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Studies on interactions of exported "plasmodium falciparum" membrane proteins

Dietz, Olivier. Studies on interactions of exported "plasmodium falciparum" membrane proteins. 2014, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10750

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Abstract

Responsible for about 700’000 annual deaths worldwide, malaria today is still one of the major health problems in developing countries. The most deadly form of human malaria called malaria tropica is caused by the apicomplexan parasite Plasmodium falciparum that is transmitted by the female Anopheles mosquito. Its pathology is associated with the asexual development of the unicellular parasite within the human red blood cell (RBC) that is devoid of all internal organelles and any protein trafficking machinery. Therefore, intraerythrocytic survival and virulence of P. falciparum strictly depend on extensive host cell refurbishments mediated by the export of parasite proteins into the erythrocyte cytosol. Although numerous of these proteins have been identified and some extensively studied, still surprisingly little is known about their functions and interactions. It could be shown that many of these proteins are transported to or via parasite-induced membranous structures in the erythrocyte cytosol termed Maurer’s clefts (MCs). While these MCs are known to have a crucial role in protein trafficking, probably by acting as secretory organelles that concentrate virulence proteins for delivery to the host cell membrane, their specific functions have yet to be determined.
With this study we aimed at bringing new insight into the functions and interactions of exported P. falciparum proteins, with intent to create a basis for the extensive interaction network of the parasite’s exportome. Therefore we not only performed interaction studies using classical approaches like co-immunoprecipitation (Co-IP), but also tried to establish the yeast mating-based split-ubiquitin system (mbSUS) as a new in vitro interaction platform for P. falciparum membrane proteins on the basis of the integral MC protein ‘membrane associated histidine rich protein 1’ (MAHRP1). To contribute to a better understanding of protein export we further identified and characterized a new MC membrane protein that we termed ‘small exported membrane protein 1’ (SEMP1).
By Co-IP experiments we identified several potential SEMP1 interaction partners, including REX1 and other membrane-associated proteins that were confirmed to co-localize with SEMP1 at the MCs. Although a number of experiments deemed the quality of the generated P. falciparum cDNA library sufficient, we were unsuccessful to identify MAHRP1-binding proteins by mbSUS high-throughput screens. However, we could show that even without a functional library the mbSUS provides a useful tool to verify P. falciparum membrane protein interactions on a one by one basis by confirming binding of SEMP1 to a protein identified as potential interaction partner by Co-IP. A combination of Co-IP and mbSUS represents a promising new strategy for the identification and confirmation of direct P. falciparum membrane protein interactions.
We showed by immunofluorescence and solubility assays that SEMP1 is early exported into the RBC cytosol upon invasion where it inserts into the MCs before it is at least partly translocated to the RBC membrane. Using conventional and conditional loss-of-function approaches we found that SEMP1 is not essential for parasite survival, gametocytogenesis, or export of the major parasite virulence factor ‘P. falciparum erythrocyte membrane protein 1’ (PfEMP1) under culture conditions. Transcriptome analysis of SEMP1-depleted parasites further showed that expression of a number of exported parasite proteins was up-regulated in its absence, including PfEMP3 and a ‘Plasmodium helical interspersed subtelomeric family’ (PHIST) protein possibly indicating a role for SEMP1 in modulation of the erythrocyte membrane skeleton.
With this thesis we contribute to a better understanding of the export of P. falciparum proteins and their interactions within the human RBC. Our findings provide a starting point for numerous follow-up studies which in the end should result in a comprehensive interaction network. By bringing new insight into the complex interactome of exported parasite proteins we will hopefully identify new intervention targets to interfere with the essential refurbishment of the host cell.
Advisors:Beck, Hans-Peter
Committee Members:Gilberger, Tim-Wolf
Faculties and Departments:09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology > Molecular Parasitology and Epidemiology (Beck)
Item Type:Thesis
Thesis no:10750
Bibsysno:Link to catalogue
Number of Pages:195 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:55
Deposited On:07 May 2014 14:26

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