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.     

Subversion of host kinases: a key network in cellular signaling hijacked by Helicobacter pylori CagA

Helicobacter pylori is a paradigm of persistent pathogens and major risk factor for developing severe diseases including adenocarcinoma in the human stomach. An important bacterial factor linked to gastric disease progression is the cag pathogenicity island‐encoded type‐IV secretion system (T4SS) effector protein CagA. Translocated CagA undergoes tyrosine phosphorylation at EPIYA‐motifs and then activates or inactivates multiple host signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent fashion. In this way, intracellular CagA acts as a ‘masterkey’ or ‘picklock’, which evolved during evolution to hijack key host cell signal transduction functions. Crucial targets of CagA represent a variety of serine/threonine and tyrosine kinases, which control major checkpoints of eukaryotic signaling. Here we review the signal transmission by translocated CagA on multiple receptor kinases (c‐Met and EGFR) and non‐receptor kinases (Src, Abl, Csk, aPKC, Par1, PI3K, Akt, FAK, GSK‐3, JAK, PAK1, PAK2 and MAP kinases), manipulating a selection of fundamental processes in the human gastric epithelium such as cell adhesion, polarity, proliferation, motility, receptor endocytosis, cytoskeletal rearrangements, apoptosis, inflammation and cell cycle progression. This enormous complexity generates a highly remarkable and puzzling scenario during H. pylori infection. The contribution of these signaling pathways to bacterial survival, persistence and gastric pathogenesis is discussed.     

The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key Hypothesis

Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type‐IV secretion system. Injected CagA becomes tyrosine‐phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high‐resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence‐associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a ‘master key’ that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin‐cytoskeletal rearrangements and the disruption of cell‐to‐cell junctions as well as proliferative, pro‐inflammatory and anti‐apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon.     

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.     

Haem oxygenase‐1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells

The cytotoxin‐associated gene A protein (CagA) plays a pivotal role in the aetiology of Helicobacter pylori‐associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c‐Src. We previously reported that the enzyme haem oxygenase‐1 (HO‐1) inhibits IL‐8 secretion by H. pylori‐infected cells. However, the cellular mechanism by which HO‐1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and haemin, two inducers of HO‐1, decrease the level of phosphorylated CagA (p‐CagA) in H. pylori‐infected gastric epithelial cells and this is blocked by either pharmacological inhibition of HO‐1 or siRNA knockdown of hmox‐1. Moreover, forced expression of HO‐1 by transfection of a plasmid expressing hmox‐1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori‐induced c‐Src phosphorylation/activation by HO‐1.Consequently, H. pylori‐induced cytoskeletal rearrangements and activation of the pro‐inflammatory response mediated by p‐CagA are inhibited in HO‐1‐expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells.     

Role of Abl and Src family kinases in actin-cytoskeletal rearrangements induced by the Helicobacter pylori CagA protein

The clinical outcome of infections with Helicobacter pylori is determined by a complex interplay of host-pathogen interactions, and persistent infection with this pathogen is the major cause of developing chronic gastritis, peptic ulcers and gastric cancer. Highly virulent strains encode a so-called type IV secretion system which translocates the CagA effector protein into gastric epithelial target cells. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinase members. CagA then binds to and activates/inactivates various signalling proteins in a phosphorylation-dependent and phosphorylation-independent manner. In this way injected CagA can act as a master key that evolved during evolution the ability to highjack multiple downstream signalling cascades. Here we review our knowledge on the tyrosine phosphorylation motifs in CagA, the recent advances in the interaction of CagA with Src and Abl tyrosine kinases and their role in signalling events leading to changes of the phosphorylation status of actin-binding proteins cortactin, ezrin and vinculin followed by actin-cytoskeletal rearrangements, cell scattering and elongation. Detailed investigation of these pathways will help to yield novel insights and to elucidate the mechanisms of H. pylori-induced pathogenesis.     

CagA phosphorylation-dependent MMP-9 expression in gastric epithelial cells

Background: Infection of cagA‐positive Helicobacter pylori is associated with increased expression of MMPs in gastric epithelial cells. The role of phosphorylated CagA in the induction of MMP‐9, a protease‐degrading basement membrane, in gastric epithelial cells has not been clearly defined yet. The aim of this study is to analyze whether the presence of CagA and its phosphorylation status play a role in increased expression of MMP‐9 in gastric epithelial cells. Materials and Methods: Induction of MMP‐9 secretion was analyzed in gastric epithelial AGS cells harboring CagA with or without EPIYA motif, which is injected by H. pylori or ectopically expressed. In addition, signaling pathways involved in the CagA‐dependent MMP‐9 production have been studied. Results: The 147C strain of H. pylori expressing tyrosine‐phosphorylated CagA (EPIYA present) induced higher MMP‐9 secretion by AGS cells than the 147A strain expressing non‐tyrosine‐phosphorylated CagA (EPIYA absent). In addition, in bacteria‐free CagA‐inducible AGS cells, expression of wild‐type CagA induced more MMP‐9 secretion than phosphorylation‐resistant CagA. Inhibition of CagA phosphorylation by the Src family kinase inhibitor PP1 downregulated CagA‐mediated MMP‐9 secretion. Knockdown of SHP‐2 phosphatase dramatically reduced MMP‐9 secretion. ERK inhibitors, PD98059 and U0126, and NF‐κB pathway inhibitors, sulfasalazine and N‐acetyl‐l ‐cysteine, also inhibited MMP‐9 expression. Conclusion: These results support a model whereby the EPIYA motif of CagA is phosphorylated by Src family kinases in gastric epithelial cells, which initiates activation of SHP‐2. In addition, they suggest that the resultant activation of ERK pathway along with CagA‐dependent NF‐κB activation is critical for the induction of MMP‐9 secretion.     

