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Towards Functional Assignment of Plasmodium Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins A thesis submitted for the degree of Doctor of Philosophy of The Australian National University and Humboldt University of Berlin by François Laurent Frederic Korbmacher Submitted July 2020 © Copyright by François Laurent Frederic Korbmacher, 2020 All Rights Reserved Declaration The research presented in this thesis, except where otherwise acknowledged, is my own original work. Research conducted in cooperation is noted. The study was performed at the Australian National University under supervision of Prof. Dr. Alex G. Maier and at the Humboldt University of Berlin under supervision of Prof. Dr. Kai Matuschewski within the frame of a Cotutelle-degree by the IRTG2290 graduate program. No portion of the work presented in this thesis has been submitted for another degree. The word count for this thesis excluding tables, figures, bibliographies and appendices is 43,156 words. François Laurent Frederic Korbmacher Berlin, July 2020 Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins Dissertation Zur Erlangung des akademischen Grades Doctor of Philosophy (PhD) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt- Universität zu Berlin und der Australian National University von François Laurent Frederic Korbmacher geb. am 20.06.1990 in Neustadt an der Weinstraße Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Bernhard Grimm Gutachter: Vorsitz: Prof. Dr. Christian Schmitz-Linneweber 1. Prof. Dr. Kai Matuschewski 2. Prof. Dr. Alex Maier 3. Prof. Tania De Koning-Ward 4. Prof. Dr. Simone Reber 5. Dr. Jandeli Niemand Tag der mündlichen Prüfung: 10.12.2020 Summary Membrane transport proteins (MTPs) transfer nutrients, metabolic products and inorganic ions across membranes. Many MTPs are essential for Plasmodium blood infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions of many MTPs still remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four selected Plasmodium MTPs, including spatio-temporal expression and phenotypical analysis of knockout mutants. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of the human malaria parasite Plasmodium falciparum. For this study, we selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4, with important roles in anti-malarial therapy. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles for Plasmodium life cycle progression. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Strikingly, in two stages, schizonts and sporozoites, FT2 displays a dual localisation. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Accordingly, very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, that is encoded by human malaria parasites, but not P. berghei and related Vinckeia parasites, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry- tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. Together, the findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion. i Zusammenfassung Membran Transport Proteine (MTPs) transferieren Nährstoffe, metabolische Produkte und anorganische Ionen über Membranen. Da etliche MTPs in den Plasmodium Blutstadien essentiell sind, geraten sie zunehmend in den Fokus der Wirkstoffentwicklung. Die eigentlichen physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika, wie zeitliche und räumliche Expressionsmuster sowie phänotypische Analysen von Mutanten untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum- spezifisches P-Typ ATPase und zwei essentielle MTPs mit wichtigen Rollen in der Malaria-Therapie (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten für Wirkstoffziele und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, eine doppelte Lokalisierung in Schizonten und Sporozoiten, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation im Vektor auf: der Großteil der ft2– Oozyten formt große, intrazelluläre und Zytoplasma verdrängende Vesikel. Demzufolge bilden sich nur sehr wenige und nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine vermeintliche Aminophospholipid P-Typ ATPase, für die kein kodierendes Gen in der P. berghei mit inbegriffenen Vinckeia Klade auftritt, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei. ii Table of contents 1 st Chapter: General Introduction ........................................................................... 1 1.1 Plasmodium and malaria ...................................................................................... 1 1.2 Life cycle of the Plasmodium parasite ................................................................ 1 1.3 Membrane Transport Proteins in Plasmodium ................................................... 4 1.4 Towards a functional classification of MTPs ...................................................... 6 1.5 Aims of the study .................................................................................................. 8 2 nd Chapter: Materials and Methods ....................................................................... 9 2.1 Materials ................................................................................................................ 9 2.1.1 Experimental organisms ............................................................................................................. 9 2.1.2 Plasmodium targeting vectors .................................................................................................... 9 2.1.3 Enzymes ...................................................................................................................................10 2.1.4 Antibodies ................................................................................................................................10 2.1.5 Media, buffers, stock and working solutions..............................................................................10 2.1.6 Laboratory equipment ..............................................................................................................11 2.1.7 Consumables ............................................................................................................................12 2.1.8 Commercial kits ........................................................................................................................13 2.1.9 Chemicals .................................................................................................................................13 2.1.10 Software and databases ............................................................................................................15 2.2 Methods ............................................................................................................... 15 2.2.1 Molecular- and microbiological methods for P. berghei and P. falciparum .................................15 2.2.1.1 Polymerase Chain Reaction (PCR).......................................................................................15
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