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DIFFERENTIAL GENE EXPRESSION of Piscirickettsia Salmonis

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DIFFERENTIAL GENE EXPRESSION of Piscirickettsia Salmonis DIFFERENTIAL GENE EXPRESSION OF Piscirickettsia salmonis STRAINS IN ADAPTIVE RESPONSE TO DIFFERENT CULTURE SYSTEMS: A STRATEGY TO IDENTIFY POSSIBLE INTRACELLULAR SURVIVAL MECHANISMS AND PATHOGENICITY DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG Vorgelegt von Tamara Scarlett Vera Sepúlveda Aus Valdivia, Chile 2013 Das Promotionsgesuch wurde eingereicht am: 1.08.2013 Die Arbeit wurde angeleitet von: Dr. Gernot Längst Dr. Jaime Figueroa Die vorgelegte Dissertation mit dem Titel DIFFERENTIAL GENE EXPRESSION OF Piscirickettsia salmonis STRAINS IN ADAPTIVE RESPONSE TO DIFFERENT CULTURE SYSTEMS: A STRATEGY TO IDENTIFY POSSIBLE INTRACELLULAR SURVIVAL MECHANISMS AND PATHOGENICITY von Tamara Vera Sepúlveda entstand unter der gemeinsamen Betreuung der Universitaet Regensburg und der Universidad Austral de Chile im Rahmen des bi- nationalen Promotionsprogramms RegenVald als Doppelpromotion. UNIVERSIDAD AUSTRAL DE CHILE FACULTAD DE CIENCIAS DIFFERENTIAL GENE EXPRESSION OF Piscirickettsia salmonis STRAINS IN ADAPTIVE RESPONSE TO DIFFERENT CULTURE SYSTEMS: A STRATEGY TO IDENTIFY POSSIBLE INTRACELLULAR SURVIVAL MECHANISMS AND PATHOGENICITY DOCTORAL THESIS TAMARA SCARLETT VERA SEPÚLVEDA VALDIVIA – CHILE 2013 DIFFERENTIAL GENE EXPRESSION OF Piscirickettsia salmonis STRAINS IN ADAPTIVE RESPONSE TO DIFFERENT CULTURE SYSTEMS: A STRATEGY TO IDENTIFY POSSIBLE INTRACELLULAR SURVIVAL MECHANISMS AND PATHOGENICITY Thesis submitted to the Faculty of Sciences of the Universidad Austral de Chile in partial satisfaction of the requirements for the degree Doctor in Science on Cellular and Molecular Biology. by TAMARA SCARLETT VERA SEPÚLVEDA Valdivia - Chile 2013 A mis padres Eflorcina y Dagoberto por su cariño y apoyo incondicional A Daniel por su amor, comprensión, ayuda y paciencia ACKNOWLEDGEMENTS I want to express my sincere gratitude to Dr. Gernot Längst for his advice, unconditional trust and support since the first day. I sincerely thanks to Dr. Jaime Figueroa for his guidance, encouragement and support during the development of my Doctoral project. I also want to express my gratitude to Dr. Gudrun Kausel for her constant support and confidence, urging me to get a double Doctoral degree at the University of Regensburg. I also thanks to Dr. Jörg Reinders and all the members of his laboratory, for their support during the development of my work. My greetings are also extended to all the members of the Genomics Functional Institute for making me feel really welcome. Finally I want to thank to all people who supported me throughout my Doctoral program, especially my family for their confidence, Carolina for giving me her unconditional friendship and Daniel my husband, for his huge patience and unconditional love. Thank you very much from my heart FUNDING Innova-Chile 07CN13: IBM-259, FONDAP 15110027: "Interdisciplinary Center for Aquaculture Research" (INCAR), CONICYT 24110194. CONICYT Doctoral Fellowship, AUSP0606 Fellowship, AUS0704 Fellowship, RegenVald Fellowship from the program, University of Regensburg, Germany, Completion Fellowship Doctoral Thesis, Department of Postgraduate Studies UACh. I INDEX INDEX .......................................................................................................................... I FIGURES INDEX ........................................................................................................ V TABLES INDEX ......................................................................................................... VII ANNEX INDEX ........................................................................................................... IX LIST OF ABBREVIATIONS ......................................................................................... X RESUMEN ............................................................................................................... XIII ABSTRACT ............................................................................................................... XV 1. INTRODUCTION ..................................................................................................... 1 1.1. PISCIRICKETTSIOSIS (SRS) .............................................................................. 2 1.2. THE BACTERIUM Piscirickettsia salmonis .......................................................... 4 1.3. MICROBICIDAL ACTIVITY OF THE PHAGOSOME ............................................ 7 1.4. INTRACELLULAR SURVIVAL STRATEGIES .................................................... 11 1.5. Gammaproteobacteria INTRACELLULAR SURVIVAL MECHANISMS .............. 14 1.5.1. Francisella tularensis ................................................................................ 