Role of type IV secretion in Helicobacter pylori pathogenesis

Helicobacter pylori is a highly successful human-specific gastric pathogen that colonizes more than half the world's population. Infection with this bacterium can induce gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent H. pylori isolates harbour the cag (cytotoxin-associated genes) pathogenicity island, a 40 kb stretch of DNA that encodes components of a type IV secretion system (T4SS). This T4SS forms a pilus for the injection of virulence factors into host target cells such as the CagA oncoprotein. This is accomplished by a specialized adhesin of the pilus surface, the CagL protein, which binds to and activates host cell integrins for subsequent delivery of CagA across the host cell membrane. Injected CagA becomes tyrosine-phosphorylated by Src and Abl family kinases and mimics a host cell protein in binding and activation of multiple signalling factors. Here we review the recent advances in the characterization of phosphorylation-dependent and phosphorylation-independent signalling activities of CagA and the T4SS which include the induction of membrane dynamics, actin cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and anti-apoptotic nuclear responses. The contribution of these signalling cascades to H. pylori pathogenesis is discussed.     

The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling

This study was performed to elucidate the host cell scaffolding and signalling molecules that Campylobacter jejuni utilizes to invade epithelial cells. We hypothesized that the C. jejuni fibronectin‐binding proteins and secreted proteins are required for cell signalling and maximal invasion of host cells. C. jejuni binding to host cells via the CadF and FlpA fibronectin‐binding proteins activated the epidermal growth factor (EGF) pathway, as evidenced by inhibitor studies and immunoprecipitation coupled with immunoblot analysis using antibodies reactive against total and active EGF receptor. Inhibitor studies revealed maximal C. jejuni host cell invasion was dependent upon PI3‐Kinase, c‐Src and focal adhesion kinase (FAK), all of which are known to participate in cytoskeletal rearrangements. Knockdown of endogenous Dock180, which is a Rac1‐specific guanine nucleotide exchange factor, using siRNA revealed that C. jejuni invasion was significantly reduced compared with cells treated with scrambled siRNA. We further demonstrated that the C. jejuni Cia proteins are, in part, responsible for Rho GTPase Rac1 recruitment and activation, as judged by immunofluorescence microscopy and Rac1 activation. Based on these data, we present a model that illustrates that C. jejuni utilizes a coordinated mechanism involving both adhesins and secreted proteins to promote membrane ruffling and host cell invasion.     

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.     

A novel NOD1‐ and CagA‐independent pathway of interleukin‐8 induction mediated by the Helicobacter pylori type IV secretion system

The type IV secretion system (T4SS) of Helicobacter pylori triggers massive inflammatory responses during gastric infection by mechanisms that are poorly understood. Here we provide evidence for a novel pathway by which the T4SS structural component, CagL, induces secretion of interleukin‐8 (IL‐8) independently of CagA translocation and peptidoglycan‐sensing nucleotide‐binding oligomerization domain 1 (NOD1) signalling. Recombinant CagL was sufficient to trigger IL‐8 secretion, requiring activation of α₅β₁ integrin and the arginine–glycine–aspartate (RGD) motif in CagL. Mutation of the encoded RGD motif to arginine‐glycine‐alanine (RGA) in the cagL gene of H. pylori abrogated its ability to induce IL‐8. Comparison of IL‐8 induction between H. pylori ΔvirD4 strains bearing wild‐type or mutant cagL indicates that CagL‐dependent IL‐8 induction can occur independently of CagA translocation. In line with this notion, exogenous CagL complemented H. pylori ΔcagL mutant in activating NF‐κB and inducing IL‐8 without restoring CagA translocation. The CagA translocation‐independent, CagL‐dependent IL‐8induction involved host signalling via integrin α₅β₁, Src kinase, the mitogen‐activated protein kinase (MAPK) pathway and NF‐κB but was independent of NOD1. Our findings reveal a novel pathway whereby CagL, via interaction with host integrins, can trigger pro‐inflammatory responses independently of CagA translocation or NOD1 signalling.     

Numb regulates cell–cell adhesion and polarity in response to tyrosine kinase signalling

Epithelial‐mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E‐cadherin (E‐cad) through its phosphotyrosine‐binding domain (PTB) and thereby regulates the localization of E‐cad to the lateral domain of epithelial cell–cell junction. Moreover, Numb engages the polarity complex Par3–aPKC–Par6 by binding to Par3 in polarized Madin‐Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E‐cad and Par3 and associates preferably with aPKC–Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF‐induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral‐to‐apicolateral translocation of E‐cad and β‐catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF‐induced cell scattering, a decrease in cell–cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell–cell adhesion and a sensor of HGF signalling or Src activity during EMT.     

