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Role of Adhesin Proteins in Chlamydia Infection

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Role of Adhesin Proteins in Chlamydia Infection Role of adhesin proteins in Chlamydia infection Inaugural dissertation for the attainment of the title of doctor in the Faculty of Mathematics and Natural Sciences at the Heinrich Heine University Düsseldorf presented by Alison Favaroni from Perugia (Italy) Düsseldorf, February 2017 from the Institute for Functional Microbial Genomics at the Heinrich Heine University Düsseldorf Published by permission of the Faculty of Mathematics and Natural Sciences at Heinrich Heine University Düsseldorf Supervisor: Prof. Dr. Johannes H. Hegemann Co-supervisor: Prof. Dr. Klaus Pfeffer Date of the oral examination: 23.03.2017 Contents Contents CONTENTS .................................................................................................. III ABBREVIATIONS ...................................................................................... XII SUMMARY .................................................................................................. XV 1. INTRODUCTION .................................................................................... 1 1.1 Taxonomy ....................................................................................................................... 1 1.2 Genome and genetics ..................................................................................................... 2 1.3 Chlamydiae cause widespread infections ..................................................................... 3 1.3.1 Chlamydial infections in animals and transmission to humans ................................ 3 1.3.2 C. pneumoniae .......................................................................................................... 4 1.3.3 C. trachomatis ........................................................................................................... 5 1.4 The infectious cycle of Chlamydia ................................................................................. 5 1.4.1 The chlamydial outer membrane complex (cOMC) ................................................. 7 1.4.2 Adhesion ................................................................................................................... 8 1.4.3 Internalization of EBs ............................................................................................. 11 1.4.4 Formation and remodeling of the inclusion ............................................................ 13 1.4.5 Differentiation of EBs into RBs.............................................................................. 14 1.4.6 RBs replication ....................................................................................................... 14 1.4.7 Re-differentiation of RBs into EBs and EBs release .............................................. 15 1.4.8 Persistence .............................................................................................................. 15 1.5 The protein secretion systems in Gram-negative bacteria ....................................... 16 1.5.1 Type 3 secretion system .......................................................................................... 16 1.5.2 Type 5 secretion system .......................................................................................... 18 1.6 Polymorphic membrane proteins (Pmps) .................................................................. 20 iii Contents 1.6.1 Pmp characteristics ................................................................................................. 20 1.6.2 Pmp expression during the infection cycle ............................................................. 24 1.6.3 Pmps functions ........................................................................................................ 27 1.6.4 Pmps processing and oligomerization .................................................................... 28 1.6.5 Pmps as vaccine candidates .................................................................................... 31 1.7 Identification of new C. pneumoniae adhesins .......................................................... 33 1.8 Functional amyloids ..................................................................................................... 34 1.9 Objectives of this work ................................................................................................ 38 2. MATERIALS .......................................................................................... 39 2.1 Materials, machines and devices ................................................................................ 39 2.2 Chemicals and reagents ............................................................................................... 40 2.3 Solutions and buffers ................................................................................................... 43 2.4 Enzymes ........................................................................................................................ 45 2.4.1 Restriction enzymes ................................................................................................ 45 2.4.2 Other enzymes ........................................................................................................ 45 2.5 Antibodies ..................................................................................................................... 45 2.5.1 Primary antibodies .................................................................................................. 45 2.5.2 Secondary antibodies .............................................................................................. 46 2.6 Kits ................................................................................................................................ 46 2.7 DNA and protein size standards ................................................................................. 46 2.8 Oligonucleotides ........................................................................................................... 47 2.8.1 Oligonucleotides for cloning .................................................................................. 47 2.8.2 Oligonucleotides for sequencing ............................................................................ 49 2.9 Plasmids ........................................................................................................................ 49 2.10 Cells and cell lines .................................................................................................... 51 2.10.1 Eukaryotic cells and cell lines .............................................................................. 51 iv Contents 2.10.2 Prokaryotic cells and cell lines ............................................................................. 51 2.11 Media ........................................................................................................................ 51 2.11.1 Media for cell culture and Chlamydia cultivation ................................................ 51 2.11.2 Media for S. cerevisiae cultivation ....................................................................... 52 2.11.3 Media for E. coli ................................................................................................... 53 3. METHODS .............................................................................................. 54 3.1 Cultivation of different organisms ............................................................................. 54 3.1.1 Cultivation of HEp-2 epithelial human cells .......................................................... 54 3.1.1.1 Cell culture medium .................................................................................................... 54 3.1.1.2 Passaging adherent cells by trypsinization .................................................................. 54 3.1.1.3 Freezing human epithelial HEp-2 cells ....................................................................... 54 3.1.2 Cultivation of Saccharomyces cerevisiae ............................................................... 55 3.1.3 Cultivation of Chlamydia ........................................................................................ 55 3.1.3.1 Infection, passaging and harvesting Chlamydia from adherent cells .......................... 55 3.1.3.2 Purification of chlamydial EBs by gastrografin gradient ............................................ 56 3.1.3.3 Determination of Chlamydia infectious rate (inclusion-forming units (IFU)/ml) ....... 56 3.1.3.4 Stationary chlamydial infection of human cells .......................................................... 57 3.1.3.5 Genomic chlamydial DNA extraction from infected cells .......................................... 57 3.1.3.6 Preparation of protein extract from Chlamydia-infected cells for SDS-PAGE analysis . ..................................................................................................................................... 58 3.1.4 Cultivation of Escherichia coli ............................................................................... 58 3.1.4.1 Production of electrocompetent E. coli cells ............................................................... 58 3.1.4.2 Production of chemical competent E. coli cells .......................................................... 59 3.2 Biomolecular methods ................................................................................................. 59 3.2.1 Polymerase chain reaction (PCR) ........................................................................... 59 3.2.1.1 PCR on chlamydial genomic DNA or from
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