Characterization of putative virulence- associated genes of Burkholderia pseudomallei Inauguraldissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald 2014 vorgelegt von Duong Tuan Linh geboren am 27. 02. 1982 in Hanoi, Vietnam Greifswald, 16.07.2014 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter 1: Prof. Dr. med. Ivo Steinmetz 2. Gutachter 2: Prof. Dr. Jan Buer Tag der Promotion: 2014-11-21 II CONTENTS CONTENTS Contents ...................................................................................................................................... III Abtract ..................................................................................................................................... VII List of tables ............................................................................................................................... IX List of figures ............................................................................................................................... X List of abbreviations ................................................................................................................ XIII 1 Introduction .................................................................................................................. 1 1.1 Burkholderia pseudomallei, the causative agent of melioidosis ............................ 1 1.1.1 Characteristics of B. pseudomallei ................................................................................ 1 1.1.2 Epidemiology and clinical features of melioidosis ......................................................... 1 1.2 Virulence factors of B. pseudomallei ......................................................................... 3 1.2.1 Overview of B. peudomallei virulence factors ................................................................ 3 1.2.2 Secretion systems of B. pseusomallei ........................................................................... 3 1.3 LysR-type transcriptional regulator family ............................................................... 6 1.3.1 Origin and evolution of LTTRs ....................................................................................... 6 1.3.2 Structure and function of LTTR family proteins ............................................................. 6 1.4 Intracellular life cycle of B. pseudomallei ................................................................. 8 1.5 In vivo B. pseudomallei infection models ................................................................. 8 1.5.1 Murine B. pseudomallei infection models ...................................................................... 9 1.5.2 Experimental vaccines against B. pseudomallei infection ............................................. 9 1.6 Aims of the study ....................................................................................................... 10 2 Materials and methods .............................................................................................. 11 2.1 Bacteria ....................................................................................................................... 11 2.1.1 Bacterial strains ........................................................................................................... 11 2.1.2 Preparation of bacterial glycerol stocks ....................................................................... 12 2.1.3 Preparation of competent cells .................................................................................... 12 2.1.4 Adjustment of bacterial dosages .................................................................................. 13 2.1.5 Growth kinetics ............................................................................................................ 13 2.1.6 Motility assays .............................................................................................................. 13 III CONTENTS 2.1.7 Biofilm assay ................................................................................................................ 13 2.1.8 Protease assay ............................................................................................................ 14 2.1.9 Antibiotic susceptibility testing ..................................................................................... 14 2.2 Plasmids ..................................................................................................................... 15 2.3 Oligonucleotides ........................................................................................................ 16 2.4 DNA manipulations .................................................................................................... 17 2.4.1 Isolation of genomic DNA of B. pseudomallei.............................................................. 17 2.4.2 Polymerase chain reaction........................................................................................... 17 2.4.3 Agarose gel electrophoresis ........................................................................................ 18 2.4.4 Purification of DNA fragment and gel extraction .......................................................... 19 2.4.5 Restriction enzymes and dephosphorylation ............................................................... 19 2.4.6 Ligation......................................................................................................................... 20 2.4.7 Heat shock transformation ........................................................................................... 20 2.4.8 Mini-preparation of DNA plasmid ................................................................................. 20 2.5 Mutagenesis and complementation of B. pseudomallei genes ............................ 20 2.5.1 Construction of knockout mutants ............................................................................... 20 2.5.2 Conjugation .................................................................................................................. 21 2.5.3 Isolation of markerless mutants ................................................................................... 21 2.5.4 Complementation ......................................................................................................... 22 2.6 Protein manipulations ............................................................................................... 23 2.6.1 Protein preparation with Trizol ..................................................................................... 23 2.6.2 Protein preparation by ribolyser method ...................................................................... 23 2.6.3 Purification of extracellular protein ............................................................................... 23 2.6.4 Bradford protein quantification ..................................................................................... 24 2.6.5 SDS polyacrylamide mini gel electrophoresis.............................................................. 24 2.6.6 Western blot ................................................................................................................. 25 2.7 Quantitation of cytoplasmic proteins by using stable isotope labelling with amino acids in cell culture ........................................................................................ 26 2.7.1 Preparation of a heavy-labelled B. pseudomallei protein standard ............................. 26 2.7.2 Sample preparation for proteomic analysis ................................................................. 26 2.7.3 Mass spectrometry and data analysis ......................................................................... 26 IV CONTENTS 2.8 RNA manipulations .................................................................................................... 27 2.8.1 RNA preparation .......................................................................................................... 27 2.8.2 DNase I treatment of RNA ........................................................................................... 27 2.8.3 Reverse transcription ................................................................................................... 27 2.8.4 Semi quantitative expression analysis ......................................................................... 28 2.9 Experiments with mammalian cells ......................................................................... 28 2.9.1 Cultivation of mammalian cell lines .............................................................................. 28 2.9.2 Generation and cultivation of primary murine bone marrow macrophages ................. 28 2.9.3 Counting, seeding and maintaining cell lines............................................................... 29 2.9.4 Infection of mammalian cells ....................................................................................... 29 2.9.5 Invasion and intracellular replication assays ............................................................... 29 2.9.6 Actin tail formation assay ............................................................................................. 30 2.10 In vivo infection experiments ................................................................................... 30 2.10.1 Bioethics......................................................................................................................