An alternative secretory pathway in the malaria parasite Plasmodium falciparum

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 i...

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Bibliographische Detailangaben
1. Verfasser: Thavayogarajah, Thuvaraka
Beteiligte: Lingelbach, Klaus (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2014
Biologie
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Zusammenfassung: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 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.
DOI:https://doi.org/10.17192/z2014.0238