Dr. N. Tegtmeyer
Research Group Tegtmeyer
Phosphorylation and signaling of injected effector proteins by host tyrosine kinases: a general mechanism in various bacterial infections
Selective interactions between tyrosine-phosphorylated proteins and their cognate, SH2-domain or PTB-domain containing ligands play key roles in mammalian signal transduction. Several bacterial pathogens use type III and type IV secretion systems to inject tyrosine kinase substrates into host target cells, which mimic their host cell counterparts. The involvement of these secretion systems in disease development is exemplified by a variety of pathogenic processes including pedestal formation (Tir of EPEC and Citrobacter rodentium), cell scattering (CagA of Helicobacter pylori), invasion (Tarp of Chlamydia trachomatis), pro-inflammatory responses and cell proliferation (BepD-F of Bartonella henselae) as well as intracellular survival (AnkA of Anaplasma phagocytophilum). Upon phosphorylation, these bacterial effector proteins recruit cellular binding partners to manipulate host cell functions. However, only a few interaction partners have been identified yet. We have recently reported the results of a global proteomic screen to systematically identify binding partners of a large variety of the known tyrosine-phosphorylated bacterial effectors by high-resolution mass spectrometry. Altogether, 39 host interactions were identified in this study, all mediated by SH2 domains (but not PTB domains), including four of the five already known interaction partners. Individual phosphorylation sites recruited a surprisingly high number of cellular interaction partners suggesting that individual phosphorylation sites can interfere with multiple cellular signaling pathways. Collectively, these results indicate that tyrosine-phosphorylation sites of bacterial effector proteins have evolved during evolution as versatile interaction modules that can recruit a rich repertoire of cellular SH2 domains. In the case of the gastric pathogen H. pylori, which is a major risk factor for the development of gastric cancer, the only known effector protein injected into host cells is the oncoprotein CagA. We followed the hierarchic tyrosine phosphorylation of CagA as a model system to study early effector phosphorylation processes by cellular c-Src and c-Abl tyrosine kinases. Translocated CagA is phosphorylated on typical Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, called EPIYA-A, EPIYA-B and EPIYA-C in Western strains of H. pylori and EPIYA-A, EPIYA-B, and EPIYA-D in East Asian strains. We found that c-Src only phosphorylated EPIYA-C and EPIYA-D, whereas c-Abl phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. Further analysis revealed that CagA molecules were phosphorylated on 1 or 2 EPIYA motifs, but never simultaneously on 3 motifs. Furthermore, none of the phosphorylated EPIYA motifs alone was sufficient for inducing AGS cell scattering and elongation. The preferred combination of phosphorylated EPIYA motifs in Western strains was EPIYA-A and EPIYA-C, either across 2 CagA molecules or simultaneously on 1. Our studies thus identify a tightly regulated hierarchic phosphorylation model for CagA starting at EPIYA-C/D, followed by phosphorylation of EPIYA-A or EPIYA-B. These results provide important insights for clinical H. pylori typing and clarify the role of phosphorylated bacterial effector proteins in pathogenesis. A detailed analysis of intracellular signaling pathways by these phosphorylated CagA and other bacterial effector proteins is currently underway.
Figure 1: Injection, phosphorylation and signal transduction by the H. pylori CagA protein.
Cortactin: an Achilles‘ heel of the actin cytoskeleton targeted by various pathogens for multiple purposes
Cell migration and invasion require the coordinated regulation of cytoskeletal architectural changes by signaling factors, including the actin-binding protein cortactin. Bacterial and viral pathogens subvert these signaling factors to promote their uptake, spread and dissemination. The involvement of cortactin in disease development is exemplified by a variety of pathogenic processes, such as pedestal formation [enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC)], invasion (Shigella, Neisseria, Rickettsia, Chlamydia, Staphylococcus and Cryptosporidium), actin-based motility (Listeria, Shigella and vaccinia virus) and cell scattering (Helicobacter). Recent progress turned our attention to how cortactin function can be regulated by serine and tyrosine phosphorylation, respectively. This has an important impact on how pathogens abuse cortactin to modulate the architecture of the host actin cytoskeleton. We could recently show that the gastric pathogen Helicobacter pylori targets cortactin by two independent and subsequent processes leading to its tyrosine dephosphorylation (at Y-421, Y-466 and Y-486) and serine phosphorylation (S-113, S-405 and S-418) to regulate epithelial cell scattering and elongation. The phosphorylation status of cortactin clearly dictates its subcellular localization and signaling partners. To investigate the specific role of individual serine residues, we produced phosphoserine-specific cortactin antibodies and use commercially available phosphotyrosine-specific cortactin antibodies for all of the above mentioned sites. In non-infected control cells we found that tyrosine-phosphorylated cortactin is in complex with N-WASP. Upon infection with H. pylori, we observed that cortactin undergoes tyrosine-dephosphorylation (a process involving CagA and Src inactivation via Csk). The interaction between N-WASP and cortactin is terminated by this process and binds to a novel interaction partner, which we have identified as the focal adhesion kinase (FAK). This interaction required the SH3 domain and phosphorylation of cortactin at S-405 (but not at S-113 or S-418) and a proline-rich sequence in FAK. Cortactin-FAK binding is associated with a profound stimulation of FAK kinase activity. Thus, using H. pylori as a model, our studies unraveled a previously unrecognized FAK activation pathway. However, cortactin is also phosphorylated at position S-113 and S-418 by H. pylori, but their role during infection is not yet clear. Experiments are currently underway to investigate how different pathogens target cortactin and manipulate downstream signaling pathways using the various serine and tyrosine phosphorylation/dephosphorylation events.
