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Bacterial Protein Systems at the Membrane Interface Structural and Biophysical Studies of the E Bacterial protein systems at the membrane interface Structural and biophysical studies of the E. coli adhesion receptor intimin and the magnesium transporter MgtA Dissertation for the degree of Ph.D. by Julia Anna Weikum Centre for Molecular Medicine Norway Nordic EMBL Partnership for Molecular Medicine University of Oslo July 2020 © Julia Anna Weikum, 2020 Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo No. 2316 ISSN 1501-7710 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. Table of contents Acknowledgments ...................................................................................................................III List of publications ................................................................................................................. IV Abbreviations ........................................................................................................................... V 1. Introduction .......................................................................................................................... 1 1.1 Pathogenic Escherichia coli .............................................................................................. 2 1.1.1 Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) .................. 2 1.2 Adhesion .......................................................................................................................... 3 1.2.1 Initial adherence and colonization .............................................................................. 5 1.2.2 Translocation of bacterial signals into the host cell via a type three secretion system 5 1.2.3 Intimate adherence and pedestal formation of A/E lesions ......................................... 6 1.2.4 Intimin ........................................................................................................................ 7 1.2.4.1 Structural features of intimin ................................................................................ 8 1.2.4.2 Inverse autotransporter ........................................................................................ 9 1.2.4.3 Autotransport mechanism of inverse autotransporters ........................................10 1.3 E. coli cell envelope .........................................................................................................11 1.3.1 Glycerophospholipids ................................................................................................12 1.3.1.1 Cardiolipin ..........................................................................................................13 1.3.1.2 Adaption of lipid composition ..............................................................................15 1.3.1.3 Lipid autooxidation ..............................................................................................15 1.3.2 Membrane proteins ...................................................................................................16 1.3.2.1 P-type ATPases ..................................................................................................17 1.3.3 Protein-lipid-interactions ............................................................................................20 1.4 Magnesium-transport in E. coli ........................................................................................22 1.4.1 Magnesium transporter A (MgtA) ..............................................................................23 1.4.1.1 The role of cardiolipin for MgtA-mediated Mg2+ transport ....................................24 1.4.1.2 Phylogenetic distribution of MgtA ........................................................................25 I 1.4.1.3 Transcriptional and translational regulation of mgtA expression..........................26 1.4.1.4 Cellular functions of MgtA ...................................................................................29 2. Aims of this thesis ..............................................................................................................31 3. Synopses of publications ...................................................................................................33 Paper I: The extracellular juncture domains in intimin adopt a constitutively extended conformation and induce restraints in the intimin reach and sphere of action ........................33 Paper II: The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species ....................................................................................................34 Paper III: The Mg2+ sensing region of MgtA resides in the C-terminus and is dependent on pH ..............................................................................................................................................35 4. Discussion ...........................................................................................................................36 5. Summary .............................................................................................................................56 6. Future perspectives ............................................................................................................58 7. References...........................................................................................................................60 Annex: Paper I-III II Acknowledgments The work presented in this PhD thesis was carried out at the Centre for Molecular Medicine (NCMM), University of Oslo, Norway from April 2017 to January 2019 and at DTU Bioengineering, Danish Technical University, Denmark from February 2019 to June 2020. Financial support was provided by NCMM core funding, the Research Council of Norway, NordForsk and DTU funding. BioCat provided travel grants for courses and national conferences attended during the PhD studies. First, I would like to thank my primary supervisor, Jens Preben Morth, who has given me the opportunity to follow my interest in protein biochemistry and pursue my PhD in his research group. I always appreciate and value his input and comments, innovative ideas and support inside as well as outside of the laboratory. I would also like to thank my co-supervisors, Ole Andreas Løchen Økstad and Reidar Lund, for their support and taking interest in my progress. Secondly, I would like to thank all my colleagues in Oslo and Copenhagen, with whom I had the opportunity and pleasure to work with. I would especially like to thank Saranya Subramani for her contributions to my PhD thesis. Thank you for always being available when I needed guidance or support. Further, I would like to thank the former members of the Morth group, Bojana Sredic, Harmonie Perdreau-Dahl and Johannes Bauer, for the friendly welcome, great scientific input and cozy coffee breaks. I also thank Lisa Gerner for her feedback on my PhD thesis. Additionally, I would like to thank my group members at DTU, Lisa Merklinger and Emilie Müller, for interesting discussions during lunch breaks and fun-filled activities outside of work. Thank you for making my year in Copenhagen a great time. Lastly, I would also like to thank Line Vejby Jægerum, who contributed to my project as a master’s student and remained a friend after. I thank my office mates and co-workers at NCMM and DTU for the scientific and non- scientific discussions in the laboratory and at the coffee machine. Further, I would like to extend my thanks to the technical and administrative staff at DTU and NCMM for their support. I would like to especially thank Nina Modahl, Anita Skolem and Elisa Bjørgo, who helped me with the administrative struggles during my relocation from Oslo to Copenhagen. I would like to thank my family for their encouragement and support in going abroad to perform my PhD studies. Further, I would like to thank my father Gerhard, my mother Liz and my sister Maria for all the valuable feedback on my PhD thesis. I thank all my friends I met in Norway, who made my time outside of the laboratory delightful, allowed me to make great memories and fall in love with the country. Lastly, I would like to thank my partner, Magnus Wannebo, for always being there for me, cheering me up and motivating me. Thank you for being you. III List of publications This thesis is based on the following scientific articles: I. Weikum J, Kulakova A, Tesei G, Yoshimoto S, Jægerum LV, Schütz M, Hori K, Skepö M, Harris P, Leo JC, Morth JP (2020). The extracellular juncture domains in the intimin passenger adopt a constitutively extended conformation inducing restraints to its sphere of action. Accepted in Scientific Reports (Nature Publishing Group). II. Weikum J, van Dyck J, Subramani S, Klebl DP, Storflor M, Muench SP, Abel S, Sobott F, Morth JP (2020). The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species. Manuscript under revision. III. Subramani S, Weikum J, Sredic B, Perdreau-Dahl H, Langbach-Hein K, Vilsen B, Morth JP (2020). The Mg2+ sensing region of MgtA resides in the C-terminus and is dependent on pH. Manuscript in preparation. IV Abbreviations A/E Attaching and effacing Arp Actin-related protein AT Autotransporter ATP Adenosine triphosphate A-domain Actuator domain BAM β-barrel assembly machinery Big Bacterial immunoglobulin-like BFP
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