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«An alternative secretory pathway in the malaria parasite Plasmodium falciparum Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften ...»

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An alternative secretory pathway in the malaria

parasite Plasmodium falciparum

Dissertation

zur Erlangung des Doktorgrades der Naturwissenschaften

(Dr. rer. nat.)

dem Fachbereich Biologie

der Philipps-Universität Marburg

vorgelegt von

Thuvaraka Thavayogarajah

aus Detmold

Marburg/Lahn 2014

Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation

angenommen am: ______________________________________

Erstgutachter Prof. Dr. Klaus Lingelbach

Zweitgutachter Prof. Dr. Anthony Holder

Tag der Disputation am:

" Imagination is more important than knowledge.

Knowledge is limited.

Imagination encircles the world. "

- Albert EinsteinFür meine Mutter

The major part of this thesis is prepared for submission:

Thavayogarajah, T., Gangopadhyay, P., Becker, K., Holder, A., Przyborski, J.M. and Lingelbach K. (2014) An alternative secretory pathway in the malaria parasite P. falciparum (manuscript in preparation)

Other publications (from undergraduate studies):

Jessica M. Vanslambrouck, Angelika Bröer, Thuvaraka Thavayogarajah, Jeff Holst, Charles G. Bailey, Stefan Bröer and John E. J. Rasko (2010) Renal imino acid and glycine transport system ontogeny and involvement in developmental iminoglycinuria.

Biochemical Journal 428 (397-407) Table of contents i Table of contents Abbreviations Summary Zusammenfassung _______________________________________________________________________________________________

1 Introduction

1 1.1 Malaria

1 1.2 The complex life-cycle of Plasmodium falciparum

2

5 1.3 The intraerythrocytic stage 1.3.1 P. falciparum-infection leads to extensive modification

–  –  –

3.2.5 Is the N-terminus of PfAK2 - containing a N-myristoylation site, a putative palmitoylation site and a polybasic cluster of amino acids - sufficient for protein secretion?

88 3.2.6 The ARF-AK2/GFP chimera is targeted to the parasite plasma membrane.............

91 3.3 The mDHFR fusion system

95 4 Discussion

98 4.1 The secretion hypothesis is based on the result of a preceding PV proteome analysis

100 4.1.1 Does the 'Met-Gly...' motif at the N-terminus of PfPrefoldin (PF3D7_0904500), PfCDPK4 (PF3D7__0717500) and PfARF1 (PF3D7_1020900) affect their subcellular localization?

100 4.1.2 Analysis of the subcellular localization of PfARF1 in P. falciparum-infected RBC

4.2 Is PfAK2 a candidate protein of an alternative secretory pathway?

104 4.2.1 How much of the N-terminus of PfAK2 is required for targeting other myristoylated proteins like PfARF1 to the PPM and beyond?

109 4.2.2 An analysis about the folding state of PfAK2 as it translocates from the parasite cytosol into the PV

110 4.2.3 A model for PfAK2 protein anchoring to the PPM and secretion

111 4.3 Concluding remarks on the analysis of PfAK2 as a candidate protein of an alternative secretory pathway in P. falciparum

113 5 Outlook

117 6 References

119 7 Appendix

135 Coding sequences (PlasmoDB, version 10.0)

135 7.1 Multiple sequence alignment (Clustal W: T-coffee)

136 7.2 Expression profile of PfAK2 (PlasmoDB, version 10.0)

139 7.3 7.4 Potential proteins of the P. falciparum genome as candidate proteins of an alternative secretory pathway

138 _______________________________________________________________________________________________

Acknowledgements Curriculum vitae Eigenständigkeitserklärung Abbreviations iv

–  –  –

Summary The malaria parasite P. falciparum invades human red blood cells (RBCs). During invasion a compartment surrounding the parasite, the so-called parasitophorous vacuole (PV), is formed. The parasite resides and develops within the PV, which protects the parasite from the host cell cytosol. During its intraerythrocytic growth the parasite exports a vast number of proteins to the host cell in order to maintain its survival within the RBC. Proteins, which are directed to the host cell cytosol and host cell membrane, respectively, therefore are challenged to cross the parasite plasma membrane, the PV and the parasitophorous vacuolar membrane (PVM). However, the secretion and export mechanisms of many parasite proteins are still not understood.

