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Proteins Involved in Host-Pathogen Recognition

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Proteins Involved in Host-Pathogen Recognition MASARYK UNIVERSITY Faculty of Science National Centre for Biomolecular Research PROTEINS INVOLVED IN HOST-PATHOGEN RECOGNITION EVA FUJDIAROVÁ Ph.D. Thesis Supervisor: Prof. RNDr. Michaela Wimmerová, Ph.D. Brno 2020 BIBLIOGRAFICKÝ ZÁZNAM Autor: MVDr. Eva Fujdiarová Masarykova univerzita, Přírodovědecká fakulta Národní centrum pro výzkum biomolekul, Název práce: Proteiny zapojené do rozpoznávání patogenu hostitelem Studijní program: Biomolekulární chemie a bioinformatika Vedoucí práce: Prof. RNDr. Michaela Wimmerová, Ph.D. Přírodovědecká fakulta, Masarykova univerzita Ústav biochemie a Národní centrum pro výzkum biomolekul - NCBR, Středoevropský technologický institut – CEITEC, Masarykova univerzita Akademický rok: 2019/2020 Počet stran: 138+165 Klíčová slova: Lektiny, sacharidy, Photorhabuds laumondii, reaktivní formy kyslíku, fenoloxidáza, multivalence BIBLIOGRAPHIC ENTRY Author: MVDr. Eva Fujdiarová National Centre for Biomolecular Research, Faculty of Science, Masaryk University Title of Thesis: Proteins involved in host-pathogen interaction Degree programme: Biomolecular chemistry and bioinformatics Field of Study: Biomolecular chemistry and bioinformatics Supervisor: Prof. RNDr. Michaela Wimmerová, Ph.D. Masaryk University, Faculty of Science, Department of Biochemistry and National Centre for Biomolecular Research Central European Institute of Technology, Masaryk University Academic year: 2019/2020 Number of Pages: 138+165 Keywords: Lectins, saccharides, Photorhabdus laumondii, reactive oxygen species, phenoloxidase, multivalency ACKNOWLEDGMENT My journey to biomolecular chemistry was not straightforward. I studied to be a veterinarian. Although I did enjoy the biochemistry classes, I threw away all my notes shortly after graduation thinking I would never need them again. I remember myself during all those lab practical seminaries thinking – I am not studying such a demanding field only to be pipetting in a lab. I wanted to be a surgeon, to save animals and gain people’s respect – as all of us did. And look at me now 10 years later – happily pipetting. In this place, I would like to thank everyone who has been there for me in this chapter of my life. I thank my parents and family for supporting my crazy idea to move to Brno and switch fields, for hiding their fears and doubts from me and telling only: you can do it, and there is always a place for you here. Their belief in me and the feeling of having a safe place to return helped me to face all challenges that doctoral study brings. I thank my supervisor professor Wimmerová for allowing me to learn and grow independently in her lab while watching my progress from distance, letting me go through all the necessary mistakes one needs to do in order to learn. But I thank her also for stepping forward whenever necessary, providing support, counsel, and insight I did not have, not letting me to walk in circles for too long. I thank Janča Vičanová and my shifu Dou Wanchun for English corrections of this text. I thank Pepa, Lenka, Janča, Jitka, Majka and all members of the Glyco group for creating the environment where it is not a shame to ask stupid questions and where co-workers become friends. I thank my friends for simply being. For traditions we have created during the years that I love so much – the movie club, Travný expeditions, TMOU!, boat trips… A special thanks to Hanka for our Dominion sessions, Andula for many hiking memories, and all people connected to “Mycelium” for making me feel I belong somewhere. Thank you all for reminding me that there are more ways to see the world and that it is important to keep things in perspective, especially when you are digging in proteins on an atomic level. And one special note for my little nephew: MITO MITO! I hereby declare that the thesis “Proteins involved in host-pathogen recognition“, was written by me under the guidance of the thesis supervisor and with the use of literature sources. Eva Fujdiarová ABSTRAKT Lektiny jsou skupina proteinů neimunitního původu, které rozeznávají sacharidy s neobyčejně vysokou specifitou. Díky této vlastnosti jsou lektiny ideálním nástrojem pro čtení “cukerného kódu”, který se nachází na povrchu všech buněk zapsán do struktury specifických cukerných epitopů. Lektiny zprostředkovávají buněčnou komunikaci na molekulární úrovni a jsou zapojeny do mnoha fyziologických i patofyziologických procesů. Patogenní bakterie a viry využívají lektiny k přichycení na hostitelskou tkáň, což je jeden z předpokladů pro rozvoj infekce. Blokace adheze patogenů specifickými lektinovými inhibitory je základem anti-adhezivní terapie, alternativního přistupu k léčbě infekcí způsobených multirezistentními bakteriálními kmeny. Tato dizertační práce je zaměřena převážně