- Tenguria, S., Ansari, S.A., Khan, N., Ranjan, A., Devi. S., Tegtmeyer. N., Lind. J., Backert. S. & Ahmed N. (2014). Helicobacter pylori cell translocating kinase (CtkA/JHP0940) is pro-apoptotic in mouse macrophages and acts as auto-phosphorylating tyrosine kinase. Int. J. Med. Microbiol. (in press)
- Heimesaat, M.M., Alutis, M., Grundmann, U., Fischer, A., Tegtmeyer, N., Böhm M., Kühl, A.A., Göbel, U.B., Backert, S. & Bereswill, S. (2014). The role of serine protease HtrA in acute ulcerative enterocolitis and extra-intestinal immune responses during Campylobacter jejuni infection of gnotobiotic IL-10 deficient mice. Front Cell Infect. Microbiol. 4:77.
- Tegtmeyer, N., Lind, J. & Backert, S. (2014). Helicobacter pylori CagL Y58/E59 mutation turns-off type IV secretion-dependent delivery of CagA into host cells. PLos One 9(6):e97782.
- Heimesaat, M.M., Fischer, A., Alutis, M., Grundmann, U., Boehm, M., Tegtmeyer, N., Göbel, U.B., Kühl, A.A., Bereswill, S. & Backert, S. (2014). The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during Campylobacter jejuni infection of infant mice. Gut. Pathog. 6:16.
- Lind, J., Backert, S., Pfleiderer, K., Berg, D.E., Yamaoka, Y., Sticht, H. & Tegtmeyer, N. (2014). Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motivs in CagA of Western-type Helicobacter pylori strains. PlosOne 9(5):e96488.
- Lind, J., Backert, S., Yamaoka, Y., Sticht, H. & Tegtmeyer, N. (2013). Variable recognition of single phosphorylated EPIYA motifs in the Helicobacter pylori CagA effector protein by phosphotyrosine antibodies. PlosOne (submitted).
- Pachathundikandi, S.K., Tegtmeyer, N. & Backert, S. (2013). Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells. Gut Microbes (submitted).
- Backert, S., Boehm, M., Wessler, S. & Tegtmeyer, N. (2013). Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both? Cell Commun Signal. (in press).
- Barden, S., Lange, S., Tegtmeyer, N., Conradi, J., Sewald, N., Backert, S. & Niemann, H. (2013) A Helical RGD Motif Promoting Cell Adhesion: Crystal Structures of the Helicobacter pylori Type IV Secretion System Pilus Protein CagL. Structure (in press).
- Patel, S.R., Smith, K., Letley, D.P., Cook, K.W., Memon, A.A., Ingram, R.J., Staples, E., Backert, S., Zaitoun, A.M., Atherton, J.C. & Robinson, K. (2013). Helicobacter pylori downregulates expression of human β-defensin 1 in the gastric mucosa in a type IV secretion-dependent fashion. Cell Microbiol. doi: 10.1111/cmi.12174.
- Tegtmeyer, N., Rivas Traverso, F., Rohde, M., Oyarzabal, O.A., Schneider-Brachert, W., Lehn, N., Berg. D.E., Fox, J.G. & Backert, S. (2013). Electron microscopic, genetic and biochemical characterization of a novel Helicobacter isolates in Bengal tigers. Plos One (PLoS One 8(8):e71220).
- Dolan, B., Naughton, J., Tegtmeyer, N., May, F.E., Clyne, M. (2012). The interaction of Helicobacter pylori with the adherent mucus gel layer secreted by polarized HT29-MTX-E12 cells. PLoS One 7, E47300
- Hirsch, C., Tegtmeyer, N., Rohde, M., Rowland, M., Oyarzabal, O.A. & Backert S. (2012). Live Helicobacter pylori in the root canal of endodontic-infected deciduous teeth. J. Gastroenterol. 47, 936-940.
- Conradi, J., Tegtmeyer, N., Huber, S., Gaus, K., Mertink, F., Gracia, S.R., Strijowski, U., Sewald, N. & Backert, S. (2012). An RGD Helper Sequence in CagL of Helicobacter pylori Assists in Interactions with Integrins and Injection of CagA. Front. Cell. Infect. Microbiol. 2, 70.
