An Alternative Secretory Pathway in the Malaria Parasite 'Plasmodium falciparum'

Table of Contents

1 Introduction

1.1 Malaria

1.2 The complex life-cycle of Plasmodium falciparum

1.3 The intraerythrocytic stage

1.3.1 P. falciparum-infection leads to extensive modification of the red blood cell

1.3.2 Novel structures and compartments in P. falciparum-infected red blood cell

1.3.3 Protein secretion mechanisms in the P. falciparum-infected red blood cell

1.4 Unconventional protein secretion

1.5 Role of fatty acid acylation of proteins in plasma membrane binding

1.5.1 Protein N-myristoylation

1.5.2 Protein-Palmitoylation

1.6 Acylated proteins as candidates of an alternative secretory pathway in P. falciparum?

1.6.1 P. falciparum ADP-ribosylation factor 1

1.6.2 P. falciparum adenylate kinase (2)

1.7 Objective

2 Materials and Methods

2.1 Materials and Chemicals

2.1.1 Appliances

2.1.2 Materials

2.1.3 Chemicals

2.1.4 Cell Culture Materials

2.1.5 Molecular Biological Kits

2.2 Enzymes

2.3 Antibodies

2.4 Solutions and buffers

2.5 Vectors and oligonucleotides

2.5.1 Vectors

2.5.2 Oligonucleotides

2.5.3 Plasmids designed for this work

2.6 Cells and Organisms

2.7 Bioinformatics

2.8 Cell culture techniques

2.8.1 In-vitro cultivation of Plasmodium falciparum

2.8.2 Synchronization of Plasmodium falciparum with Sorbitol

2.8.3 Enrichment of trophozoite-stage parasites via Gelafundin flotation

2.8.4 High enrichment of late-stage parasites using a high gradient magnetic field

2.8.5 Transfection and Co-transfection of Plasmodium falciparum

2.8.6 Cryopreservation of Plasmodium falciparum-infected erythrocytes

2.8.7 Thawing of cryopreserved Plasmodium falciparum-infected erythrocytes

2.9 Molecularbiological methods

2.9.1 Cultivation of Escherichia coli

2.9.2 Preparation of electrocompetent bacterial cells (E. coli strain TOP10)

2.9.3 Mini- and Maxipreparation for isolation of plasmid DNA

2.9.4 Transformation of E. coli cells

2.9.5 Isolation of genomic DNA from Plasmodium falciparum

2.9.6 Isolation of mRNA from Plasmodium falciparum

2.9.7 Quantification of nucleic acid

2.9.8 Reverse transcriptase PCR

2.9.9 Polymerase chain reaction

2.9.10 In-vitro site-directed mutagenesis

2.9.11 Agarose gel electrophoresis

2.9.12 Purification of DNA

2.9.13 Ethanol precipitation of DNA

2.9.14 Restriction of DNA

2.9.15 Ligation of DNA

2.9.16 Screening for positive clones with colony PCR

2.9.17 Sequencing of DNA

2.9.18 Generation of plasmid constructs for transfection

2.10 Biochemical methods

2.10.1 Cell fractionation of Plasmodium falciparum-infected red blood cells

2.10.2 Streptolysin O permeabilization of Plasmodium falciparum-infected erythrocytes

2.10.3 Saponin lysis of Plasmodium falciparum-infected erythrocytes

2.10.4 Protease protection assay

2.10.5 SDS-PAGE

2.10.6 Semi-Dry-Immunoblotting

2.11 Fluorescence microscopy

2.11.1 Live cell imaging

2.11.2 Immunofluorescence assay

2.11.3 Image processing with Image J

2.12 Experimental design

3 Results

3.1 Selected candidate proteins

3.1.1 PfARF1 shows a different subcellular localization upon removal of the N-myristoylation site in P. falciparum-infected red blood cells

3.1.2 PfARF1 shows co-localization with marker proteins of the compartments of the secretory pathway

3.1.3 PfARF1 is not secreted into the PV in the blood-stage according to biochemical analyses

3.2 Is PfAK2 secreted beyond the parasite plasma membrane?

3.2.1 A multiple sequence alignment

3.2.2 A putative palmitoylation site at the N-terminus of PfAK2

3.2.3 The PfAK2G2AC4A expressing parasites localize to the parasite cytosol

3.2.4 Is a third motif involved in the secretion process of PfAK2?

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?

