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Dissertation DISSERTATION Titel der Dissertation „Characterization of potential vaccine antigens from Moraxella catarrhalis and nontypeable Haemophilus influenzae with focus on iron metabolism“ Verfasserin Mag.rer.nat. Margarita Smidt angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr.rer.nat.) Wien, 2011 Studienkennzahl lt. Studienblatt: A 091 441 Dissertationsgebiet lt. Studienblatt: Genetik - Mikrobiologie Betreuer: Univ. Prof. Dr. Alexander von Gabain ____________________________________________________________Table of contents ACKNOWLEDGEMENTS.................................................................................................VII 1 ABSTRACT ..................................................................................................................... 1 2 KURZFASSUNG............................................................................................................. 3 3 INTRODUCTION ........................................................................................................... 5 3.1 OTITIS MEDIA – A POLYMICROBIAL AND MULTIFACTORIAL DISEASE ...............................5 3.1.1 Otitis media disease and pathogens causing otitis media..........................................5 3.1.2 Interactions of M. catarrhalis and NTHI with the immune system and mechanisms of immune evasion....................................................................................7 3.1.3 Risk factors for otitis media............................................................................................9 3.1.4 The burden of otitis media and current treatment....................................................10 3.2 VACCINES AGAINST OTITIS MEDIA: ACHIEVEMENTS AND CHALLENGES........................11 3.3 ANIMAL MODELS FOR OTITIS MEDIA AND CORRELATES OF PROTECTION ......................12 3.4 EVALUATION OF VACCINE ANTIGENS FOR THE PREVENTION OF OTITIS MEDIA.............14 3.4.1 Potential vaccine antigens and virulence factors of M. catarrhalis ..........................14 3.4.2 Potential vaccine antigens and virulence factors of NTHI.......................................16 3.5 THE IMPORTANCE OF IRON FOR PATHOGENESIS...............................................................17 3.5.1 Iron metabolism in bacteria – NTHI and M. catarrhalis............................................18 3.5.2 Iron‐regulated proteins as potential vaccine candidates..........................................20 3.6 DEFINING THE ANTIGENOME OF M. CATARRHALIS AND NTHI.......................................21 3.7 AIM OF THE STUDIES ...........................................................................................................24 4 MATERIALS AND METHODS.................................................................................. 25 4.1 BACTERIAL STRAINS AND PLASMID CONSTRUCTS ...........................................................25 4.2 GENERAL METHODS ............................................................................................................26 4.2.1 SDS‐Polyacrylamide gel electrophoresis....................................................................26 4.2.2 Agarose gel electrophoresis..........................................................................................26 I ____________________________________________________________Table of contents 4.2.3 Western blot analysis ....................................................................................................26 4.2.4 Southern blot analysis...................................................................................................27 4.2.5 Isolation of genomic DNA............................................................................................27 4.2.6 Isolation of RNA ............................................................................................................28 4.2.7 PCR ..................................................................................................................................28 4.3 M. CATARRHALIS GROWTH CONDITIONS...........................................................................28 4.3.1 Autoagglutination of M. catarrhalis .............................................................................29 4.4 NTHI GROWTH CONDITIONS ............................................................................................29 4.5 PREPARATION OF BACTERIAL LYSATES ..............................................................................30 4.6 PROTEOMIC ANALYSIS ........................................................................................................30 4.6.1 Preparation of whole membranes from M. catarrhalis and NTHI ...........................30 4.6.2 Preparation of outer membrane vesicles from M. catarrhalis...................................30 4.6.3 Preparation of the outer membrane from NTHI .......................................................30 4.6.4 Mass spectrometry.........................................................................................................31 4.7 CLONING OF ANTIGENS FOR RECOMBINANT EXPRESSION ...............................................31 4.8 RECOMBINANT EXPRESSION OF ANTIGENS IN E. COLI......................................................32 4.9 GENERATION OF MOUSE HYPERIMMUNE SERA AGAINST M. CATARRHALIS AND NTHI RECOMBINANT PROTEINS ...................................................................................................33 4.10 DETERMINATION OF ANTIBODY TITRES BY ELISA...........................................................33 4.11 FLOW CYTOMETRY...............................................................................................................33 4.12 M. CATARRHALIS GENE DELETION MUTANTS ....................................................................34 4.12.1 Generation of M. catarrhalis gene deletion mutants ..................................................34 4.12.2 Antibiotic sensitivity test ..............................................................................................34 4.12.3 M. catarrhalis complementation using plasmid pEMCJH04‐KAN..........................34 4.13 CHARACTERIZATION OF MSP22.........................................................................................35 4.13.1 Cloning of msp22 for expression in M. catarrhalis......................................................35 II ____________________________________________________________Table of contents 4.13.2 Purification of Msp22 from M. catarrhalis...................................................................35 4.13.3 Determination of heme‐dependent peroxidase activity...........................................36 4.14 GENERATION OF NTHI GENE DELETION MUTANT HXUAΔ .............................................37 4.15 PROTECTION STUDIES .........................................................................................................37 4.15.1 Intranasal immunization ..............................................................................................37 4.15.2 Mouse challenge ............................................................................................................37 4.15.3 Euthanasia, tissue collection and bacterial culture ...................................................38 4.16 MICROARRAY ANALYSIS.....................................................................................................38 4.16.1 NimbleGen 4x72K array design...................................................................................38 4.16.2 cDNA synthesis and labeling.......................................................................................38 4.16.3 Hybridization of labeled cDNA with 4x72K array and array scanning.................39 4.16.4 Evaluation of Microarray data.....................................................................................39 4.16.5 Quantitative RT‐PCR.....................................................................................................40 4.17 PRIMER ORDER AND SEQUENCING.....................................................................................40 5 RESULTS M. CATARRHALIS..................................................................................... 41 5.1 THE MEMBRANE PROTEOME OF M. CATARRHALIS SHOWS OVERLAP WITH THE ANTIGENOME .......................................................................................................................41 5.2 M. CATARRHALIS GROWTH IS SIGNIFICANTLY DIMINISHED DURING IRON‐LIMITING CONDITIONS AND CAN BE RESTORED BY IRON REPLETION ..............................................44 5.3 OUTER MEMBRANE VESICLES ISOLATED FROM M. CATARRHALIS CULTURES CONTAIN IRON‐REPRESSIBLE AND IRON‐INDUCIBLE PROTEINS .......................................................45 5.4 IN VIVO AND IN VITRO EVALUATION OF M. CATARRHALIS CANDIDATE ANTIGENS ........48 5.4.1 Recombinant expression of M. catarrhalis proteins in E. coli....................................48 5.4.2 Identification of three protective candidates .............................................................49 5.4.3 In vitro expression of M. catarrhalis candidate antigens............................................52 III ____________________________________________________________Table of contents 5.5 CHARACTERIZATION OF M. CATARRHALIS GENE DELETION MUTANTS...........................53 5.5.1 Generation and confirmation of gene deletion mutants in M. catarrhalis ..............53 5.5.2 M. catarrhalis gene deletion mutants do not show increased sensitivity to selected antibiotics........................................................................................................................57
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