Identification of a Novel Mycobacterial Gene Involved in the Synthesis of a Phenolic Glycolipid and its Role in the Prevention of Phagosome Maturation Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Nirmal Robinson aus Nagercoil, Indien Bonn 2007 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn. 1. Referent: Priv. Doz. Dr. med. G. Plum 2. Referent: Prof. Dr. rer. nat. B. Wiedemann Tag der Promotion: 09. 11. 2007 Contents Abstract……………………………………………………………………………….. 4 1 Introduction....................................................................................................................... 6 1.1 Tuberculosis.......................................................................................................................... 6 1.1.1 TB Pathogenesis............................................................................................................................. 6 1.2 Virulent Mycobacteria Inhibit Phagosome Maturation................................................... 8 1.2.1 Phagosome Maturation .................................................................................................................. 8 1.2.2 Methods to Study Phagosome Maturation ..................................................................................... 9 1.2.3 Intracellular Signals Targeted by Virulent Mycobacteria to Inhibit Phagosome Maturation....... 11 1.3 Genetic Tools to Study Mycobacterial Virulence Factors.............................................. 20 1.3.1 Gene Expression Analysis ........................................................................................................... 20 1.3.2 Comparative Genomics................................................................................................................ 22 1.3.3 Generation of Mutations in Specific Genes ................................................................................. 23 1.3.4 Analysis of Mutant Library.......................................................................................................... 24 1.4 Mycobacterial Factors Disrupting Phagosome Maturation........................................... 25 1.5 Unique Cell Envelope and Lipids of Mycobacteria ........................................................ 27 1.6 Analysis of Mycobacterial Lipids ..................................................................................... 32 1.7 Mycobacterium marinum as a Model ................................................................................ 34 2 Objectives......................................................................................................................... 36 3 Materials and Methods.................................................................................................... 37 3.1 Materials ............................................................................................................................. 37 3.1.1 Instruments Used ......................................................................................................................... 37 3.1.2 Chemicals, Media and other Accessories Used............................................................................ 39 3.1.3 Oligonucleotides .......................................................................................................................... 45 3.1.4 Plasmids ....................................................................................................................................... 47 3.1.5 Bacterial Strains........................................................................................................................... 48 3.1.6 Cell Lines..................................................................................................................................... 49 3.2 Methods............................................................................................................................... 49 3.2.1 Culturing Human Monocyte Derived Macrophages (HMDM).................................................... 49 3.2.2 Screening for Mutants Incompetent in Retarding Phagosome Maturation .................................. 50 3.2.3 Southern Blot Analysis ................................................................................................................ 52 3.2.4 Cloning by Homologous Recombination using “Infusion Kit” ................................................... 54 3.2.5 Western Blot Analysis ................................................................................................................. 55 1 3.2.6 Rab5-, Rab7- and Rab11-GFP Adenoviral Vector Construction ................................................. 56 3.2.7 Transfection of HMDM and Mouse Dendritic Cells with Rab-GFP Adenoviral Vectors ........... 58 3.2.8 Staining using Antibodies and Immunofluorescence Microscopy............................................... 58 3.2.9 Staining of Mycobacterium with FITC or TRITC ....................................................................... 59 3.2.10 Growth Rate of M. marinum ........................................................................................................ 59 3.2.11 Flowcytometric Analysis of Cells Expressing EGFP-Rab........................................................... 60 3.2.12 Trans-Complementation of the Mutant P1................................................................................... 60 3.2.13 Radiolabelling of Mycobacterium................................................................................................ 61 3.2.14 Quantitative Analysis of Mutants Reaching the Phagolysosmes ................................................. 61 3.2.15 Mice Infection.............................................................................................................................. 61 3.2.16 Mycolic Acid Extraction and Analysis ........................................................................................ 62 3.2.17 Non-Polar Lipid Analysis ............................................................................................................ 63 3.2.18 Extraction of a Lipid Species from WT M. marinum Missing in Mutant P1 ............................... 63 3.2.19 Structural Elucidation of the Lipid Missing in Mutant P1 by Mass and NMR Spectrometry...... 64 3.2.20 Coating of Beads with Mycobacterial Lipids............................................................................... 64 3.2.21 Characterisation of Phagosomes Containing Lipid Coated Beads. .............................................. 65 3.2.22 Cloning, Expression and Purification of PmiA-Protein ............................................................... 66 3.2.23 Capturing Proteins Interacting with PmiA................................................................................... 67 4 Results.............................................................................................................................. 69 4.1 Magnetic Separation of Phagolysosomes from Subcellular Components..................... 69 4.2 Screening for M. marinum Mutants Permitting Phagosome Maturation..................... 70 4.3 Defining Transposon Insertion Sites by Sequence and Southern Blot Analysis .......... 71 4.4 Adenoviral Vectors to Track the Endocytic / Phagocytic Pathway............................... 75 4.5 Immunofluorescence Microscopy of HMDM Infected with Mutants ........................... 78 4.6 Mutant P1 is Attenuated in HMDM................................................................................. 81 4.7 Bioinformatics Analysis of Transposon Inserted Gene of Mutant P1........................... 82 4.8 Trans-complementation of P1 and Co-elution of 14C Labelled Bacterial Cells with Lysosomal Marker............................................................................................................. 83 4.9 In vivo Survival of P1 in Mice ........................................................................................... 85 4.10 Histopathology of Infected Organs................................................................................... 87 4.11 Non-Cording Phenotype and Lipid Profile...................................................................... 89 4.12 Characterization of the Lipid Missing in Mutant P1...................................................... 91 4.13 M. marinum PGL Inhibits Phagosome Maturation ........................................................ 96 2 4.14 Functional Characterization of the Gene pmiA............................................................... 98 5 Discussion...................................................................................................................... 100 5.1 Mycobacterial Genes Possibly Involved in the Inhibition of Phagosome Maturation100 5.1.1 Genes Belonging to PE/PPE Gene Family................................................................................. 100 5.1.2 Membrane Transporters ............................................................................................................. 102 5.1.3 Porins ......................................................................................................................................... 103 5.1.4 β-Propeller Protein....................................................................................................................