The current study focuses on the discovery of an alternative secretory pathway to the ER/Golgi route in the malaria parasite P. falciparum in infected RBCs. Two proteins appeared to be promising candidates of an alternative secretory pathway: the PfADPribosylation factor 1 (ARF1) and the Pfadenylate kinase 2 (AK2). Both proteins contained a N-myristoylation site at their N-terminus, which is indicative for Nmyristoylation. N-myristoylation is a co-translational modification of a protein, whereby a fatty acid (myristate) is irreversibly attached to the glycine residue at the N-terminus of a protein via the PfN-myristoyltransferase (NMT). A preceding proteomic analysis of the parasitophorous vacuole and a reporter construct study proposed for both PfARF1 (determined by a proteomic study) and PfAK2 (determined by a reporter construct study) PV localization although both proteins lacked a signal peptide. That’s why it was hypothesized whether or not N-myristoylation would drive protein secretion across the parasite plasma membrane (PPM). The subcellular localization of the PfARF1/GFP parasites and the PfAK2/GFP parasites, respectively, were analyzed via epifluorescence microscopy and biochemical methods. In parallel, another batch of reporter constructs were generated and analyzed, where the N-myristoylation site of PfARF1 (this study) and PfAK2 (Ma et al., 2012), respectively, was removed (PfARF1G2A/GFP and PfAK2G2A/GFP). Live cell imaging showed that the fusion protein ARF1/GFP was localized as dot-like structures in the parasite. In contrast, the phenotype of the fusion Summary viii protein of the PfARF1G2A/GFP parasites showed an evenly distributed signal in the parasite cytosol. Further analysis of the subcellular localization of the PfARF1 strongly supports its localization to compartments of the early secretory pathway of the parasite, but no localization in the PV. In contrast, the fusion protein PfAK2/GFP localized to a ring-like structure around the parasite indicating PV localization. The PfAK2G2A/GFP parasites showed a cytosolic localization of the fusion protein (Ma et al., 2012).





Biochemical analyses revaled that the fusion protein PfAK2/GFP was secreted into the PV when the N-myristoylation site was present. Furthermore, it could be shown that the N-terminus of the PfAK2 protein is sufficient for parasite plasma membrane targeting, stable membrane anchoring and subsequent protein translocation across the PPM. A possible role of the early secretory pathway in PfAK2 trafficking and the folding state of PfAK2 prior to translocation across the PPM was also examined. However, the exact mechanism how PfAK2 is translocated across the PPM still remains elusive.

Zusammenfassung ix Zusammenfassung Der Malariaerreger P. falciparum befällt rote Blutkörperchen im Menschen. Bei der Invasion der Erythrozyten formt der Parasit eine sogenannte parasitophore Vakuole (PV), die ihn dann umgibt. Der Parasit verbleibt und entwickelt sich in dieser PV, die ihn vom Zytosol der Wirtszelle schützt. Während des intraerythrozytären Wachstums exportiert der Parasit eine hohe Anzahl seiner eigenen Proteine in die Wirtszelle um sein Überleben in der Wirtszelle zu sichern. Proteine, die entweder in das Zytosol oder der Membran der Wirtszelle exportiert werden, müssen zunächst die Plasmamembran des Parasiten (PPM), die PV und die anliegende parasitophore Vakuolenmembran (PVM) passieren. Der Sekretions- und Exportmechanismus vieler Parasitenproteine ist jedoch noch immer unbekannt.

Ziel dieser Arbeit ist es alternative Sekretionswege zum klassichen ER/Golgi Sekretionsweg im Malariaparasiten P. falciparum aufzudecken. Zwei Proteine schienen geeignete Kandidaten eines alternativen Sekretionsweges zu sein: der PfADPRibosylierungsfaktor 1 (ARF1) und die PfAdenylat kinase 2 (AK2). Beide Proteine besitzen eine N-myristoylierungsstelle am N-terminus was auf eine N-myristoylierung des jeweiligen Proteins hindeutet. N-myristoylierung ist eine ko-translationale Modifizierung eines Proteins, wobei eine Fettsäure (Myristat) irreversibel am Glycinrest am N-terminus eines Proteins durch die PfN-myristoyltransferase (NMT) angehängt wird. Eine vorangegangene Proteomuntersuchung der parasitophoren Vakuole und eine Untersuchung mit Reporterkonstrukten ergab für PfARF1 (Proteomuntersuchung) und PfAK2 (Analyse der Reporterkonstrukte) eine PV Lokalisation, obwohl beiden ein Signalpeptid fehlt. Deshalb wurde die Hypothese aufgestellt, dass N-myristoylierung womöglich an der Proteinsekretion über die Plasmamembran des Parasiten beteiligt sein könnte. Demnach wurde die subzelluläre Lokalisation der PfARF1/GFP Parasiten und der PfAK2/GFP Parasiten mithilfe von Epifluoreszenzmikroskopie und biochemischen Methoden untersucht. Parallel dazu wurden Reporterkonstrukte generiert und analyisert, bei denen die N-myristoylierungsstelle von PfARF1 (diese Arbeit) und PfAK2 (Ma et al., 2012) entfernt wurden (PfARF1G2A/GFP und PfAK2G2A/GFP). Beim live cell imaging war das Fusionsprotein ARF1/GFP als punktförmige Struktur im Parasiten Zusammenfassung x erkennbar. Der Phänotyp des Fusionsproteins der PfARF1G2A/GFP Parasiten dagegen zeigte ein zytosolisches Signal im Parasiten. Weitere Analysen im Hinblick auf die subzelluläre Lokalisation des PfARF1 deuten auf eine Lokalisation dieses Proteins mit Kompartimenten des frühen Sekretionsweges des Parasiten hin jedoch auf keine Lokalisation in der PV. Im Gegensatz dazu war das Fusionsprotein PfAK2/GFP als ringförmige Struktur sichtbar was auf eine PV Lokalisation hindeutet. Die PfAK2G2A/GFP Parasiten zeigten hingegen eine zytosolische Lokalisation des Fusionsproteins (Ma et al., 2012). Biochemische Untersuchungen konnten zeigen, dass das Fusionsprotein PfAK2/GFP in Anwesenheit der N-myristoylierungsstelle in die PV sekretiert wurde. Des Weiteren konnte gezeigt werden, dass der N-terminus von PfAK2 das Protein zur Plasmamembran führt und eine stabile Membranverankerung hervorruft bevor die Translokation über die Plasmamembran des Parasiten erfolgt. Eine mögliche Rolle des frühen Sekretionsweges im Transport von PfAK2 und der Faltungszustand von PfAK2 vor der Translokation über die Parasitenplasmamembran wurden ebenfalls untersucht. Dennoch ist der genaue Mechanismus der Proteintranslokation über die Plasmamembran des Parasiten nicht bekannt.

