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Moonlighting Proteins of Lactobacillus Crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Helsingin yliopiston digitaalinen arkisto Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host Veera Kainulainen General Microbiology Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public examination in the Auditorium 1041, at Viikki Biocenter (Viikinkaari 5, Helsinki) on January 20th, 2012, at 12 noon. Helsinki 2012 Supervisor Professor Timo K. Korhonen General Microbiology Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Rewievers Professor Per Saris Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki Doctor Minja Miettinen Valio Ltd. Research and Development Fermented Dairy Opponent Docent Pekka Varmanen Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki Custos Dennis Bamford Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki ISSN 1799-7372 ISBN 978-952-10-7566-7 (pbk.) ISBN 978-952-10-7567-4 (PDF) ;http://ethesis.helsinki.fi Unigrafia Helsinki 2012 TO PASI AND KAMILLA PREFACE This study was carried out at the Department of Biosciences, Faculty of Biological and Environmental Sciences, in the University of Helsinki. I am grateful to Professor Timo Korhonen, Head of the division and Professor Kielo Haahtela, Head of the department, for the opportunity to perform this work and providing excellent working and educational facilities. I want to express my deepest gratitude to Professor Timo Korhonen for supervising this work. His knowledge, advices and visions were priceless. I cincerely wish to thank Professor Per Saris and Dr. Minja Miettinen for the rapid and kind reviewing of the thesis. Your comments were very valuable! I also want to acknowledge Professor Per Saris and Doc. Pentti Kuusela, members of my PhD thesis follow-up group, for encourage and advices. My co-writers and collaborators are grately thanked for their contribution. I also want to thank all the present and the former members of Timå’s research group, especially Ritva, Benita, Katri, Maiju and Riikka who encouraged me during last months of my thesis writing. Laboratory staff, especially Raili, is thanked for excellent assistance throughout this work. My warm thanks belong to Timo Lehti for sharing the stress of thesis writing. All my friends, especially Päivi, Hansu and Jaana, and colleagues from Finnish Gymnastic Federation are acknowlidged for keeping me attached to the world outside of lab. I am grateful to my parents and brothers for support, understanding (and babysitting during the last months of writing). This work has never been completed without you. Most of all I want to thank Pasi and Kamilla for their love and care. You two are the best thing in my life! Veera, September 2011 5 CONTENTS Preface ........................................................................................................................................5 List of original publications .....................................................................................................7 Summary ....................................................................................................................................8 1 Introduction ���������������������������������������������������������������������������������������������������������������������9 2 Moonlighting proteins in Gram-positive bacteria ......................................................11 2.1 Definition of a moonlighting protein ...............................................................................11 2.2 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ................................................15 2.2.1 Bacteria and the mammalian plasminogen system ..........................................................17 2.2.2 Adhesive and virulence functions of GAPDH ................................................................18 2.3 Enolase ............................................................................................................................19 2.4 Other glycolytic enzymes ...............................................................................................21 2.5 Nonglycolytic moonlighting enzymes .............................................................................23 2.6 Molecular chaperones ......................................................................................................23 2.7 Other proteins .................................................................................................................24 2.8 Moonlighting proteins in commensal bacteria ................................................................25 3 Secretion and cell wall anchoring of lactobacillar surface proteins ..........................28 3.1 The cell wall and anchoring of surface proteins ..............................................................28 3.2 Protein Secretion mechanisms in lactobacilli ..................................................................29 3.2.1 Hypothetized mechanisms for secretion of moonlighting proteins .................................30 4 Aims of the study ...........................................................................................................31 5 Materials and methods ..................................................................................................32 6 Results and discussion ...................................................................................................35 6.1 Identification of enolase, GAPDH, GS and GPI in buffer extract and cell wall of L. crispatus (I, IV) ..........................................................................................................35 6.1.1 Properties of enolase, GAPDH, GS and GPI (I, II, III, IV) .............................................37 6.2 Enolase, GAPDH, GS and GPI are released from cell surface at neutral pH and by high ionic strength (I, II, IV) ......................................................................................40 6.3 Enhancement of the release by epithelial cathelidicin LL-37 (IV) ................................42 6.4 Interaction of enolase and GAPDH with lipoteichoid acids (II) .....................................43 6.5 Reassociation of enolase, GAPDH, GS and GPI to cell surface at low pH (II, IV) ��������44 6.6 Functions of lactobacillar enolase, GAPDH, GS and GPI (I, II, III, IV) ........................45 6.6.1 Interaction of lactobacilli with the human plasminogen system (I, II, III, IV) ...............45 6.6.2 Lactobacillar moonlighting proteins as adhesins (III, IV) ...............................................49 7 Conclusions ....................................................................................................................51 8 References.......................................................................................................................53 6 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following publications. The original publications are reprinted with the kind permission of the copyright holders. I Veera Hurmalainen*, Sanna Edelman*, Jenni Antikainen, Marc Baumann, Kaarina Lähteenmäki, Timo K. Korhonen. Extracellular proteins of Lactobacillus crispatus enhance activation of human plasminogen. Microbiology, 2007, 153: 1112-1122 II Jenni Antikainen, Veera Kuparinen, Kaarina Lähteenmäki, Timo K. Korhonen. pH-dependent association of enolase and glyceraldehydes-3-phosphate dehydrogenase of Lactobacillus crispatus with the cell wall and lipoteichoid acids. Journal of Bacteriology, 2007, 189: 4539-4543 III Jenni Antikainen, Veera Kuparinen, Kaarina Lähteenmäki, Timo K. Korhonen. Enolases from Gram-positive bacterial pathogens and commensal lactobacilli share functional similarity in virulence-associated traits. FEMS Immunology & Medical Microbiology, 2007, 51: 526-534 IV Veera Kainulainen, Vuokko Loimaranta, Anna Pekkala, Sanna Edelman, Jenni Antikainen, Riikka Kylväjä, Maiju Laaksonen, Jukka Finne, Liisa Laakkonen, Timo K. Korhonen. Glutamine synthetase and Glucose-6-phosphate isomerase are adhesive moonlighting proteins of Lactobacillus crispatus released by epithelial cathelicidin LL-37. Manuscript submitted for publication. The publications are referred to in the text by their roman numerals. Veera Kainulainen´s contribution to the articles: I Laboratory experiments except IEM pictures and cloning of GAPDH, participated in planning and writing II Initial observations about release of proteins to neutral buffer, participated in cell wall association and LTA- binding experiments and planning III Cloning of lactobacillar genes, protein purification and plasminogen binding and activation experiments, enzyme activity measurements, participated in ECM binding experiments, participated in planning IV Laboratory experiments except mutanolysin extraction and fractioning the extract, participated in planning and writing 7 Summary SUMMARY Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional
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