Stability of Helicobacter pylori CagA oncoprotein in human gastric epithelial cells

Upon delivery into gastric epithelial cells, Helicobacter pylori cytotoxin-associated gene A (CagA) binds and deregulates cellular proteins such as Src homology 2 domain-containing protein tyrosine phosphatase 2 and partitioning-defective 1 (PAR1), thereby acting as an epigenetic oncoprotein that promotes early phases of gastric cancer development. To elucidate the spatial and temporal contribution of CagA to carcinogenesis, it is crucial to know the stability of CagA in host cells. Here we show that the biological half-life of CagA is about 200 min in gastric epithelial cells. Furthermore, deletion of the PAR1-binding sequence accelerates CagA degradation. Thus, CagA is a relatively short half-life protein whose stability may be modulated through complex formation with PAR1.     

CagA of Helicobacter pylori interacts with and inhibits the serine‐threonine kinase PRK2

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation, CagA interferes with various host–cell signalling pathways. Most notably this toxin is involved in the disruption of apical–basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration and cell morphological changes. These are processes that also play an important role in epithelial‐to‐mesenchymal transition and cancer cell invasion. In fact, CagA is considered as the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine–threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP‐2. Additionally, CagA was described to affect the activity of Src family kinases and C‐terminal Src kinase (Csk) suggesting that interference with multiple cellular kinase‐ and phosphatase‐associated signalling pathways is a major function of CagA. Here, we describe the effect of CagA on protein kinase C‐related kinase 2 (PRK2), which acts downstream of Rho GTPases and is known to affect cytoskeletal rearrangements and cell polarity. CagA interacts with PRK2 and inhibits its kinase activity. Because PRK2 has been linked to cytoskeletal rearrangements and establishment of cell polarity, we suggest that CagA may hijack PRK2 to further manipulate cancer‐related signalling pathways.     

Attenuation of Helicobacter pylori CagA x SHP-2 signaling by interaction between CagA and C-terminal Src kinase

Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "hummingbird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.     

Tertiary structure-function analysis reveals the pathogenic signaling potentiation mechanism of Helicobacter pylori oncogenic effector CagA

The Helicobacter pylori type IV secretion effector CagA is a major bacterial virulence determinant and critical for gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA localizes to the inner face of the plasma membrane, where it acts as a pathogenic scaffold/hub that promiscuously recruits host proteins to potentiate oncogenic signaling. We find that CagA comprises a structured N-terminal region and an intrinsically disordered C-terminal region that directs versatile protein interactions. X-ray crystallographic analysis of the N-terminal CagA fragment (residues 1-876) revealed that the region has a structure comprised of three discrete domains. Domain I constitutes a mobile CagA N terminus, while Domain II tethers CagA to the plasma membrane by interacting with membrane phosphatidylserine. Domain III interacts intramolecularly with the intrinsically disordered C-terminal region, and this interaction potentiates the pathogenic scaffold/hub function of CagA. The present work provides a tertiary-structural basis for the pathophysiological/oncogenic action of H. pylori CagA.     

SagA of CagA in Helicobacter pylori pathogenesis

Much attention has recently been given to the role of the Helicobacter pylori CagA protein, the only as yet identified H. pylori protein that is delivered into the host gastric epithelial cells by a type IV secretion system, in the development of H. pylori-associated diseases, including gastric carcinoma. This review summarizes the latest advances in our understanding of pathogenic actions of H. pylori CagA, particularly focusing on the molecular mechanisms underlying CagA entry into the host cells as well as CagA-mediated perturbation of host cell signaling involved in proliferation, motility, differentiation, and polarity, which contributes malignant transformation of mammalian cells.     

Conformational Analysis of Isolated Domains of Helicobacter pylori CagA

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation - a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.     

Focal adhesion kinase is a substrate and downstream effector of SHP-2 complexed with Helicobacter pylori CagA

Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the "hummingbird phenotype." Here we show that CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection.     

Attenuated CagA oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon

Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically.     

Grb2 is a key mediator of helicobacter pylori CagA protein activities

CagA delivered from Helicobacter pylori into gastric epithelial cells undergoes tyrosine phosphorylation and induces host cell morphological changes. Here we show that CagA can interact with Grb2 both in vitro and in vivo, which results in the activation of the Ras/MEK/ERK pathway and leads to cell scattering as well as proliferation. Importantly, this ability of CagA is independent from the tyrosine phosphorylation, which occurs within the five repeated EPIYA sequences (PY region) of CagA. However, the PY region appears to be indispensable for the Grb2 binding and induction of the cellular responses. Thus, intracellular CagA via its binding to Grb2 may act as a transducer for stimulating growth factor-like downstream signals which lead to cell morphological changes and proliferation, the causes of H. pylori-induced gastric hyperplasia.     