Dynamics of the Cag‐type IV secretion system of Helicobacter pylori as studied by bacterial co‐infections

Many pathogenic Gram‐negative bacteria possess type IV secretion systems (T4SS) to inject effector proteins directly into host cells to modulate cellular processes to their benefit. The human bacterial pathogen Helicobacter pylori, a major aetiological agent in the development of chronic gastritis, duodenal ulcer and gastric carcinoma, harbours the cag‐T4SS to inject the cytotoxin associated Antigen (CagA) into gastric epithelial cells. This results in deregulation of major signalling cascades, actin‐cytoskeletal rearrangements and eventually gastric cancer. We show here that a pre‐infection with live H. pylori has a dose‐dependent negative effect on the CagA translocation efficiency of a later infecting strain. This effect of the ‘first’ strain was independent of any of its T4SS, the vacuolating cytotoxin (VacA) or flagella. Other bacterial pathogens, e.g. pathogenic Escherichia coli, Campylobacter jejuni, Staphylococcus aureus, or commensal bacteria, such as lactobacilli, were unable to interfere with H. pylori's CagA translocation capacity in the same way. This interference was independent of the β1 integrin receptor availability for H. pylori, but certain H. pylori outer membrane proteins, such as HopI, HopQ or AlpAB, were essential for the effect. We suggest that the specific interference mechanism induced by H. pylori represents a cellularresponse to restrict and control CagA translocation into a host cell to control the cellular damage.     

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.     

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.     

Importance of EGF receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating cytotoxin VacA

Helicobacter pylori is the causative agent of gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent strains carrying both the cag pathogenicity island ( cag PAI) and the vacuolating cytotoxin VacA are key players in disease development. The ca gPAI encodes a type IV secretion system (T4SS) which forms a pilus for injection of the CagA protein into gastric epithelial cells. Injected CagA undergoes tyrosine phosphorylation and induces actin-cytoskeletal rearrangements involved in host cell scattering and elongation. We show here that the CagA-induced responses can be inhibited in strains expressing highly active VacA. Further investigations revealed that VacA does not interfere with known activities of phosphorylated CagA such as inactivation of Src kinase and cortactin dephosphorylation. Instead, we demonstrate that VacA exhibits inactivating activities on the epidermal growth factor receptor EGFR and HER2/Neu, and subsequently Erk1/2 MAP kinase which are important for cell scattering and elongation. Inactivation of vacA gene, downregulation of the VacA receptor RPTP-α, addition of EGF or expression of constitutive-active MEK1 kinase restored the capability of H. pylori to induce the latter phenotypes. These data demonstrate that VacA can downregulate CagA's effects on epithelial cells, a novel molecular mechanism showing how H. pylori can avoid excessive cellular damage.     

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.     

PAR1b takes the stage in the morphogenetic and motogenetic activity of Helicobacter pylori CagA oncoprotein

Helicobacter pylori CagA oncoprotein is critically involved in gastric carcinogenesis. Upon delivery into gastric epithelial cells via type IV secretion, CagA induces an extremely elongated cell-shape known as the hummingbird phenotype, which is associated with massive changes in actin cytoskeleton and elevated motility. With the notion that the hummingbird phenotype reflects pathogenic/oncogenic activity of CagA, many studies have focused on the mechanism through which CagA induces the morphological change. Once delivered, CagA interacts with host proteins such as oncogenic phosphatase SHP2 and polarity-regulating kinase PAR1b. Whereas the essential role of the CagA-SHP2 interaction in inducing the hummingbird phenotype has been extensively investigated, involvement of the CagA-PAR1b interaction in the morphological change has remained uncertain. Recently, we found that the CagA-PAR1b interaction, which inhibits PAR1b kinase activity, influences the actin cytoskeletal system and potentiates the magnitude of the hummingbird phenotype. We also found that PAR1b inactivates a RhoA-specific GEF, GEF-H1, via phosphorylation and thereby inhibits cortical actin and stress fiber formation. Collectively, these findings indicate that CagA-mediated inhibition of PAR1b promotes RhoA-dependent actin-cytoskeletal rearrangement and thereby strengthens the hummingbird phenotype induced by CagA-stimulated SHP2 during infection with H. pylori cagA-positive strains.     

Expression of CEACAM1 or CEACAM5 in AZ‐521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin‐associated genes pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c‐Src and c‐Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ‐521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA‐dependent fashion. In contrast, we discovered that AZ‐521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin‐β₁ or carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ‐521 cells readily express integrin‐β₁, but overexpression of integrin‐β₁ constructs did not restore the T4SS defect. We further show that AZ‐521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ‐521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ‐521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori‐infected AGS cells. Our results suggest the existence of an integrin‐β₁‐ and CEACAM1‐ or CEACAM5‐dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.