Functional Characterization of Actin Sequestering Proteins in Plasmodium berghei [Elektronische Ressource] / Marion Hliscs. Gutachter: Richard Lucius ; Kai Matuschewski ; Elena A. Levashina

Marion Hliscs

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2012
• Nombre de lectures: 105
• Langue: Deutsch
• Poids de l'ouvrage: 15 Mo

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Functional Characterization of Actin Sequestering
Proteins in Plasmodium berghei

D i s s e r t a t i o n

Zur Erlangung des akademischen Grades

d o c t o r r e r u m n a t u r a l i u m

(Dr. rer. nat.)

im Fach Biologie

eingereicht an der

Mathematischen-Naturwissenschaftlichen Fakultät I

Der Humboldt-Universität zu Berlin

von

Dipl. Biol. Marion Hliscs



Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Jan-Hendrik Olberts


Dekan der Mathematischen-Naturwissenschaftlichen Fakultät I

Prof. Dr. Andreas Herrmann


1. Gutachter: Prof. Dr. Richard Lucius

2. Gutachter: Prof. Dr. Kai Matuschewski

3. Gutachter: Ph. D., D. Habil. Elena A. Levashina



Tag der mündlichen Prüfung: 17.08.2011






























EIDESSTATTLICHE ERKLÄRUNG

Hiermit erkläre ich an Eides statt, die vorliegende Dissertation selbstständig angefertigt und
keine anderen als die angegebenen Hilfsmittel verwendet zu haben.

Ich erkläre hiermit, dass ich an keiner anderen Universität ein Prüfungsverfahren beantragt
bzw. die Dissertation in dieser oder anderer Form bereits anderweitig als Prüfungsarbeit
verwendet oder einer anderen Fakultät als Dissertation vorgelegt habe.

Die vorliegende Arbeit wurde am Max Planck Institut für Infektionsbiologie, Berlin und am
Hygiene Institut, Abteilung Parasitologie der Karls-Ruprechts Universität, unter Leitung von
Prof. Dr. Kai Matuschewski durchgeführt.






Berlin, den 05.07.2011 Marion Hliscs
























ACKNOWLEDGEMENTS

In the first place I would like to express my gratitude my supervisor Prof. Dr. Kai
Matuschewski, who gave me the big opportunity to work in his lab. His tremendous and
inventive support as well as his great openness regarding new ideas account importantly for
my achieved work. He generously provided international congress participation and
internship, which were always an exciting challenge and motivated me even more.

I would like to thank Prof. Dr. Richard Lucius for his support as my doctoral thesis supervisor
at the Humboldt University and his collaboration and generosity, for using the nice
microscope.
I would like to thank Prof. Dr. Michael Lanzer for the opportunity to start my work at the
department of parasitology at the hygiene institute of Heidelberg.

I am grateful to Julia Sattler and Herwig Schüler for the great innovative and fruitful
collaboration, for sharing knowledge and thoughts as well as antibodies and proteins. I wish
we had more time working together.
I would like to thank to Nishith Gupta and Martin Blume for the prosperous collaboration and
thereby bringing together research of two apicomplexan parasites.
I would like to thank to Elena Levashina and Guilla Costa for open-mindedness and testing
mutant parasites and hopefully continuation of collaboration.
I would like to thank to Marcelo Jacobs-Lorena for kindly providing the CAP380 antibody.

It is a pleasure to thank all present and past members of the Matuschewskis laboratory for
motivation, helpfulness and also many funny moments that created a friendly working
environment: Katja B., Kaja M., Johanna, Caro, Johannes, Alyssa, Elyzana, Fifi, Jan,
Sanketha, Manuel, Taco, Faustin, Diana, Vittoria. Also, a special thanks to Georgina
Montagna, Olivier Silvie and Markus Ganter for inspiring discussions and supervision.
I would like to thank Ulrike Abuabed for the nice intricate transmission electron microscope
pictures, which really contribute to the understanding what is going on in the parasite.


Additionally, I would like to thank Freddy Frischknecht, who provided the initial steps for
working with this interesting parasite, and Stephan Hegge, Michael Kudriashev and Luis
Barniol for the great time back in Heidelberg.

A big thank is for my dear friends, whose big support and appreciation over the last time
helped me to manage this work; Nadja, Jana, Barbara, Anne, Wiebke, Stine, Katja and Anna.

Martin, I deeply thank you for your appreciation, patience and encouragement over the last
time, which helped me to manage this work. I am happily looking forward sharing future
plans with you.