The Helicobacter pylori CagA protein induces cortactin dephosphorylation and actin rearrangement by c-Src inactivation

The gastric pathogen Helicobacter pylori translocates the CagA protein into epithelial cells by a type IV secretion process. Translocated CagA is tyrosine phosphorylated (CagA(P-Tyr)) on specific EPIYA sequence repeats by Src family tyrosine kinases. Phos phorylation of CagA induces the dephosphorylation of as yet unidentified cellular proteins, rearrangements of the host cell actin cytoskeleton and cell scattering. We show here that CagA(P-Tyr) inhibits the catalytic activity of c-Src in vivo and in vitro. c-Src inactivation leads to tyrosine dephosphorylation of the actin binding protein cortactin. Concomitantly, cortactin is specifically redistributed to actin-rich cellular protrusions. c-Src inactivation and cortactin dephosphorylation are required for rearrangements of the actin cytoskeleton. Moreover, CagA(P-Tyr)-mediated c-Src inhibition downregulates further CagA phosphorylation through a negative feedback loop. This is the first report of a bacterial virulence factor that inhibits signalling of a eukaryotic tyrosine kinase and on a role of c-Src inactivation in host cell cytoskeletal rearrangements.     

c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs

The human pathogen Helicobacter pylori colonizes the mucous layer of the stomach. During parasitic infection, freely swimming bacteria adhere to the gastric epithelial cells and trigger intracellular signalling pathways. This process requires the translocation of the effector protein CagA into the host cell through a specialized type IV secretion system encoded in the cag pathogenicity island. Following transfer, CagA is phosphorylated on tyrosine residues by a host cell kinase. Here, we describe how the tyrosine phosphorylation of CagA is restricted to a previously identified repeated sequence called D1. This sequence is located in the C-terminal half of the protein and contains the five-amino-acid motif EPIYA, which is amplified by duplications in a large fraction of clinical isolates. Tyrosine phosphorylation of CagA is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. In addition, we observed that two members of the src kinases family, c-Src and Lyn, account for most of the CagA-specific kinase activity in host cell lysates. Thus, CagA translocation followed by tyrosine phosphorylation at the EPIYA motifs promotes a growth factor-like response with intense cytoskeletal rearrangements, cell elongation effects and increased cellular motility.     

Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo.

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.     

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.     

Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche.     

Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA⁺/VacA⁺ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.     

Helicobacter pylori infection and CagA protein translocation in human primary gastric epithelial cell culture

BACKGROUND: Increasing evidence has shown that Helicobacter pylori CagA protein translocation into gastric epithelial cells plays an important role in the development of gastric inflammation and malignancy. Translocated CagA undergoes tyrosine phosphorylation in gastric adenocarcinoma cell line cells, and CagA involves disruption of cellular apical-junction complex in Madin-Darby canine kidney cells. METHODS: To elucidate whether these events take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. RESULTS: Our results demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeletal rearrangement and the disruption of tight junctions in primary gastric epithelial cells. CONCLUSIONS: This study provides direct evidence of the modulation of gastric epithelial cells by CagA protein translocation, and advances our understanding of the pathogenesis of H. pylori infection.     

The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin

Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of the CagA bacterial protein into gastric epithelial cells has been linked to an increased risk of peptic ulcer disease and gastric carcinoma. Upon translocation, CagA is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated CagA disrupts adhesion of AGS cells to the extracellular matrix. Owing to the inactivation of c-Src via CagA interaction, vinculin is dephosphorylated at tyrosine residues, 100 and 1065, by corresponding phosphatases. Vinculin dephosphorylation disturbs the interaction and recruitment of the actin-related protein 2/3 (Arp2/3) complex by p34Arc, resulting in a reduction of focal adhesion complexes. These defects can be mimicked by downregulating vinculin using RNA interference in non-infected cells. Tyrosine dephosphorylation of vinculin results in severe cellular deficiencies in cell-matrix adhesion, cell spreading and wound repair. We hypothesize that CagA-mediated inactivation of vinculin is a key step in the mechanism by which H. pylori induces damage to the gastric epithelium and represents an important step in disease development.