Uncovering interactions between exported Plasmodium falciparum and human erythrocyte cytoskeleton proteins in the process of host cell remodeling

Warncke, Jan Dominic. Uncovering interactions between exported Plasmodium falciparum and human erythrocyte cytoskeleton proteins in the process of host cell remodeling. 2018, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_13080

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The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria, an infectious tropical disease of global public health importance. Despite efforts and means to prevent or treat this disease, there are still over 200 million cases and almost half a million deaths annually attributed to P. falciparum. Transmitted to the human host by female Anopheles mosquitos serving as vector, the parasite eventually invades erythrocytes and starts asexual replication. This stage causes the clinical symptoms of malaria.
The red blood cell is an interesting choice of a host cell for the intracellular parasite P. falciparum as it lacks a nucleus, protein transport machinery, and its nutrient channels are inactive. To survive within this host environment, the parasite therefore has to remodel its host cell. The extensive host cell remodelling of human erythrocytes during the course of P. falciparum infection is facilitated by a large number of proteins which the parasite exports into its host cell cytoplasm. The function of the majority of these proteins remains elusive. Existing data suggests that some of these exported parasite proteins target the host cytoskeleton and modulate its properties, as apparent in changed mechanical properties of the host cell.
The aim of this project was to identify interactions between host cytoskeleton and exported parasite proteins and to create a protein interaction network of the remodeled cytoskeleton. Identifying the key players and essential interactions in the process of host cell remodelling will lead to the identification of new targets in the fight against the malaria parasite. To this end, a number of exported proteins belonging to the PHIST family were selected. All selected PHIST proteins were exported into the host cell with most of them localizing in proximity to the erythrocyte cytoskeleton or membrane. The promiscuous biotin ligase BirA* (BioID) was fused to these proteins and upon addition of biotin proteins in the proximity were labelled with biotin. This allowed the pull-down using streptavidin-beads and identification of potential interaction partners of these transgenic, exported proteins by mass spectrometry. Based on the results from this study and additional data from previous projects, I generated a network of potential protein-protein interactions at the erythrocyte cytoskeleton.
A standard approach to verify potential protein interactions is to perform reverse protein pull-downs. Because erythrocytes lack a nucleus, the classical transgenic approach to add molecular tags to erythrocyte proteins or to modify them in any way is not possible. To circumvent this holdback and to facilitate immunoprecipitations with erythrocyte proteins as bait, I generated parasite lines which expressed and exported different tagged human cytoskeleton proteins. These transgenic human proteins were designed to be exported and to be soluble within the cytosol of the infected erythrocyte. It was expected that these proteins would bind to their putative endogenous parasite binding partners while these are transported to their final destination within the host cell. These transgenic human proteins can then be used for immunoprecipitations to identify these binding partners. I tested several export sequences and showed that each of them resulted in efficient export of the intracellular loop of band 3 (residues 1-379) and the full-length band 4.1. In both of these cell lines, the majority of the protein was soluble in the host cytosol. Due to time constraints, these cell lines could not be further analyzed in detail.
While little is known about the function and role of exported proteins in host cell remodeling during asexual developmental stages, even less is known about these proteins and their functions during gametocyte development of P. falciparum. Until recently, it was difficult to obtain high numbers of gametocytes, making it difficult to study host cell remodeling in these stages. The availability of a transgenic cell line from the Voss lab at Swiss TPH, in which high sexual conversion rates can be induced, provides a great opportunity to study these interactions in gametocytes. Taking advantage of this cell line we characterized GEXP02, a member of the PHIST protein family which is expressed and exported in gametocytes. We confirmed the expression pattern and localized GEXP02 at the periphery of the gametocyte-infected erythrocyte. By immuno-precipitation and mass spectrometry we could identify cytoskeleton proteins as well as other exported proteins as potential interaction partners. Based on co-labelling of GEXP02 with PFI1780w and PF3D7_0424600, two other PHIST proteins, we could confirm these as likely interaction partners. In GEXP02 knock-out parasites, no obvious detrimental effect or phenotype could be observed in asexual parasites or during gametocyte development nor throughout the mosquito stages or in liver hepatocyte infectivity. Although no function could be assigned to this protein, our study is one of the first to characterize in great detail an exported protein in gametocytes and shows that the erythrocyte cytoskeleton is targeted by exported parasite proteins also during gametocyte development.
Furthermore, within the context of this present study, I conducted two extensive literature reviews. In one review I collected information on the functionally elusive PHIST family in the genus Plasmodium. The review on the PHIST protein family presents an in-depth overview on this protein family. It acts as a reference work for quick, but detailed information on these proteins that are thought to be involved in cytoskeleton remodelling. The other review concerned protein-protein interactions involved in host cytoskeleton remodeling of P. falciparum. By combining pieces of existing information, new insights were gained in this review. I could show that each stage of the intraerythrocytic life cycle presents different challenges to the intracellular survival of the parasite. Consequently, P. falciparum remodels its host cell differently in the various stages to meet the specific needs.
In summary, this thesis provides new insight into host cell remodeling by the malaria parasite, shows the importance of exported proteins in this process, and offers a new tool in the study of interactions between erythrocyte cytoskeleton and exported parasite proteins.
Advisors:Beck, Hans-Peter and Matuschewski, Kai
Faculties and Departments:09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Former Units within Swiss TPH > Molecular Parasitology and Epidemiology (Beck)
UniBasel Contributors:Beck, Hans-Peter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13080
Thesis status:Complete
Number of Pages:1 Online-Ressource (xvi, 194 Seiten)
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Last Modified:08 Feb 2020 15:09
Deposited On:11 Jun 2019 13:26

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