UCC Library and UCC researchers have made this item openly available. Please let us know how this has helped you. Thanks! Title Molecular analysis of virulence mechanisms associated with adherent- invasive Escherichia coli (AIEC) Author(s) O'Driscoll, Adam Publication date 2012-10 Original citation O'Driscoll, A. 2012. Molecular analysis of virulence mechanisms associated with Adherent-Invasive Escherichia coli (AIEC). PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2012, Adam O'Driscoll http://creativecommons.org/licenses/by-nc-nd/3.0/ Item downloaded http://hdl.handle.net/10468/952 from Downloaded on 2021-09-25T13:37:32Z Molecular analysis of virulence mechanisms associated with Adherent- Invasive Escherichia coli (AIEC) Adam O‟Driscoll A thesis submitted for the degree of Doctor of Philosophy National University of Ireland, Cork Department of Microbiology October 2012 Head of Department: Prof Gerald F. Fitzgerald Supervisor: Dr David Clarke Contents Declaration............................................................................................................................... i Acknowledgements ................................................................................................................ ii List of abbreviations ............................................................................................................. iii Abstract ................................................................................................................................. vii Chapter 1.0: Introduction ..................................................................................................... 1 1.1 Introduction to Crohn’s Disease ..................................................................................... 1 1.1.1 Gene polymorphisms associated with Crohn‟s Disease .............................................. 4 1.1.2 Crohn‟s Disease and Autophagy ................................................................................. 5 1.1.2.1 Autophagy-related 16-like 1 protein like complex (ATG16L1) ........................... 6 1.1.3 NOD2/CARD15 .......................................................................................................... 9 1.1.3.1 NOD2/CARD15 function ..................................................................................... 9 1.1.4 Interleukin 23 Receptor (IL23-R) ............................................................................. 13 1.1.5 Environmental risk factors for Crohn‟s disease ........................................................ 15 1.1.6 Bacterial involvement in Crohn‟s disease ................................................................. 16 1.1.6.1 Defective mucosal barrier function and microbial clearance ............................. 18 1.1.6.2 Microbial diversity ............................................................................................. 20 1.1.6.3 Association of specific microbes with Crohn‟s disease ..................................... 22 1.1.6.4 Crohn‟s disease-associated E. coli ..................................................................... 23 1.2 Escherichia coli: commensals and pathogens .............................................................. 24 1.2.1 Pathotypes of Escherichia coli .................................................................................. 25 1.2.1.1 Enteropathogenic E. coli (EPEC) ....................................................................... 27 1.2.1.2 Enterohaemorrhagic E. coli (EHEC).................................................................. 28 1.2.1.3 Enterotoxigenic E. coli (ETEC) ......................................................................... 29 1.2.1.4 Enteroaggregative E. coli (EAEC) ..................................................................... 30 1.2.1.5 Enteroinvasive E. coli (EIEC) ............................................................................ 31 1.2.1.6 Uropathogenic E. coli (UPEC) ........................................................................... 34 1.2.1.7 Neonatal meningitis E.coli (NMEC) .................................................................. 35 1.3 Adherent-invasive Escherichia coli (AIEC) and Crohn’s disease .............................. 36 1.3.1 AIEC and the Epithelial Barrier ................................................................................ 37 1.3.1.1 Type 1 pili and flagella mediated adhesion/invasion to epithelial cells. ............ 39 1.3.1.2 Type 1 fimbriae and Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM-6) expression .............................................................................................. 41 1.3.1.3 AIEC and M-cells: A potential entry point. ....................................................... 43 1.3.1.4 Outer membrane vesicles (OMVs) contribute to AIEC invasion ...................... 45 1.3.2 Replication in macrophages. ..................................................................................... 47 1.3.2.1 AIEC replicates in mature phagolysosomes ...................................................... 48 1.3.2.2 The periplasmic oxidoreductase DsbA and the RNA binding protein Hfq are required for intramacrophage replication of AIEC strain LF82. .................................... 52 1.3.2.3 Defects in autophagy enhance intracellular replication of AIEC strain LF82 ... 53 1.4 Comparative genomics. ................................................................................................ 54 1.5 Pathoadaptive mutations .............................................................................................. 56 1.6 Objectives of this study ................................................................................................ 57 Chapter 2.0 Materials and Methods ................................................................................... 58 2.1 Bacterial strains and Plasmids ...................................................................................... 58 2.2 Cell lines and cell culture............................................................................................... 58 2.2.1 J774.A1 murine macrophages ................................................................................... 58 2.2.2 C2Bbe1 epithelial cells ............................................................................................. 58 2.2.3 Acanthamoeba polyphaga ......................................................................................... 58 2.3 Growth conditions .......................................................................................................... 59 2.4 Overnight Starter Cultures ........................................................................................... 59 2.5 Kits and Enzymes ........................................................................................................... 64 2.6 Electrophoresis ............................................................................................................... 65 2.7 Genomic DNA Extraction ............................................................................................. 65 2.8 Primer List ...................................................................................................................... 66 2.9 Electrocompetent Escherichia coli ................................................................................ 67 2.10 Construction of HM605 Tn5 mutant library ............................................................. 67 2.11 Screening for motility minus mutants in E. coli HM605 Tn5 mutant library ........ 67 2.12 Screening for hydrogen peroxide sensitive (H2O2) mutants in E. coli HM605 Tn5 mutant library ...................................................................................................................... 67 2.13 Arbitrarily primed PCR .............................................................................................. 68 2.14 Minimum inhibitory concentration (MIC) determination of E. coli HM605 to hydrogen peroxide ............................................................................................................... 69 2.15 Sensitivity of E. coli strains to hydrogen peroxide. ................................................... 69 2.16 Catalase test .................................................................................................................. 70 2.17 Bacterial stress tolerance ............................................................................................. 70 2.18 Identification of hydrogen peroxide-sensitive mutant strains of E. coli HM605 .... 70 2.19 Determination of fim switch orientation .................................................................... 70 2.20 Yeast aggregation assay ............................................................................................... 73 2.21 Flp recombinase-mediated marker excision from chromosomally-inserted DNA . 73 2.22 Construction of deletion mutants in E. coli using P1 transduction ......................... 75 2.22.1: Preparation of Lysogens ........................................................................................ 75 2.22.2: Preparation of high titre P1 c/kml lysate ................................................................ 75 2.22.3 Transduction ........................................................................................................... 76 2.23 Colony PCR for kanamycin cassette .........................................................................