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Listeria Sensu Stricto Specific Genes Involved in Colonization of the Gastrointestinal Tract by Listeria Monocytogenes

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Listeria Sensu Stricto Specific Genes Involved in Colonization of the Gastrointestinal Tract by Listeria Monocytogenes TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Mikrobielle Ökologie Characterization of Listeria sensu stricto specific genes involved in colonization of the gastrointestinal tract by Listeria monocytogenes Jakob Johannes Schardt Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. rer.nat. Siegfried Scherer Prüfende der Dissertation: 1. apl.Prof. Dr.rer.nat. Thilo Fuchs 2. Prof. Dr.med Dietmar Zehn Die Dissertation wurde am 18.01.2018 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 14.05.2018 angenommen. Table of contents Table of contents ___________________________________________________________ I List of figures _______________________________________________________________ V List of tables ______________________________________________________________ VI List of abbreviations ________________________________________________________ VII Abstract __________________________________________________________________ IX Zusammenfassung __________________________________________________________ X 1 Introduction ______________________________________________________________ 1 1.1 The genus Listeria ____________________________________________________________ 1 1.1.1 Listeria sensu stricto ________________________________________________________________ 1 1.1.2 Listeria sensu lato __________________________________________________________________ 2 1.2 The pathogenic L. monocytogenes _______________________________________________ 3 1.2.1 Human listeriosis ___________________________________________________________________ 3 1.2.2 L. monocytogenes as an intracellular model pathogen _____________________________________ 4 1.2.3 Animal models for in vivo studies on listeriosis ___________________________________________ 6 1.3 Pathophysiology of listeriosis___________________________________________________ 7 1.3.1 The transition between environmental and pathogenic lifestyle _____________________________ 8 1.3.2 The GI phase of infection ____________________________________________________________ 8 1.3.2.1 Survival under acidic conditions in the stomach ______________________________________ 9 1.3.2.2 Osmotolerance _________________________________________________________________ 9 1.3.2.3 Bile resistance _________________________________________________________________ 9 1.3.2.4 Colonization resistance _________________________________________________________ 10 1.3.2.5 Oxygen availability in the gut and facultative anaerobic metabolism _____________________ 11 1.3.3 Crossing of the intestinal barrier and subsequent systemic disease __________________________ 13 1.3.3.1 Invasion of the intestinal epithelium_______________________________________________ 13 1.3.3.2 Dissemination to distant organs __________________________________________________ 14 1.3.3.3 Liver and spleen as the main targets for dissemination ________________________________ 16 1.4 Aim of this thesis ___________________________________________________________ 16 2 Material & Methods ______________________________________________________ 18 2.1 Material ___________________________________________________________________ 18 2.1.1 Chemicals, enzymes, and commercial kits ______________________________________________ 18 2.1.2 Equipment and consumables ________________________________________________________ 18 2.1.3 Bacterial strains ___________________________________________________________________ 18 2.1.4 Plasmids _________________________________________________________________________ 19 2.1.5 Oligonucleotides __________________________________________________________________ 20 2.1.6 Media and Buffers _________________________________________________________________ 22 2.1.7 Supplements _____________________________________________________________________ 23 2.2 Microbiology _______________________________________________________________ 24 2.2.1 Culture conditions _________________________________________________________________ 24 2.2.1.1 Bacterial glycerol stocks _________________________________________________________ 24 2.2.1.2 Growth on plates ______________________________________________________________ 24 2.2.1.3 Overnight cultures _____________________________________________________________ 24 I 2.2.1.4 Growth for RNA isolation ________________________________________________________ 24 2.2.2 Growth analysis ___________________________________________________________________ 24 2.2.2.1 Growth in liquid media _________________________________________________________ 24 2.2.2.2 Growth analysis using the Bioscreen C _____________________________________________ 25 2.2.2.3 Growth analysis on selective agar plates ___________________________________________ 25 2.3 Molecular biology ___________________________________________________________ 25 2.3.1 Preparation of nucleic acids _________________________________________________________ 25 2.3.1.1 Plasmid isolation ______________________________________________________________ 25 2.3.1.2 Isolation of genomic DNA _______________________________________________________ 25 2.3.1.3 Isolation of bacterial RNA _______________________________________________________ 26 2.3.1.4 cDNA synthesis ________________________________________________________________ 28 2.3.1.5 Determination of nucleic acid concentrations, purity and integrity ______________________ 29 2.3.1.6 DNA sequence analysis _________________________________________________________ 29 2.3.1.7 Purification of DNA ____________________________________________________________ 29 2.3.2 Agarose gel electrophoresis _________________________________________________________ 29 2.3.3 Polymerase chain reaction __________________________________________________________ 30 2.3.3.1 Standard PCR _________________________________________________________________ 30 2.3.3.2 qRT-PCR _____________________________________________________________________ 31 2.3.4 Enzymatic modification of nucleic acids ________________________________________________ 32 2.3.4.1 DNA digestion by restriction endonucleases ________________________________________ 32 2.3.4.2 Dephosphorylation of plasmids ___________________________________________________ 32 2.3.4.3 DNA ligation __________________________________________________________________ 33 2.3.5 Genetic modification of bacteria _____________________________________________________ 33 2.3.5.1 Preparation of CaCl2-competent cells ______________________________________________ 33 2.3.5.2 Transformation _______________________________________________________________ 33 2.3.5.3 Preparation of electrocompetent cells _____________________________________________ 34 2.3.5.4 Electroporation _______________________________________________________________ 34 2.3.5.5 Construction of in-frame deletion mutants _________________________________________ 34 2.3.6 NGS methods _____________________________________________________________________ 35 2.3.6.1 De novo genome sequencing of L. kieliensis sp. nov. __________________________________ 35 2.3.6.2 Transcriptomics of L. monocytogenes EGDe _________________________________________ 36 2.3.7 Bioinformatics ____________________________________________________________________ 37 2.3.7.1 Genome assembly and annotation ________________________________________________ 37 2.3.7.2 Phylogenetic analysis ___________________________________________________________ 37 2.3.7.3 Genome comparison of different Listeria spp. _______________________________________ 37 2.3.7.4 Analysis of transcriptome sequencing data _________________________________________ 38 2.3.7.5 Bioinformatic protein analyses ___________________________________________________ 39 2.4 Cell culture ________________________________________________________________ 39 2.4.1 Cell lines _________________________________________________________________________ 39 2.4.2 Culture conditions _________________________________________________________________ 39 2.4.3 Subcultivation of cells ______________________________________________________________ 40 2.4.4 Freezing of cells ___________________________________________________________________ 40 2.4.5 Thawing and seeing of cells __________________________________________________________ 40 2.4.6 Cell counting _____________________________________________________________________ 40 2.4.7 Mycoplasma detection _____________________________________________________________ 41 2.4.8 Glycerol stocks for infection _________________________________________________________ 41 2.4.9 Adhesion assay ___________________________________________________________________ 41 2.4.10 Intracellular replication assay _______________________________________________________ 42 2.4.11 Comparative analysis of adhesion and invasion _________________________________________ 42 2.5 Mouse infections ___________________________________________________________ 42 2.5.1 Mouse strain _____________________________________________________________________ 42 2.5.2 Basic mouse care and maintenance ___________________________________________________ 43 II 2.5.2.1 Accommodation _______________________________________________________________ 43 2.5.2.2 Food and water _______________________________________________________________
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