Aus dem Max von Pettenkofer-Institut Lehrstuhl für Medizinische Mikrobiologie und Krankenhaushygiene der Ludwig-Maximilians-Universität München Vorstand: Prof. Dr. med. Sebastian Suerbaum Exploring the role of Mucispirillum schaedleri in enteric Salmonella enterica serovar Typhimurium infection Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Simone Herp aus Offenburg 2018 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuerin: Prof. Dr. Barbara Stecher-Letsch Zweitgutachterin: Prof. Dr. Gabriele Rieder Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 19.02.2019 i Eidesstattliche Erklärung Ich, Simone Herp, erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema: Exploring the role of Mucispirillum schaedleri in enteric Salmonella enterica serovar Typhimurium 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. München, den 07.03.2019 Simone Herp ii Table of Contents Table of Contents Table of Contents ........................................................................................................................ iii List of abbreviations .................................................................................................................. vii List of publications ...................................................................................................................... x Summary ..................................................................................................................................... xi Zusammenfassung ................................................................................................................... xiv 1 Introduction .......................................................................................................................... 1 1.1 The intestinal microbiota: keeper of homeostasis and health............................................ 1 1.1.1 Mechanisms of colonization resistance (CR) ........................................................... 1 1.1.2 SCFAs are important for our intestinal health ........................................................... 3 1.1.3 Host produced bile acids are modified by the intestinal microbiota ........................... 3 1.2 Mucispirillum schaedleri , a commensal bacterium in the murine gut ................................. 4 1.3 Salmonella enterica serovar Typhimurium ( S.Tm) ............................................................ 6 1.3.1 S.Tm is a major cause of gastroenteritis .................................................................. 6 1.3.2 Experimental models for studying S.Tm induced colitis ............................................ 6 The streptomycin mouse model ....................................................................... 6 Gnotobiotic low complex microbiota models ..................................................... 7 1.3.3 The Salmonella pathogenicity island 1 type 3 secretion system (SPI1-T3SS) .......... 9 1.3.4 The gut is an oxygen limited environment .............................................................. 10 Sensing of environmental oxygen by S.Tm .................................................... 10 Utilization of alternative electron acceptors during inflammation: advantage of S. Tm over the commensal microbiota ........................................................................... 10 S. Tm creates a favorable environment by induction of an inflammatory response ... 11 1.4 Role of the intestinal mucus layer: protection and supply of nutrients ............................. 13 1.4.1 Architecture of the intestinal mucus layer............................................................... 13 1.4.2 Agr2 -/- mice, lacking secreted MUC2, are protected against S. Tm induced colitis .. 13 2 Aims of this PhD thesis ...................................................................................................... 15 3 Materials and Methods ....................................................................................................... 16 3.1 Materials ....................................................................................................................... 16 3.1.1 Media and buffer ................................................................................................... 16 3.1.2 Strains and plasmids ............................................................................................. 22 Strains ........................................................................................................... 22 iii Table of Contents Plasmids ....................................................................................................... 24 3.1.3 Oligonucleotides ................................................................................................... 25 3.1.4 Chemicals and antibiotics ...................................................................................... 30 3.1.5 Antibodies ............................................................................................................. 33 3.1.6 Devices and specific materials .............................................................................. 34 3.2 Methods ........................................................................................................................ 36 3.2.1 Anaerobic cultivation of M. schaedleri (ASF457) .................................................... 36 3.2.2 Preparation of cryostocks from anaerobic cultures ................................................. 36 3.2.3 Construction of Salmonella enterica serovar Typhimurium ( S. Tm) mutants by λ Red recombination .................................................................................................................... 36 PCR fragment ............................................................................................... 36 Electroporation of PCR product in S. Tm pKD46 ............................................ 37 P22 phage transduction ................................................................................. 38 Generation of S.Tm avir ∆nr3 ............................................................................... 38 Generation of S. Tm ∆moaA ............................................................................... 39 3.2.4 Fluorescence In Situ Hybridization (FISH) ............................................................. 39 3.2.5 Immunofluorescence staining ................................................................................ 39 3.2.6 Luciferase assay ................................................................................................... 40 3.2.7 Hematoxylin and eosin staining (HE staining) ........................................................ 40 3.2.8 Lipocalin-2 (LCN2) Elisa ........................................................................................ 41 3.2.9 Invasion assay of S. Tm in HuTu80 cells ............................................................... 41 3.2.10 gDNA extraction from Gram negative bacteria ................................................... 42 3.2.11 gDNA extraction from feces and cecal content ................................................... 42 QIAamp Fast DNA Stool Kit (Qiagen) ............................................................ 42 gDNA extraction (Turnbaugh et al. , 2009) ...................................................... 43 3.2.12 Hydrolysis-probe based quantitative real-time PCR (qPCR) ............................... 44 3.2.13 Animal experiments ........................................................................................... 45 Mice used in this study .................................................................................. 45 Handling of gnotobiotic mice .......................................................................... 45 Colonization of gnotobiotic mice with M. schaedleri ........................................ 46 Growth of S. Tm for mouse infections ............................................................ 46 Infection of mice with a single S. Tm strain ..................................................... 46 Competitive infection experiments ................................................................. 47 Measuring the replicative capacity of S. Tm in vivo ......................................... 47 Analysis of short chain fatty acids (SCFAs) in cecal content ........................... 47 Quantification of bile acids in cecal content .................................................... 48 iv Table of Contents Transcriptome analysis .................................................................................. 48 3.2.14 Statistical analysis ............................................................................................. 49 4 Results ................................................................................................................................ 50 4.1 Studying the effect of M. schaedleri on the development of S. Tm induced colitis