- Boehm, M., Hoy, B., Rohde, M., Tegtmeyer, N., Bæk, K.T., Oyarzabal, O.A., Brøndsted, L., Wessler, S. & Backert, S. (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 Pathog. 25, 3.
- Mueller, D., Tegtmeyer, N., Brandt, S., Yamaoka, Y., De Poire, E., Sgouras, D., Wessler, S., Torres, J., Smolka, A. & Backert, S. (2012). Hierarchic phosphorylation of the Helicobacter pylori CagA effector protein by Src and Abl tyrosine kinases. J. Clin. Inves. 122, 1553-1566.
- Wiedemann, T., Hofbaur, S., Tegtmeyer, N., Huber, S., Sewald, N., Wessler, S., Backert, S. & Rieder G. (2012). Helicobacter pylori CagL-dependent induction of gastrin expression via a novel αvβ5-integrin-integrin linked kinase signalling complex. Gut 61, 986-996.
- Backert, S. & Tegtmeyer, N. (2012). Helicobacter pylori CagA tertiary structure reveals functional insights. Cell Host Microbe 12, 3-5.
- Boehm, M., Krause, M., Rohde, M., Tegtmeyer, N., Takahashi, S., Oyarzabal, O.A & Backert, S. (2011). Major host factors in host cell invasion of Campylobacter jejuni: Role of fibronectin, Integrin Beta1, FAK, Tiam-1, and DOCK180 in activating Rho GTPase Rac1. Front. Cell. Infect. Microbiol. 1, 17.
- Krause, M., Boehm, M., Rohde, M., Tegtmeyer, N., Takahashi, S., Buday, L., Oyarzabal, O.A. & Backert, S. (2011). The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2. Cell Commun. Signal. 9, 32.
- Tegtmeyer, N., Wittelsberger, R., Hartig, R., Martinez-Quiles, N. & Backert, S. (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. & Backert, S. (2011). Role of Abl and Src family kinases in actin-cytoskeletal rearrangementsinduced by the Helicobacter pylori CagA protein. Eur. J. Cell Biol. 90, 880-890.
- Tegtmeyer, N., Wessler, S. & Backert S. (2011). Role of the cag-pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis. FEBS J. 278, 1190-1202.
- Backert, S., Clyne, M. & Tegtmeyer, N. (2011). Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori. Cell Commun. Signal. 9, 28-35.
- Hoy, B., Löwer, M., Weydig, C., Carra, G., Tegtmeyer, N., Geppert, T., Plattner, P., Sewald, N., Backert, S., Schneider, G. & Wessler, S. (2010). Helicobacter pylori HtrA is a novel secreted virulence factor which cleaves E- Cadherin to disrupt intercellular adhesion. EMBO Reports 11, 798-804.
- Tegtmeyer, N., Hartig, R., Delahay, R.M., Rohde, M., Brandt, S., Conradi, J., Takahashi, S., Smolka, A.J., Sewald, N. & Backert, S. (2010). A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation. J. Biol. Chem. 285, 23515-23526.
- Backert, S., Tegtmeyer, N. & Selbach, M. (2010). The versatility of Helicobacter pylori CagA effector protein functions: The master key hypothesis. Helicobacter 15, 163-176.
- Olofsson, A., Vallström, A., Petzold, K., Tegtmeyer, N., Schleucher, J., Carlsson, S., Haas, R., Backert, S., Wai, S.N., Gröbner, G. & Arnqvist, A. 2010). Biochemical and functional characterization of Helicobacter pylori vesicles. Mol. Microbiol. 77, 1539-1555.
- Backert, S., Kenny, B., Gerhard, R., Tegtmeyer, N. & Brandt, S. (2010). PKA-mediated phosphorylation of EPEC-Tir at serine residues 434 and 463: A novel pathway in regulating Rac1 GTPase function. Gut Microbes 1: 94-99.
- Backert, S. & Tegtmeyer, N. (2010). The Versatility of the Helicobacter pylori Vacuolating Cytotoxin VacA in Signal Transduction and Molecular Crosstalk. Toxins 2: 69-92.
- Tegtmeyer, N., Zabler, D., Schmidt, D., Hartig, R., Brandt, S. & Backert, S. (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. Cell. Microbiol. 11, 488-505.
- Tegtmeyer, N. & Backert, S. (2009). Bacterial type III effectors inhibit cell lifting by targeting integrin-linked kinase. Cell Host Microbe 5; 514-516.
- Franco, A.T., Friedman, D.B., Nagy, T.A., Romero-Gallo, J., Krishna, U., Kendall, A., Israel, D.A., Tegtmeyer N., Washington, M.K. & Peek, R.J. Jr. (2009). Delineation of a carcinogenic Helicobacter pylori proteome. Mol. Cell Proteomics 8, 1947-1958.
- Ziegelin, G., Tegtmeyer, N., Lurz, R., Hertwig, S., Hammerl, J., Appel B. & Lanka, E. (2005). The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli. J. Bacteriol. 187, 3445-3454.