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

3.3 The mDHFR fusion system

4 Discussion

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

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?

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?

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?

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

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

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

5 Outlook

Research Objectives and Key Topics

This thesis investigates the existence of unconventional secretory pathways in the malaria parasite Plasmodium falciparum, focusing on how proteins lacking classical secretion signals are exported beyond the parasite plasma membrane. By analyzing specific candidate proteins and the role of N-myristoylation and palmitoylation, the research aims to elucidate mechanisms of membrane anchoring and protein translocation during the parasite's intraerythrocytic development.

• Mechanisms of unconventional protein secretion in P. falciparum.
• Role of N-myristoylation and protein palmitoylation in membrane targeting.
• Subcellular localization of candidate proteins like PfARF1 and PfAK2.
• Impact of N-terminal motif modifications on protein trafficking.
• Evaluation of the mDHFR fusion system for analyzing protein folding states.

Excerpt from the Book

Summary of Chapters

1 Introduction: Provides an overview of malaria, the parasite's life cycle, and current models of protein secretion in P. falciparum.

2 Materials and Methods: Details the experimental setups, cultivation techniques, molecular cloning methods, and biochemical assays used throughout the study.

3 Results: Documents the analysis of candidate proteins, demonstrating that N-myristoylation is crucial for membrane targeting and secretion for specific parasite proteins.

4 Discussion: Evaluates the findings in the context of alternative secretory pathways and the role of N-terminal motifs in protein trafficking.

5 Outlook: Proposes future studies, particularly regarding the validation of potential drug targets and further elucidation of secretion mechanisms.

Keywords

Plasmodium falciparum, N-myristoylation, protein palmitoylation, unconventional protein secretion, parasitophorous vacuole, PfARF1, PfAK2, malaria parasite, intracellular trafficking, protein membrane anchoring, secretory pathway, parasite plasma membrane, mDHFR system, cellular biology.

Frequently Asked Questions

What is the primary focus of this dissertation?

The research focuses on discovering and understanding alternative, unconventional protein secretion pathways in the malaria parasite Plasmodium falciparum during its intraerythrocytic stage, specifically looking beyond the classical ER/Golgi secretion route.

What are the central themes of the work?

Central themes include the role of N-terminal lipid modifications like N-myristoylation and palmitoylation in targeting parasite proteins to membranes and their subsequent translocation across the parasite plasma membrane into the parasitophorous vacuole.

What is the core research objective?

The core objective is to determine whether N-myristoylation acts as a signal for the secretion of specific parasite proteins that lack classical signal peptides, using GFP-reporter constructs and biochemical localization assays.

Which scientific methods are employed?

The study utilizes molecular cloning to generate reporter constructs, epifluorescence microscopy for live-cell imaging and co-localization studies, and biochemical techniques such as hypotonic, SLO, and saponin lysis combined with protease protection assays.

What is the scope of the main body?

The main body investigates several candidate proteins, with a detailed analysis of PfARF1 and PfAK2, testing their localization and the effect of mutating their N-terminal acylation sites.

What defines the candidate proteins studied?

The proteins were selected based on their N-terminal glycine residues (predictive of N-myristoylation) and the absence of canonical signal sequences, making them likely candidates for unconventional secretory mechanisms.

How does removing the N-myristoylation site affect PfARF1?

Removing this site (G2A mutation) leads to a shift from dot-like structures within the parasite cytosol to a completely cytosolic distribution, indicating that N-myristoylation is essential for membrane association and correct subcellular targeting.

What role does the N-terminus of PfAK2 play?

The N-terminus of PfAK2, which contains both N-myristoylation and potential palmitoylation sites, is shown to be sufficient for membrane anchoring and facilitating translocation beyond the parasite plasma membrane.