Persistent infection by Yersinia pseudotuberculosis Kemal Avican Department of Molecular Biology Umeå Centre for Microbial Research (UCMR) Laboratory for Molecular Infection Sweden (MIMS) Umeå University Umeå 2015 Copyright © Kemal Avican Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-335-9 ISSN: 0346-6612 New Series No: 1748 Cover Picture: Transcriptome of Yersinia pseudotuberculosis Cover Design: Kemal Avican Electronic version is avaliable at http://umu.diva-portal.org/ Printed by: Print & Media Umeå, Sweden 2015 To my parents… ! Table of contents Abstract ................................................................................................... i List of Abbreviations ............................................................................. ii Papers in This Thesis ........................................................................... iv 1! Introduction ...................................................................................... 1! 1.1! Emergence of Bacterial Pathogens ................................................................ 3! 1.1.1! Emergence of pathogenic properties ............................................................................ 3! 1.1.1.1! Bacteria–protozoa interactions .............................................................................. 3! 1.1.1.2! Genome plasticity .................................................................................................. 5! 1.2! Bacterial Pathogenesis .................................................................................... 6! 1.2.1! Bacterial Pathogenic Mechanisms ................................................................................ 6! 1.2.1.1! Adherence ............................................................................................................. 7! 1.2.1.2! Invasion ................................................................................................................. 8! 1.2.1.3! Bacterial Camouflage ............................................................................................ 8! 1.2.1.4! Damage to the Host ............................................................................................ 10! 1.3! Host Defense Against Bacterial Infection .................................................... 12! 1.3.1! Barriers ....................................................................................................................... 12! 1.3.1.1! Skin and Mucosal Surfaces ................................................................................. 12! 1.3.1.2! Antimicrobial Proteins ......................................................................................... 13! 1.3.1.3! Microbiota ............................................................................................................ 14! 1.3.2! Cellular Innate Immune Response ............................................................................. 15! 1.3.2.1! Recognition of Bacteria ....................................................................................... 16! 1.3.2.2! Cytokines and Chemokines ................................................................................ 17! 1.3.2.3! Complement System ........................................................................................... 17! 1.3.2.4! Phagocytosis and Killing of Bacteria ................................................................... 18! 1.3.3! From the Innate to the Adaptive Immune System ...................................................... 19! 1.4! Bacterial Adaptation to Host Stresses .......................................................... 19! 1.4.1! Sensing the Stress ...................................................................................................... 19! 1.4.2! General Stress Response ........................................................................................... 20! 1.4.3! Temperature ............................................................................................................... 20! 1.4.4! Acidic Stress ............................................................................................................... 21! 1.4.5! Oxidative and Nitrosative Stress ................................................................................. 21! 1.4.6! Osmotic Stress ........................................................................................................... 22! 1.4.7! Oxygen Stress ............................................................................................................ 22! 1.5! Persistent Bacterial Infections ...................................................................... 23! 1.5.1! Helicobacter pylori ...................................................................................................... 24! 1.5.2! Mycobacterium tuberculosis ....................................................................................... 24! 1.5.3! Salmonella .................................................................................................................. 25! 1.6! Yersinia as a New Model for Persistence ..................................................... 26! 1.6.1! Yersinia pathogens ..................................................................................................... 27! 1.6.1.1! Pathogenesis and Adaptation of Enteric Yersinia ............................................... 27! 1.6.1.2! Adherence ........................................................................................................... 28! 1.6.1.3! T3SS ................................................................................................................... 29! 1.6.1.4! T6SS ................................................................................................................... 30! 1.6.1.5! LPS ..................................................................................................................... 30! 1.7! Identification of Pathogenicity Factors ......................................................... 31! 1.7.1! Biochemical Approaches ............................................................................................ 31! 1.7.2! Molecular Genetic Approaches ................................................................................... 32! 1.7.2.1! Transposon Mutagenesis .................................................................................... 32! 1.7.2.2! Identification of In Vivo–Expressed Genes ......................................................... 32! 1.7.2.2.1! Signature-tagged Mutagenesis .................................................................... 33! 1.7.2.2.2! In Vivo Expression Technology ................................................................... 33! 1.7.3! Genomic Approaches ................................................................................................. 33! 1.7.3.1! Microarrays ......................................................................................................... 33! 1.7.3.2! Next-generation Sequencing Technology ........................................................... 34! 1.7.3.2.1! Genome Sequencing by NGS ...................................................................... 34! 1.7.3.2.2! Metagenomics ............................................................................................. 35! 1.7.3.2.3! Transposon Insertion Sequencing ............................................................... 35! 1.7.3.2.4! ChiP-seq ...................................................................................................... 35! 1.7.3.2.5! RNA-seq ...................................................................................................... 36! 2! Objectives of This Thesis ............................................................. 41! 3! Results and Discussion ................................................................ 42! 3.1! Monitoring Y. pseudotuberculosis Infection in Mice ................................... 42! 3.2! Persistent Infection of Y. pseudotuberculosis in Cecum ........................... 42! 3.2.1! A Mouse Model for Persistent Y. pseudotuberculosis Infection .................................. 43! 3.2.2! Cytokine Expression During Persistency .................................................................... 44! 3.3! Transcriptome of Persistent Y. pseudotuberculosis ................................... 44! 3.3.1! Transcriptome of Persistent Y. pseudotuberculosis is Similar to 26°C Growth .......... 44! 3.3.2! Transcriptional Reprogramming for Persistence ........................................................ 45! 3.3.3! Persistent Y. pseudotuberculosis is Influenced by Environmental Cues .................... 45! 3.4! Complex RNA-Populations can be Resolved by RNA-seq ......................... 46! 3.5! ArcA, Fnr, FrdA, and WrbA are Required for Persistent Infection ............. 47! 3.6! Yersinia Infections Alter Microbial Composition in Cecum ........................ 47! 3.7! RfaH is Required for Establishment of Infection ......................................... 48! 4! Main Findings in This Thesis ........................................................ 49! 5! Future Perspectives ...................................................................... 50! 1.1! Search for Novel Targets for Antimicrobials ................................................ 50! 5.1! Switch for Reprogramming ...........................................................................