14 1.5.2. Legionella pneumophila ........................................................................... 16 1.5.3. Coxiella burnetii ........................................................................................ 18 1.6. HYPOTHESIS AND OBJETIVES ....................................................................... 21 2. MATERIALS AND METHODS ............................................................................... 23 2.1. MATERIALS ....................................................................................................... 23 2.1.1. Equipment and Instruments ..................................................................... 23 2.1.2. Chemical reagents ................................................................................... 24 2.1.3. Solutions .................................................................................................. 26 II 2.1.4. Software and on line tools ........................................................................ 27 2.1.5. Biological material .................................................................................... 27 2.2. METHODS ......................................................................................................... 28 2.2.1. Culture biological material ........................................................................ 28 2.2.1.1. Cell culture .......................................................................................... 28 2.2.1.2. Bacterial culture in cell line ................................................................. 28 2.2.1.3. Bacterial culture in solid medium ........................................................ 28 2.2.1.4. Bacterial culture in liquid medium ....................................................... 29 2.2.2. Proteomic analysis of P. salmonis ............................................................ 29 2.2.2.1. Purification of P. salmonis propagated in cell line SHK-1 ................... 29 2.2.2.2. Indirect Immunofluorescence .............................................................. 30 2.2.2.3. Immunocytochemistry ......................................................................... 30 2.2.2.4. Total protein extraction of P. salmonis ................................................ 31 2.2.2.5. Protein quantification .......................................................................... 31 2.2.2.6. Total protein electrophoresis in one dimension (SDS-PAGE) ............. 32 2.2.2.7. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) ...... 32 2.2.2.8. Two-Dimensional difference gel electrophoresis (2D-DIGE) .............. 33 2.2.2.9. Lava purple staining ........................................................................... 33 2.2.2.10. Silver staining ................................................................................... 33 2.2.2.11. Sample preparing for nano LC-MS-MS ............................................. 34 2.2.3. Transcriptome analyses of P. salmonis .................................................... 34 2.2.3.1. RNA isolation from bacteria grown on supplemented TSB ................. 34 2.2.3.2. RNA isolation from bacteria grown on cell line SHK1 ......................... 35 2.2.3.3. Denaturing agarose gel electrophoresis ............................................. 35 2.2.3.4. Sequencing transcriptome of LF89 and IBM040 ................................. 36 2.2.3.5. Primer design ..................................................................................... 36 2.2.3.6. DNA isolation from bacteria grown on supplemented TSB ................. 39 2.2.3.7. First strand cDNA ............................................................................... 39 2.2.3.8. Polymerase chain Reaction PCR ....................................................... 39 2.2.3.9. Electrophoretic fractionation on agarose gels ..................................... 40 2.2.3.10. qPCR assay ...................................................................................... 40 III 3. RESULTS .............................................................................................................. 41 3.1. PART 1: OPTIMIZATION OF THE SYSTEM UNDER STUDY ........................... 41 3.1.1. Designing a liquid culture medium for optimal growth of P. salmonis ....... 41 3.1.2. Identification of optimal conditions of infection in cell line SHK1 .............. 49 3.1.3. Purification of bacteria from an infected cell line ...................................... 53 3.2. PART 2: PROTEOMIC ANALYSIS OF LF89 STRAIN CULTIVATED IN CELL- FREE MEDIUM VERSUS GROWN IN SHK1CELL LINE .......................................... 55 3.2.1 Differential expression of proteins in P. salmonis, grown in a cell-free medium or
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