Steffen Backert

Helicobacter exploits integrin for type IV secretion and kinase activation

Integrins are important mammalian receptors involved in normal cellular functions as well as pathogenesis of chronic inflammation and cancer. We propose that integrins are exploited by the gastric pathogen and type-1 carcinogen Helicobacter pylori for injection of the bacterial oncoprotein cytotoxin-associated gene A (CagA) into gastric epithelial cells. Virulent H. pylori express a type-IV secretion pilus that injects CagA into the host cell; CagA then becomes tyrosine-phosphorylated by Src family kinases. However, the identity of the host cell receptor involved in this process has remained unknown. Here we show that the H. pylori CagL protein is a specialized adhesin that is targeted to the pilus surface, where it binds to and activates integrin α5β1 receptor on gastric epithelial cells through an arginine-glycine-aspartate motif. This interaction triggers CagA delivery into target cells as well as activation of focal adhesion kinase and Src. Our findings provide insights into the role of integrins in H.-pylori-induced pathogenesis. CagL may be exploited as a new molecular tool for our further understanding of integrin signalling.

Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus

Helicobacter pylori is one of the most common bacterial pathogens, infecting about 50% of the world population. The presence of a pathogenicity island (PAI) in H. pylori has been associated with gastric disease. We present evidence that the H. pylori protein encoded by the cytotoxin‐associated gene A (cagA) is translocated and phosphorylated in infected epithelial cells. Two‐dimensional gel electrophoresis (2‐DE) of proteins isolated from infected AGS cells revealed H. pylori strain‐specific and time‐dependent tyrosine phosphorylation and dephosphorylation of several 125–135 kDa and 75–80 kDa proteins. Immunoblotting studies, matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), cell fractionation and confocal microscopy demonstrated that one of the 125–135 kDa proteins represents the H. pylori CagA protein, which is translocated into the host cell membrane and the cytoplasm. Translocation of CagA was dependent on functional cagA gene and virulence (vir) genes of a type IV secretion apparatus composed of virB4, virB7, virB10, virB11 and virD4 encoded in the cag PAI of H. pylori. Our findings support the view that H. pylori actively translocates virulence determinants, including CagA, which could be involved in the development of a variety of gastric disease.

Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor‐like (‘hummingbird’) phenotype. In this study, using a cagA mutant complemented with wild‐type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti‐phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA–green fluorescent protein (GFP) fusion proteins and two‐dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C‐terminus. Site‐directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).

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 worlds 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 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 (CagAP‐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 CagAP‐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, CagAP‐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.

Role of the cag pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis

Helicobacter pylori is a very successful human‐specific bacterium worldwide. Infections of the stomach with this pathogen can induce pathologies, including chronic gastritis, peptic ulcers and even gastric cancer. Highly virulent H. pylori strains encode the cytotoxin‐associated gene (cag)‐pathogenicity island, which expresses a type IV secretion system (T4SS). This T4SS forms a syringe‐like pilus structure for the injection of virulence factors such as the CagA effector protein into host target cells. This is achieved by a number of T4SS proteins, including CagI, CagL, CagY and CagA, which by itself binds the host cell integrin member β1 followed by delivery of CagA across the host cell membrane. A role of CagA interaction with phosphatidylserine has also been shown to be important for the injection process. After delivery, CagA becomes phosphorylated by oncogenic tyrosine kinases and mimics a host cell factor for the activation or inactivation of some specific intracellular signalling pathways. We review recent progress aiming to characterize the CagA‐dependent and CagA‐independent signalling capabilities of the T4SS, which include the induction of membrane dynamics, disruption of cell–cell junctions and actin‐cytoskeletal rearrangements, as well as pro‐inflammatory, cell cycle‐related and anti‐apoptotic transcriptional responses. The contribution of these signalling pathways to pathogenesis during H. pylori infections 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.

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion

Mammalian and prokaryotic high‐temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell‐adhesion protein E‐cadherin. E‐cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small‐molecule inhibitor that efficiently blocks HtrA activity, E‐cadherin cleavage and intercellular entry of H. pylori.

Specific Entry of Helicobacter pylori into Cultured Gastric Epithelial Cells via a Zipper-Like Mechanism

ABSTRACT Although Helicobacter pylori has generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number of H. pylori colonies recovered was lower than that for Salmonella enterica serovar Typhimurium X22, Escherichia coli expressing InvA, and Yersinia enterocolitica YO:9 grown at 25°C but higher than that for Neisseria gonorrhoeae VP1 and Y. enterocolitica YO:9 grown at 37°C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. Internalized H. pylori was bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry of H. pylori in a sensitive and dose-dependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry of H. pylori into AGS cells. Collectively, these results demonstrate that the entry of H. pylori into AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events.

Functional Analysis of the Helicobacter pylori cag Pathogenicity Island Reveals Both VirD4-CagA-Dependent and VirD4-CagA-Independent Mechanisms

ABSTRACT The type IV secretion machinery encoded by the cag pathogenicity island (PAI) of Helicobacter pylori has been implicated in a series of host responses during infection. Here, we analyzed the function of 12 cag PAI genes from both cag I and cag II loci, including the complete virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, and virD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering (“hummingbird”) phenotype upon H. pylori infection of AGS gastric epithelial cells. For the first time, we have complemented individual cag PAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on all virB/D genes and several other genes of the cag PAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model.