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Establishment of a Gnotobiotic Mouse Model to Investigate the Mechanisms of Colonization Resistance Against Salmonella Enterica Serovar Typhimurium (S

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Establishment of a Gnotobiotic Mouse Model to Investigate the Mechanisms of Colonization Resistance Against Salmonella Enterica Serovar Typhimurium (S Aus dem Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie Lehrstuhl: Bakteriologie der Ludwig-Maximilians-Universität München Vormaliger Direktor: Prof. Dr. Dr. Jürgen Heesemann Kommissarischer Vorstand: Prof. Dr. Rainer Haas Thema der Dissertation: Establishment of a gnotobiotic mouse model to investigate the mechanisms of colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm) and study of the protective role of the intestinal mucus layer during S. Tm infection Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Sandrine Brugiroux aus Firminy, Frankreich 2016 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Berichterstatter: Prof. Dr. Bärbel Stecher Mitberichterstatter: Prof. Dr. Heinrich Jung Mitbetreuung durch den promovierten Mitarbeiter: …………………………………………………… Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 10.10.2016 Eidesstattliche Erklärung Ich, Sandrine Brugiroux, erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema: “Establishment of a gnotobiotic mouse model to investigate the mechanisms of colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm) and study of the protective role of the intestinal mucus layer during S. Tm infection” selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. Nîmes, 20.10.2016 Sandrine Brugiroux Ort, Datum Unterschrift To all the mice sacrificed for science. Table of contents List of figures ..............................................................................................................................................xi List of tables .............................................................................................................................................. xiv List of abbreviations ................................................................................................................................ xvii List of publications ..................................................................................................................................... xx Summary ................................................................................................................................................... xxi Zusammenfassung .................................................................................................................................. xxiii 1. Introduction ......................................................................................................................................... 1 1.1. The mammalian gut microbiota .................................................................................................. 1 1.1.1. Composition of the microbiota in humans and mice ........................................................... 1 1.1.2. The effects of the microbiota on its host ............................................................................. 2 1.1.2.1. Education of the immune system.................................................................................... 2 1.1.2.2. Influence of the gut microbiota on gut morphology and nutrition .................................. 3 1.1.3. Environmental and host factors influencing the intestinal microbiota composition ............ 4 1.1.3.1. Diet ................................................................................................................................ 4 1.1.3.2. Antibiotic treatment........................................................................................................ 5 1.1.3.3. Inflammation and infection ............................................................................................ 5 1.1.3.4. Host genotype ................................................................................................................ 6 1.2. Analytical tools to study the gut microbiota composition and function ....................................... 6 1.2.1. High-throughput amplicon sequencing and quantitative PCR of bacterial 16S rRNA genes ............................................................................................................................................ 7 1.2.2. Meta-omics analyses .......................................................................................................... 7 1.2.3. Fluorescence in situ hybridization ...................................................................................... 8 1.2.4. Bacterial isolation and culture ............................................................................................ 8 Table of contents 1.3. Salmonella enterica serovar Typhimurium - a model human pathogen to study microbiota- pathogen interaction in the gut .................................................................................................... 9 1.3.1. Mechanisms of Salmonella Typhimurium pathogenesis ..................................................... 9 1.3.2. Salmonella Typhimurium outcompetes the gut microbiota and benefits from the gut inflammation .................................................................................................................... 12 1.4. Colonization resistance ............................................................................................................. 13 1.4.1. Direct effects of the gut microbiota on the enteric pathogens ........................................... 14 1.4.2. Indirect effects on pathogen colonization ......................................................................... 15 1.4.3. Mouse models developed to study the colonization resistance and the underlined mechanisms ...................................................................................................................... 15 1.5. The intestinal mucus layer......................................................................................................... 16 1.5.1. Composition of the mucus layer ....................................................................................... 16 1.5.2. Interactions of the mucus layer with the gut microbiota and the enteropathogens ............ 17 1.5.3. Mouse models developed to study the role of the mucus layer ......................................... 18 1.5.4. The protein disulfide isomerase AGR2 and its functions .................................................. 19 2. Objectives .......................................................................................................................................... 21 3. Materials and Methods ...................................................................................................................... 22 3.1. Materials ................................................................................................................................... 22 3.1.1. Chemicals, Consumables and Instruments ........................................................................ 22 3.1.2. Oligonucleotides, probes and antibodies .......................................................................... 26 3.1.3. Plasmids and strains ......................................................................................................... 32 3.1.4. Media and buffers ............................................................................................................. 34 3.2. Methods .................................................................................................................................... 41 3.2.1. Bacterial culture methods ................................................................................................. 41 3.2.1.1. Cryoconservation of bacteria ........................................................................................ 41 3.2.1.2. Bacterial culture conditions .......................................................................................... 43 Table of contents 3.2.1.3. Streptomycin halo assay ............................................................................................... 43 3.2.2. Bacterial strains ................................................................................................................ 44 3.2.2.1. Salmonella strains ........................................................................................................ 44 3.2.2.2. The Altered Schaedler Flora strains ............................................................................. 44 3.2.2.3. Isolation of the Oligo-MM strains ................................................................................ 44 3.2.2.4. Preparation of bacterial inocula as frozen stocks .......................................................... 46 3.2.3. Molecular biology methods .............................................................................................. 46 3.2.3.1. Genomic DNA extraction ............................................................................................. 46 3.2.3.2. RNA extraction and microarray analysis .....................................................................
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