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Diplomarbeit DIPLOMARBEIT Design and development of an immune dependent system for the detection of antibodies against R. conorii, R. helvetica and R. slovaca and screening for specific antibodies. zur Erlangung des akademischen Grades Magister der Naturwissenschaften (Mag. rer.nat.) Verfasserin / Verfasser: Zutz Christoph Matrikel-Nummer: 0200576 Studienrichtung /Studienzweig A441 (lt. Studienblatt): Betreuerin / Betreuer: Prof. Stanek Wien, im 14.04.2009 Table of contents 1. Abbreviations ..................................................................................................................................1 2. Abstract ...........................................................................................................................................3 3. Introduction ....................................................................................................................................5 3.1. History of rickettsiology ..........................................................................................................5 3.1.1. Rickettsia rickettsii agent of the RMSF ........................................................................5 3.2. Phylogeny of rickettsia ............................................................................................................6 3.2.1. Genus Rickettsia ..........................................................................................................6 3.2.2. Phylogenetic markers pf rickettsiae and techniques...................................................8 3.2.3. RFLP .............................................................................................................................8 3.2.4. MST..............................................................................................................................8 3.2.5. A phylogenetic tree for rickettsiae ..............................................................................9 3.3. Morphology...........................................................................................................................11 3.3.1. Mitochondrial ancestor role of the genus Rickettsia ....................................................12 3.3.2. Cell wall structure..........................................................................................................13 3.3.3. Replication mechanism .................................................................................................14 3.3.4. Cell migration ................................................................................................................15 3.4. Metabolism ...........................................................................................................................17 3.4.1. Energy Transport in rickettsia........................................................................................17 3.4.1.1. ATP/ ADP transport ...............................................................................................17 3.4.1.2. AMP transport .......................................................................................................18 3.4.1.3. NAD+ transport......................................................................................................18 3.4.2. Polyamine metabolism in rickettsiae ............................................................................19 3.4.3. Amino acid transport and metabolism in rickettsiae ....................................................20 3.4.3.1. Proline ...................................................................................................................20 3.4.3.2. Serine.....................................................................................................................20 3.4.3.3. Glutamine..............................................................................................................21 3.4.3.4. Other amino acids .................................................................................................21 3.4.4. Glycolysis and TCA cycle in rickettsia ............................................................................21 3.4.5. Potassium acquisition in rickettsia ................................................................................22 3.4.6. Genome of rickettsiae ...................................................................................................22 3.4.6.1. Rickettsia bellii.......................................................................................................24 3.4.6.2. Selfish repeats .......................................................................................................24 page I 3.4.6.3. Evolution in rickettsiae or reductive genome........................................................24 3.4.6.4. metK gene in the process of degeneration ...........................................................26 3.4.6.5. Evolution of the rickettsial mother........................................................................26 3.5. Clinical relevance and pathogenesis .....................................................................................27 3.5.1. Pathogenesis of rickettsiae............................................................................................27 3.5.2. Adherence to the host cell ............................................................................................27 3.5.3. Invasion of host cells .....................................................................................................28 3.5.4. Medical relevance .........................................................................................................29 3.5.5. Treatment of rickettsioses.............................................................................................30 3.6. Aim ........................................................................................................................................31 3.6.1. Rickettsia conorii ...........................................................................................................31 3.6.1.1. Rickettsia conorii complex.....................................................................................32 3.6.2. Rickettsia helvetica........................................................................................................32 3.6.2.1. First confirmed cases of Rickettsioses by R.helvetica............................................33 3.6.3. Rickettsia slovaca ..........................................................................................................34 3.6.3.1. First confirmed cases with Rickettsia slovaca........................................................34 4. Material and Methods...................................................................................................................35 4.1. Genomic material..................................................................................................................35 4.1.1. Human serum samples..................................................................................................35 4.2. Methods ................................................................................................................................35 4.2.1. In silico methods............................................................................................................35 4.2.1.1. Selecting the antigen of interest for the primer design ........................................35 4.2.1.2. Assorting a panel of test sera ................................................................................36 4.2.1.3. Biostatistics............................................................................................................36 4.2.2. DNA manipulation techniques ......................................................................................37 4.2.2.1. PCR ........................................................................................................................37 4.2.2.2. Real time PCR ........................................................................................................37 4.2.3. DNA purification and separation methods....................................................................38 4.2.3.1. Separation by agarose gel electrophoresis............................................................38 4.2.3.2. Purification of DNA from agarose gel....................................................................39 4.2.3.3. Purification of plasmidic DNA from cell culture ....................................................39 4.2.3.4. Sequencing of the DNA fragments ........................................................................40 4.2.4. Cloning and expression techniques...............................................................................40 4.2.4.1. A‐Addition .............................................................................................................40 4.2.4.2. Ligation with pQE‐30 UA .......................................................................................41 page II 4.2.4.3. Bacterial strain.......................................................................................................41 4.2.4.4. The vector plasmid and the inhibition plasmid of the expression system ............41 4.2.4.5. Preparation of competent E. coli...........................................................................42 4.2.4.6. Transformation of competent M15 cells...............................................................43 4.2.4.7. E.coli culture growth and inducing
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