This 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 PfADP-ribosylation factor 1 (ARF1) and the Pfadenylate kinase 2 (AK2).
Both proteins contained an N-myristoylation site at their N-terminus, which is indicative for N-myristoylation. 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.
Inhaltsverzeichnis (Table of Contents)
- Abbreviations
- Summary
- Zusammenfassung
- Introduction
- Malaria
- The complex life-cycle of Plasmodium falciparum
- The intraerythrocytic stage
- P. falciparum-infection leads to extensive modification of the red blood cell.
- Novel structures and compartments in P. falciparum-infected red blood cell.....
- Protein secretion mechanisms in the P. falciparum-infected red blood cell.....
- Unconventional protein secretion.
- Role of fatty acid acylation of proteins in plasma membrane binding..
- Protein N-myristoylation..
- Protein-Palmitoylation
- Acylated proteins as candidates of an alternative secretory pathway in P. falciparum? .
- Objective.....
- Materials and Methods.
- Materials and Chemicals.
- Enzymes
- Antibodies.
- Solutions and buffers...
- Vectors and oligonucleotides
- Vectors.....
- Oligonucleotides.
- Plasmids designed for this work.
- Cells and Organisms...........
- Bioinformatics...........
- Cell culture techniques.
- In-vitro cultivation of Plasmodium falciparum.
- Synchronization of Plasmodium falciparum with Sorbitol
- Enrichment of trophozoite-stage parasites via Gelafundin flotation.
- High enrichment of late-stage parasites using a high gradient magnetic field..
- Transfection and Co-transfection of Plasmodium falciparum.........
Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)
This dissertation explores the existence of an alternative secretory pathway in the malaria parasite Plasmodium falciparum. The main objective is to investigate the potential role of fatty acid acylation of proteins in this unconventional protein secretion pathway.
- The complex life-cycle of Plasmodium falciparum
- Unconventional protein secretion mechanisms in P. falciparum-infected red blood cells
- The role of fatty acid acylation in protein targeting and localization
- Identification of potential candidate proteins involved in the alternative secretory pathway
- Investigating the molecular mechanisms of protein transport and secretion in P. falciparum
Zusammenfassung der Kapitel (Chapter Summaries)
The introduction provides an overview of malaria, the complex life-cycle of P. falciparum, and the intraerythrocytic stage of the parasite's development. It focuses on the extensive modifications the red blood cell undergoes during infection and the unconventional protein secretion mechanisms employed by P. falciparum. The chapter also delves into the role of fatty acid acylation in plasma membrane binding, particularly examining N-myristoylation and palmitoylation. The chapter concludes by introducing the potential candidates for an alternative secretory pathway in P. falciparum and outlining the objectives of the dissertation.
The "Materials and Methods" chapter details the experimental techniques and materials used in this research. It covers aspects such as cell culture techniques, molecular biology tools, and bioinformatics analyses. Specific methods discussed include in-vitro cultivation and synchronization of P. falciparum parasites, transfection procedures, and protein analysis techniques.
Schlüsselwörter (Keywords)
The central theme of this dissertation revolves around the study of an alternative secretory pathway in the malaria parasite Plasmodium falciparum. Key focus areas include unconventional protein secretion, fatty acid acylation, protein targeting, protein localization, N-myristoylation, palmitoylation, and the identification of potential candidate proteins involved in this novel pathway. The work aims to unravel the complex mechanisms of protein transport and secretion within P. falciparum, offering insights into the parasite's biology and potential avenues for therapeutic intervention.
- Quote paper
- Thuvaraka Thavayogarajah (Author), 2014, An Alternative Secretory Pathway in the Malaria Parasite 'Plasmodium falciparum', Munich, GRIN Verlag, https://www.grin.com/document/369414
