the significance of H. pylori as a human pathogen. Gastric cancer is a highly lethal disease when diagnosed in the United States, and establishment of H. pylori as a risk factor for this malignancy permits an approach to identify persons at increased risk; however, infection with this organism is extremely common and most colonized persons never develop cancer. Thus, techniques to identify high-risk subpopulations must use other biological markers. Analytical tools now exist, including genome sequences (H. pylori and human), measurable phenotypes (CagA phosphorylation), and practical animal models, to discern the fundamental biological basis of H. pylori–associated neoplasia, which should have direct clinical applications. It is important to gain more insight into the pathogenesis of H. pylori–induced gastric adenocarcinoma, not only to develop more effective treatments for this common cancer, but also because it might serve as a paradigm for the role of chronic inflammation in the genesis of other malignancies that arise within the gastrointestinal tract.