From great heart I thank my family Eva and Reiner Hliscs, Thomas with family and Rudolf
with family, which always supported me in any condition and their encouragement and love
enables me to proceed to and to break new grounds.


SUMMARY

Plasmodium spp. are apicomplexan parasites that rely on an obligate intracellular life-style.
They depend on an evolutionary conserved actin-dependent molecular motor machinery that
facilitates their motility, host cell invasion and egress. Furthermore parasite actin is implicated
in endocytosis during blood stages. However, the molecular mechanisms that are involved in
the spatiotemporal regulation of actin-turnover are not yet understood. In this work I report
implications of the actin-regulators adenylyl cyclase-associated protein (C-CAP), profilin and
actin depolymerization factor 1 and 2 (ADF1, ADF2) in distinct and previously unanticipated
cellular processes during the life cycle of in the rodent malarial parasite Plasmodium berghei.
By using a reverse genetic approach I tagged the endogenous C-CAP genetic locus to a
fluorescent reporter. C-CAPmCherry localizes in the cytosol, which is consistent with its
proposed G-actin sequestering activity. Gene deletion demonstrates that the G-actin binding
protein C-CAP is entirely dispensable for the pathogenic blood stages and ookinete
development. In in vitro motility assays C-cap(-) ookinetes exhibit a modestly impaired
motility that indicates involvement of C-CAP in actin-turnover. Nevertheless, these parasites
are able to infect the mosquito host. Unexpectedly however, the c-cap(-) mutant fails to
complete oocyst maturation. Defects that emerge in this unique extracellular and non-motile
replication phase include attenuation of oocyts growth, absence of oocyst compartmentation
and impeded nuclear divisions. These findings indicate previously unrecognized functions of
the C-CAP protein in the malarial parasites. Successful trans-species complementation with
the C. parvum C-CAP ortholog, rescues the c-cap(-) phenotype and proves functional
redundancy between apicomplexan CAP proteins. Furthermore, the actin regulator profilin
fails to rescue the defects of c-cap(-) parasites, despite sharing its actin sequestering activity
with C-CAP. Taken together, C-CAP is the first G-actin sequestering protein of Plasmodium
species that is not required for motility but performs essential functions during oocyst
maturation instead.
Characterization of the actin regulators profilin, ADF1 and ADF2 revealed dramatic
transcriptional down-regulation and the absence of the profilin protein in sporozoites. To test
whether G-actin binding proteins interfere with sporozoite functions, I ectopically
overexpressed of profilin and C-CAP stage-specifically in sporozoites. In these recombinant
parasites, but not in control lines, demonstrate that overexpression abolishes salivary gland
invasion and arrests their lifecycle.
1Based on these unexpected findings and the available literature data, I developed a
“minimalistic model” for actin regulation in sporozoites that predicts ADF1 as the main actin-
turnover regulating factor.
2ZUSAMMENFASSUNG

Plasmodien spp. sind einzellige eukaryotische Parasiten, welche sich mittels eines evolutionär
konservierten und aktinabhängigen molekularen Motors fortbewegen. Diese spezielle Art der
Fortbewegung ermöglicht dem Parasiten einen aktiven Wirtszellein- und -austritt und damit
die Etablierung seines pathogenen Lebenszyklusses. Des Weiteren ist bekannt, dass Aktin an
der Endozytose in den parasitären Blutstadien beteiligt ist. Die molekularen Mechanismen,
welche das Aktinfließgleichgewicht zeitlich und räumlich regulieren, sind bisher für
Plasmodien nicht vollkommen verstanden.
In dieser Arbeit werden die G-Aktin (globuläres Aktin) bindenden Regulatoren, wie das
Zyclase- assoziierte Protein (C-CAP), das Profilin sowie die Aktin depolymerizierenden
Faktoren 1 und 2 (ADF1, ADF2) in Plasmodium berghei charakterisiert.
Mittels genetischer Geninaktivierung zeige ich, dass das C-CAP Gen nicht für die
Entwicklung von pathogenen Blutstadien und Ookineten in P. berghei notwendig ist. In vitro
Motilitätsuntersuchungen an Ookineten bestätigen eine verminderte Geschwindigkeit der c-
cap(-) Mutanten, was auf einen aktinabhängigen Prozess hindeutet. Die Fähigkeit den Wirt
(Moskito) zu infizieren wird dadurch jedoch nicht aufgehoben. Mit Hilfe d