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Cellular Targets of Pseudomonas Aeruginosa Toxin Exoenzyme S

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Cellular Targets of Pseudomonas Aeruginosa Toxin Exoenzyme S UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series no. 851 ISSN: 0346-6612 ISBN: 91-7305-505-0 - From the Departments of Molecular Biology Umeå University, Umeå, Sweden CELLULAR TARGETS OF PSEUDOMONAS AERUGINOSA TOXIN EXOENZYME S Akademisk avhandling som med vederbörligt tillstånd från rektorsämbetet vid Umeå Universitet för avläggande av doktorsexamen i cellbiologi offentligen kommer att försvaras i hörsal Betula, byggnad 6M, Norrlands Universitetssjukhus fredagen den 31 oktober 2003, klockan 09.00 av Maria Henriksson Fakultetsopponent: Professor Lars Rönnstrand, Institutionen för Experimentell Klinisk Kemi, Lunds Universitet, MAS ABSTRACT Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S Maria Henriksson, Department of Molecular Biology, Umeå University, Sweden Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP- ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro. The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo. 2 TABLETABLE OF OF CONTENTS CONTENTS ABSTRACT .............................................................................................................................. 2 PAPERS IN THIS THESIS..................................................................................................... 5 ABBREVIATIONS .................................................................................................................. 6 INTRODUCTION....................................................................................................................7 Pseudomonas aeruginosa.......................................................................................................... 7 Infection and virulence........................................................................................................... 7 The Ras superfamily .............................................................................................................. 10 Ras superfamily members .................................................................................................... 11 Signalling through Ras........................................................................................................... 12 Guanine nucleotide Exchange Factors (GEFs) for Ras........................................................ 13 GTPase Activating Proteins (GAPs) for Ras ....................................................................... 14 Downstream of Ras .............................................................................................................. 15 The Raf pathway .............................................................................................................. 15 The PI-3 kinase pathway.................................................................................................. 17 The RalGDS pathway....................................................................................................... 18 Signals from RhoA, Rac1 and Cdc42 ................................................................................... 18 The signalling molecules Rap1 and Rap2 ............................................................................ 21 The abundant protein 14-3-3................................................................................................. 21 ExoS, ExoT and eukaryotic signal transduction ................................................................. 22 ExoS, ExoT and their ADP-ribosylated target molecules.................................................... 22 ExoS, ExoT and the GAP activity........................................................................................ 23 ExoS and 14-3-3................................................................................................................... 23 The Yersinia pseudotuberculosis model system.................................................................... 24 AIM.......................................................................................................................................... 26 RESULT AND DISCUSSION............................................................................................... 27 Ras inhibition by ADP-ribosylation in vivo (Paper I) ......................................................... 27 ExoT elicits cytotoxicity without interfering with Ras (Paper II) ..................................... 28 3 ExoS acts towards members of the Ras superfamily in vivo (paper III)........................... 29 ExoS activity on Ras subfamily ........................................................................................... 30 ExoS activity on Rho subfamily........................................................................................... 31 14-3-3 binding is required for the inhibition of Ras by ExoS(Papers IV and V) ............. 32 CONCLUSIONS.....................................................................................................................33 ACKNOWLEDGEMENT..................................................................................................... 34 REFERENCES....................................................................................................................... 36 4 PAPERSPAPERS IN IN THIS THIS THESIS THESIS This thesis is based on the following publications referred to in the text by their roman numerals (I-V). Paper I Henriksson, M.L., Rosqvist, R., Telepnev, M., Wolf-Watz, H. and Hallberg, B. (2000). Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras. Biochem J. 347, 217-222. Paper II Sundin C, Henriksson, M.L., Hallberg, B., Forsberg, A., Frithz-Lindsten, E. (2001). Exoenzyme T of Pseudomonas aeruginosa elicits cytotoxicity without interfering with Ras signal transduction. Cell Microbiol. Apr;3(4):237-46. Paper III Henriksson, M.L., Sundin, C., Jansson, A.L., Forsberg, Å. Palmer, R.H. and Hallberg, B. (2002). Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo. Biochem J. 367, 617-628. Paper IV Henriksson, M.L., Trollér, U. and Hallberg, B. (2000). 14-3-3 proteins are required for the inhibition of Ras by exoenzyme S. Biochem J. 349, 697-701. Paper V Henriksson, M.L., Francis, M., Peden, A., Aili, M., Stefansson, K., Palmer, R.H., Aitken, A. and Hallberg, B. (2002). A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo. Eur J Biochem 269(20), 4921-4929. 5 ABBREVIATIONSABBREVIATIONS ADP adenosine diphosphate cAMP cyclic adenosine monophosphate CNF1 Cytotoxic Necrotizing Factor 1 Crk CT10-regulator of kinase C-TAK1 Cdc25C-associated kinase 1 EGF Epidermal Growth Factor Erk Extracellular signal-regulated kinase E-son Elofsson F-actin filamentous actin FAS Factor Activating exoenzyme S G-actin globular (monomeric) actin GAP GTPase Activating Proteins GDI Guanine nucleotide Dissociation factors GDP guanine diphosphate GEF Guanine nucleotide Exchange Factor Grb2 growth factor receptor-bound protein 2 GRF Guanine nucleotide Releasing Factor GRP Guanine nucleotide Releasing Protein GSK3 Glycogen Synthase Kinase 3 GTP guanine triphosphate KSR Kinase Suppressor of Ras LPA lysophosphatidic acid MAPK Mitogen-Activated Protein Kinase MEK Mitogen-activated protein kinase/Erk Kinase MP1 MEK Partner 1 NAD Nicotinamide Adenine Dinucleotide NF1 Neurofibromin 1 PAK p21-associated kinase PDGF Platelet-Derived Growth Factor PDK PI-3K Dependent Kinase PH pleckstrin homology PI-3K phosphoinositide 3-kinase PI-4-P5K phosphatidyl inositol 4-phosphate 5-kinase PKB Protein Kinase B PLD phospholipase
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