na studium lektinů z bakterie Photorhabdus laumondii. Tato bakterie má komplexní životní cyklus, který zahrnuje fázi mutualismu s mikroskopickou hlísticí rodu Heterorhabditis a také fázi patogenity vůči hmyzu. Bakterie ve svém genomu kóduje lektiny, jejichž přesná role v životním cyklu bakterie není dosud známá. Detailní charakterizace lektinů, jejich struktury a vazebných vlastností, stejně tak i studium delečních bakteriálních mutantů, může na tuto otázku odpovědět. Obsahem dizertační práce je také testování účinnosti multivalentních cukerných inhibitorů, zacílených na blokaci lidských patogenů jako Pseudomonas aeruginosa, Burkholderia cenocepacia a Photorhabdus asymbiotica. Text dizertační práce je rozdělen na teoretický úvod a praktickou část. Teoretický úvod je zaměřený na problematiku lektinů, antiadhezivní terapie a rodu Photorhabdus. V praktické části práce jsou popsány tři projekty, do kterých jsem byla v rámci studia zapojena a jsou v ní shrnuty dosažené výsledky. ABSTRACT Lectins are a group of proteins of non-immune origin that recognize carbohydrates with extremely high specificity. Due to this property, lectins are the ideal tool for reading a glyco code which is found on the surface of every cell and is encoded in the structure of specific sugar epitopes. Lectins mediate cellular communication at the molecular level and are involved in many physiological and pathophysiological processes. Pathogenic bacteria and viruses use lectins to attach to host tissues, which is one of the prerequisites for infection development. Blocking the adhesion of pathogens by specific lectin inhibitors is the basis of anti-adhesion therapy, an alternative approach to the treatment of infections caused by multi-resistant bacterial strains. This thesis is focused mainly on the study of lectins from Photorhabdus laumondii. This bacterium has a complex life cycle that includes the phase of mutualism with the microscopic nematode of the genus Heterorhabditis and also the phase of pathogenicity towards insects. The bacterium encodes lectins in its genome. Their exact role in the bacterial life cycle is not yet known. The detailed characterization of lectins, their structure, and binding properties, as well as the study of deletion bacterial mutants, may answer this question. The content of this thesis is also testing the effectiveness of multivalent sugar inhibitors aimed at blocking human pathogens such as Pseudomonas aeruginosa, Burkholderia ceanocepatia, and Photorhabdus asymbiotica. The text of the thesis is divided into a theoretical introduction and a practical part. The theoretical introduction is focused on lectins, anti-adhesion therapy, and Photorhabdus genus. In the practical part of the thesis, there are three projects, which I was involved in during the study, described and the results are summarized. ABBREVIATIONS 3OMG 3-O-methyl-D-glucose AFP alpha-fetoprotein AUC analytical ultracentrifugation CA15-3 carbohydrate antigen associated with breast cancer CRDs carbohydrate-binding domains CRP C-reactive protein CuAAC copper-catalyzed azide-alkyne cycloaddition DMSO dimethyl sulfoxide EPNs entomopathogenic nematodes fMLF N-formyl-L-methionyl-L-leucyl-L-phenylalanine HA hemagglutinin HBSS Hank`s balanced salt solution HIA hemagglutination inhibition assay IJ infective juvenile IPTG isopropyl-β-D-1-thiogalactoside ITC isothermal titration calorimetry LCA Lens culinaris agglutinin MIC minimal inhibitory concentration MBL mannose-binding lectin MGMR 3,6-O-Me2-D-Glcβ1-4(2,3-O-Me2)-L-Rhaα MRSA methicillin-resistant Staphylococcus aureus RNA ribonucleic acid ROS reactive oxygen species SAP serum amyloid P SNR signal to noise ratio sp2 linker -O-(p-C6H4)-O-CH2CH2NH2 SPR surface plasmon resonance STD NMR saturation transfer difference nuclear magnetic resonance P1 primary variant of Photorhabdus (phase one) P2 secondary variant of Photorhabdus (phase two) PCR polymerase chain reaction PHA Phaseolus vulgaris agglutinin PMA phorbol 12-myristate 13-acetate PRSA penicillin-resistant Staphylococcus aureus PO phenoloxidase pPO prophenoloxidase UTIs urinary tract infections VRSA vancomycin-resistant Staphylococcus aureus WGA wheat germ agglutinin TABLE OF CONTENT BIBLIOGRAFICKÝ ZÁZNAM ................................................................................. 3 BIBLIOGRAPHIC ENTRY ........................................................................................ 5 ACKNOWLEDGMENT ............................................................................................. 7 ABSTRAKT .............................................................................................................. 11 ABSTRACT .............................................................................................................. 13 ABBREVIATIONS ..................................................................................................
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