1 Introduction 1

1 Introduction

1.1 Malaria Since ancient times a disease today referred to as malaria ('mal' 'aria' meaning 'bad air') has been noted to have a detrimental effect on people’s life quality, impeding population growth and affecting settling patterns throughout human history (Carter and Mendis, 2002; Sallares et al., 2004). Today malaria is recognized as one of the largest, life-threatening, infectious diseases in the world, caused by a eukaryotic parasite of the genus Plasmodium and transmitted by a bite of an infected female Anopheles mosquito.

Although the causative agent of this disease was identified in the late 19th century malaria continues to be a major health problem in tropical and subtropical parts of the world, where billions of people are still exposed to this deadly disease (Fig. 1) (WHO 2011; cdc). In 2010 approximately 216 million clinical cases were reported by the World Health Organisation with 80 % occurring in African regions alone. Also 90 % of the 655 000 cases of deaths by malaria were registered in Africa (WHO 2011). In malaria-endemic regions pregnant women and children under the age of five (86 % of malaria deaths) succumb to this lethal disease more frequently than other groups of people as stated by the WHO in 2011. Countries, in which malaria is prevalent are also the ones suffering from a high poverty rate and a low economic growth making malaria prevention control and treatment more difficult (Gallup and Sachs, 2001; Sachs and Malaney, 2002).

Figure 1.1 Malaria distribution1 Introduction 2

To date five different species of the genus Plasmodium – P. vivax, P. ovale, P. malariae, P. falciparum and P. knowlesi - are known to cause malaria in humans.

While P. vivax and P. ovale, the causative agents of tertian malaria, and P. malariae, the causative agent of quartian malaria, are less likely to lead to severe forms of malaria outbreaks in humans, P. falciparum infections are mostly responsible for the high morbidity and mortality rates in endemic regions of Africa (cdc). Only recently P. knowlesi, a Plasmodium species known to infect macaque monkeys with malaria, was discovered to cause malaria in humans as well. (Singh et al., 2004; Cox-Singh et al., 2008).

Since the early 20th century many projects emerged to fight this deadly disease involving the use of the synthetic insecticide dichlorodiphenyl-trichloroethane (DDT) to prevent transmission by mosquitoes and the chemical compound chloroquine, known to inhibit the development of the blood-stage parasite. However, the increasing resistance in the mosquitoes and the Plasmodium-species, respectively, and the adverse effect of DDT on the environment made these attempts over the period of time unfruitful (cdc). Nevertheless, many malaria eradication programmes, especially in the early 21st century, are determined to reduce the high malaria casualties by the use of bed-nets treated with insecticide, artemisin-based combination therapies, etc. In fact the combination of these various methods has reduced the number of malaria cases of deaths by around 33 % since 2000 as registered in African regions, which are monitored by the WHO (WHO 2011). However, the number of malaria infections is still high and strains resistant to the available current anti-malarial drugs are already occurring. That is why continuous study on the biology of the parasite, to eventually develop an efficient vaccine and developments of new drugs against malaria, still needs to be continued.



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