Analysis of exported proteins and parasite-induced host cell rigidity changes during the Plasmodium falciparum intraerythrocxytic life cycle

Abstract

Malaria is an infectious disease of global public health importance. Despite a number of successful efforts to fight the disease, there are still over 200 million cases and 445,000 deaths each year. Plasmodium falciparum, a protozoan parasite, is the causative agent of severe malaria. The parasite is transmitted by female Anopheles mosquitos form human to human and has different life stages inside both hosts. However, the only stages that are accountable for clinical symptoms of the disease are asexually replicating stages in the red blood cells. Red blood cells are highly specialized cells lacking a nucleus, organelles and certain cellular mechanisms. Therefore, the parasite has to extensively remodel the erythrocyte to ensure survival. The remodeling is achieved by exporting a large number of proteins that have various functions. Those functions include nutrient uptake and host immune response evasion. A crucial part of the refurbishment process is the establishment of a protein trafficking system that ensures correct delivery of exported proteins to their final destination. Maurer’s clefts are new organelles established by the parasite in the cytosol of infected red blood cells. They are presumably involved in protein trafficking acting as sorting stations. A number of parasite exported proteins have been shown to localize permanently or transiently at Maurer’s clefts. Among the latter is PfEMP1, the major virulence factor of P. falciparum. PfEMP1 is exported from the parasite, transported to Maurer’s clefts and finally inserted in the infected erythrocyte membrane. The surface exposed domain of the protein is able to confer binding to endothelial receptors. Thus, infected red blood cells can sequester in the vasculature and avoid passage through the spleen where they would potentially be removed from circulation and eliminated. The aim of this project was to expand our knowledge of P. falciparum exported proteins, their function and interactions. In this thesis I have characterized three exported proteins, namely PF3D7_0702500, MESA, and STARP. In order to investigate these proteins transgenic parasite lines were generated and analyzed. By establishing cell lines expressing tagged proteins, expression and localization studies as well as co-immunoprecipitation experiments for the identification of protein interaction partners could be performed. Protein functional analysis was achieved by phenotypical characterization of knock out cell lines. Thereby the main readouts were growth, transport, surface presentation and anchoring of PfEMP1, and infected red blood cell deformability. We propose a function of PF3D7_0702500 in correct PfEMP1 display at the infected red blood cell surface, while the functions of STARP and MESA remain elusive. Upon P. falciparum infection, the physical properties, e.g. cell deformability and membrane flexibility of an erythrocyte change dramatically. These changes are controlled by the parasite and are important for the adaptation to the host environment. Those biomechanical changes can be potentially exploited as a drug target, e.g. with drugs that reduce iRBC deformability, especially in circulating ring stages, enhancing the filtering function of the spleen to remove those iRBCs. Using microsphiltration to assess infected red blood cell deformability, we performed a study on the effect of the spiroindolone drug KAF246 on the rigidity of P. falciparum infected red blood cells. We were able to correlate these results with in vivo experiments where we observed P. berghei parasites accumulating in the mouse spleen rapidly after KAF246 treatment. In summary, this thesis contributes to a better understanding of the protagonists of erythrocyte remodeling upon P. falciparum infection and gives new insights into parasite induced host cell changes during the pathology relevant stages. The findings of this thesis facilitate further studies and may eventually lead to the identification of potential new malaria intervention strategies.