The project focuses on Chaperone mediated autophagy (CMA). CMA normally contributes to the degradation of cytosolic proteins in the lysosome, but also might have a role in nutrition of T. gondii as this parasite is sequestered in a parasitophorous vacuole. During CMA substrate proteins that contain the KFERQ targeting motif are detected by a cytosolic chaperone. This chaperone binds to the substrate protein and mediates the delivery of the substrate protein to the surface of the lysosome, which binds to the lysosome-associated membrane protein type 2A (LAMP-2A) receptor. LAMP-2A plays a role in the translocation of substrates across the lysosomal membrane. Thus, we hypothesize that T. gondii can use CMA for nutrient uptake. Therefore, the student will maintain in vitro cultures of obligatory intracellular T gondii strains and learn different molecular biology and imaging techniques. These consist of PCR, immunofluorescence assays, gel electrophoresis, western blots and confocal microscopy. Also the student will learn to maintain, cultivate and cryopreserve eukaryotic HFF cell line. Other skills consist of cell culture techniques of mammalian cells such as preparing single cell suspensions from different organs, how to process samples for ELISA and how to do flow cytometric analysis.
The student will also be involved in another project focusing on parasite co-infections with the protozoan Toxoplasma gondii and the helminth Heligmosomoides polygyrus. The aim of this project is to focus on the cross-regulation of immune responses of the two species, with the use of animal experiments and analysis of immunological parameters of mice. The student would be able to learn all cell culture techniques (e. g. preparing single cell suspensions from different organs such as lymph nodes, intestine and brain). Also the student will learn how to stain and handle cell samples for subsequent flow cytometric analysis.