Functional role of integrin alpha5betha1 and the Helicobacter pylori CagL protein in type IV secretion and signal transduction
Final Report Abstract
Integrins are important mammalian receptors involved in many normal cellular functions as well as the pathogenesis of chronic inflammation and cancer. We proposed that integrins are exploited by the gastric pathogen and type-1 carcinogen Helicobacter pylori. Virulent H. pylori express a type-IV secretion system (T4SS) pilus that injects the CagA protein, which then becomes tyrosine-phosphorylated by Src and other kinases. We have recently identified the integrin member α5β1 as the host cell receptor involved in T4SS functions with the H. pylori CagL protein as ligand. The present project was designed to investigate the role of CagL in binding to integrin α5β1 and to identify novel CagL targets with emphasis on how these interactions hijack cellular signalling pathways. We could first show that recombinant CagL mimics important functions of the extracellular matrix protein fibronectin (a natural ligand of integrin α5β1) with regard to cell binding, focal adhesion formation and cell spreading in vitro. The role of CagL in the specific host cell responses such as the induction of membrane and cytoskeletal dynamics for injection of bacterial effector proteins as well as nuclear responses were examined in detail both in vitro and during infection. We have demonstrated that CagL is a novel specialised adhesin which is targeted to the T4SS pilus surface, where it binds to integrin α5β1 through RGD and FEANE helper motifs. This interaction triggered CagA delivery into target cells as well as activation of focal adhesion kinase FAK and Src which is an elegant mechanism for the bacteria to ensure phosphorylation of translocated CagA directly at the injection site. We further characterised the CagL→integrin α5β1 pathway and could show that this interaction also triggers various downstream signalling events leading to the activation of other host receptors, including EGF receptor and Her3/ErbB3, but also ERK kinases and the actin-binding protein cortactin. It was found that the activation of EGF receptor involves the ADAM17 protease, which is released from the integrin complex by CagL and then becomes activated leading to the generation of HB-EGF - the growth factor activating EGF receptor. We also identified in this project two novel interaction partners of CagL, the integrin members αVβ3 and αVβ5. The interaction of CagL with αVβ5 was characterised in detail, exhibiting a specific pathway via ILK and ERK kinases to target nucear responses resulting in elevated expression of the hormone gastrin. Alterations in gastrin expression are of general interest in medicine because, in addition to the role of gastrin in stimulating gastric acid secretion, it can regulate the growth and differentiation of gastric epithelial cells. Changes in these parameters might be relevant in the pathogenesis of gastric cancer. Our findings were also remarkable in another regard. In healthy tissues of polarised gastric epithelial cells, integrins are normally only present on basolateral but not apical surfaces. We therefore also aimed to find out how the T4SS could actually approach the integrins during infection. In this project, we identified a novel secreted virulence factor of the bacteria, the serine protease HtrA. HtrA was found to cleave-off the extracellular domain of the junctional protein and tumor suppressor E-cadherin in vitro and during infection. We also demonstrated that E-cadherin shedding (and maybe other targets of HtrA) disrupts epithelial barrier functions allowing H. pylori to access the intercellular space between neighboring epithelial cells and reach basolateral surfaces. This novel mechanism could explain how the bacteria can make access to the basolateral integrins. Taken together, with this project we contributed significantly to our current understanding of signalling cascades induced by the H. pylori T4SS, CagL and translocated CagA protein. This has important impact on molecular mechanisms in gastric pathogenesis including signalling processs which may lead to the development of gastric diseases including cancer in the human stomach. Better understanding of these processes at the molecular level may allow developing novel treatment regimes in future.
Publications
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(2009) Host cell interactome of tyrosine-phosphorylated bacterial proteins. CELL host & microbe 5: 397-403
Selbach, M., Paul, F.E., Brandt, S., Guye, P., Daumke, O., Backert, S., Dehio, C., Mann, M.
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(2009) 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. Cellular Microbiology 11: 488-505
Tegtmeyer, N., Zabler, D., Schmidt, D., Hartig, R., Brandt, S., Backert, S.
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(2010) A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation. Journal of Biological Chemistry 285: 23515-23526
Tegtmeyer, N., Hartig, R., Delahay, R.M., Rohde, M., Brandt, S., Conradi, J., Takahashi, S., Smolka, A.J., Sewald, N., Backert, S.
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(2010) Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H,K H,K ATPase alpha subunit. Gastroenterology 139: 239-248
Saha, A., Backert, S., Hammond, C.E., Gooz, M., Smolka, A.J.
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(2010) Helicobacter pylori HtrA is a novel secreted virulence factor which cleaves E- cadherin to disrupt intercellular adhesion. EMBO Reports 11: 798-804
Hoy, B., Löwer, M., Weydig, C., Carra, G., Tegtmeyer, N., Geppert, T., Plattner, P., Sewald, N., Backert, S., Schneider, G., Wessler, S.
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(2011) Serine phosphorylation of cortactin by ERK1/2 controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori. CELL host & microbe 9: 520-531
Tegtmeyer, N., Wittelsberger, R., Hartig, R., Martinez-Quiles, N., Backert, S.
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(2012) An RGD Helper Sequence in CagL of Helicobacter pylori Assists in Interactions with Integrins and Injection of CagA. Frontiers Cellular Infection Microbiology 2:70
Conradi, J., Tegtmeyer, N., Huber, S., Gaus, K., Mertink, F., Gracia, S.R., Strijowski, U., Sewald, N. Backert, S.
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(2012) Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrin alphaV beta3. Amino Acids 43:219-232
Conradi, J., Huber, S., Gaus, K., Mertink, F., Gracia, S.R., Strijowski, U., Backert, S., Sewald, N.
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(2012) Distinct roles of HtrA proteases from different Gram-negative bacteria in cleaving the junctional protein and tumor suppressor E-cadherin. Journal of Biological Chemistry 287: 10115-10120
Hoy, B., Geppert, T., Reisen, F., Boehm, M., Plattner, P., Gadermaier, G., Sewald, N., Ferreira, F., Briza, P., Schneider, G., Backert, S., Wessler, S.
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(2012) Helicobacter pylori CagL-dependent induction of gastrin expression via a novel αvβ5-integrin-integrin linked kinase signalling complex. Gut 61:986-996
Wiedemann T, Hofbaur S, Tegtmeyer N, Huber S, Sewald N, Wessler S, Backert S., Rieder G.
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(2012) Hierarchic phosphorylation of the Helicobacter pylori CagA effector protein by Src and Abl tyrosine kinases. Journal of Clinical Investigation 122:1553-1566
Müller, D., Tegtmeyer, N., Brandt, S., Yamaoka, Y., De Poire, E., Sgouras, D., Wessler, S., Torres, J., Smolka, A., Backert, S.
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(2012) Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin. Gut Pathogens 25:3
Boehm, M., Hoy, B., Rohde, M., Tegtmeyer, N., Bæk, K.T., Oyarzabal, O.A., Brøndsted, L., Wessler, S., Backert, S.
