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Proteome Characterization of Brachyspira Strains

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Proteome Characterization of Brachyspira Strains ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Department of Cellular Biology, Physiology and Immunology Doctoral Program in Immunology Proteome characterization of Brachyspira strains. Identification of bacterial antigens. Doctoral Thesis Mª Vanessa Casas López Bellaterra, July 2017 Department of Cellular Biology, Physiology and Immunology Doctoral Program in Immunology Proteome characterization of Brachyspira strains. Identification of bacterial antigens. Doctoral thesis presented by Mª Vanessa Casas López To obtain the Ph.D. in Immunology This work has been carried out in the Proteomics Laboratory CSIC/UAB under the supervision of Dr. Joaquin Abián and Dra. Montserrat Carrascal. Ph.D. Candidate Ph.D. Supervisor Mª Vanessa Casas López Dr. Joaquin Abián Moñux CSIC Research Scientist Department Tutor Ph.D. Supervisor Dra. Dolores Jaraquemada Pérez de Dra. Montserrat Carrascal Pérez Guzmán CSIC Tenured Scientist UAB Immunology Professor Bellaterra, July 2017 “At My Most Beautiful” R.E.M. from the album “Up” (1998) “And after all, you’re my wonderwall” Oasis from the album “(What´s the Story?) Morning Glory” (1995) Agradecimientos A mis directores de tesis, por su tiempo, sus ideas y consejos. Por participar en esta aventura bacteriana que ha sido complicada en muchos aspectos. A toda la gente con la que he compartido laboratorio estos años. De todos y cada uno he aprendido algo. A todas las personas que han contribuido a esta tesis, con su trabajo o con sus ánimos. Por los paseos, llamadas, abrazos, notas en post-its, y eterna paciencia. A miña familia, por lembrarme sempre o que realmente é importante. INFINITAS GRACIAS!!! “…The older I get, the more I appreciate the difference between the results of scientific investigations and the methods by which the results are obtained. The results constitute the body of scientific knowledge- they are science. But, as has been noted by others, the facts of science exist in nature and are waiting to be discovered; if not found by one investigator, they will be found by another. The methods, however, are the creations of individual scientists; they are more art than science. For this reason, and depending on one's mood, they may be even more admirable than the science they make possible…” NORMAN H. HOROWITZ One-gene-one-enzyme: Remembering biochemical genetics. Protein Sci. 4, 1017–1019 (1995). Index Abbreviations ................................................................................................................................... 1 1. Introduction ................................................................................................................................. 3 1.1 Brachyspira ............................................................................................................................. 5 1.1.1 Spirochaetes .................................................................................................................... 5 1.1.1.1 Brachyspira hyodysenteriae ....................................................................... 6 1.1.1.2 Brachyspira pilosicoli .................................................................................. 8 1.1.2 Diseases ......................................................................................................................... 11 1.1.2.1 Swine dysentery ....................................................................................... 11 1.1.2.2 Human intestinal spirochetosis ................................................................ 12 1.1.3 Treatment and control .................................................................................................. 13 1.1.3.1 Antibiotics ................................................................................................. 13 1.1.3.2 Vaccines .................................................................................................... 13 1.1.4 Antimicrobial resistance ................................................................................................ 15 1.2 Bacterial proteomics ............................................................................................................. 17 1.2.1 Proteomes and subproteomes ...................................................................................... 17 1.2.1.1 Surface proteome ..................................................................................... 18 1.2.1.2 Extracellular proteome ............................................................................. 19 1.2.1.3 Immunoproteome .................................................................................... 22 1.2.2 Bacterial PTMs ............................................................................................................... 24 1.2.2.1 Acetylation ............................................................................................... 25 1.2.2.2 Phosphorylation ....................................................................................... 26 1.2.2.3 Methylation .............................................................................................. 27 1.2.2.4 Other PTMs............................................................................................... 28 1.2.3 Antimicrobial resistance and proteomics...................................................................... 28 1.3 Brachyspira and proteomics ................................................................................................. 29 1.3.1 Flagellar proteins ........................................................................................................... 30 1.3.2 Outer membrane proteins ............................................................................................ 32 2. Justification ................................................................................................................................ 35 3. Paper#1 Bacterial Proteome ..................................................................................................... 41 4. Paper#2 Exposed Proteome ...................................................................................................... 87 5. Paper#3 Immunoproteome ..................................................................................................... 109 6. General Discussion ................................................................................................................... 127 6.1 Providing Experimental evidence for Brachyspira proteins ................................................ 129 6.2 Post-translational Modifications ......................................................................................... 131 6.2.1 The case of acetylation ................................................................................................ 134 6.3 Vsp proteins ........................................................................................................................ 138 6.4 Immunoreactive proteins.................................................................................................... 141 6.4.1 Immunoreactive proteins common to both species.................................................... 141 6.4.2 B. pilosicoli immunoreactive proteins dataset is enriched in HSPC ............................. 143 6.4.3 B. hyodysenteriae immunoreactive proteins .............................................................. 144 6.4.4 Immunoreactive proteins proposed as vaccine components ..................................... 148 7. Conclusions ............................................................................................................................... 151 8. Bibliography .............................................................................................................................. 155 Abbreviations 2D Two-dimensional ACP Acetyl-Phosphate ELISA Enzyme-linked immunosorbent assay FASP Filter-aided sample preparation GAPDH Glyceraldehyde-3-phosphate dehydrogenase GTA Gene transfer agent HIS Human intestinal spirochetosis HPLC High-performance liquid chromatography HSP Heat shock protein HSPC Heat shock protein complex KEGG Kyoto Encyclopedia of Genes and Genomes LC-MS/MS Liquid chromatography coupled to tandem mass spectrometry LOS Lipooligosaccharides LPS Lipopolysaccharide MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight MGF Mascot generic format OMP Outer membrane protein OMV Outer membrane vesicle ORF Open reading frame PAGE Polyacrylamide gel electrophoresis PEPCK Phosphoenolpyruvate carboxykinase PIS Pig intestinal spirochetosis PSM Peptide spectrum match PTM Post-translational modification SAS Single aminoacid substitution SERPA Serological proteome analysis Vsp Variable surface protein 1 1. Introduction Introduction 1.1 Brachyspira 1.1.1 Spirochaetes The species of the genus Brachyspira (formerly Treponema or Serpulina) are anaerobic bacteria classified within the phylum Spirochaetes (Stanton, 2006). Spirochaetes (aka spirochetes)
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