The project leader was appointed as a mentor of four completed MSc and BSc theses of three students from Biotechnical faculty (University of Ljubljana), and one biotechnology student from the University of Rijeka, Croatia [COBISS.SI-ID 4033103], [COBISS.SI-ID 4090191], [COBISS.SI-ID 4065615], [COBISS.SI-ID 3993935]. All these theses are tightly connected to the thematic of the project J4-7162, and deal with investigation of interactions of aegerolysins from fungi and bacteria with artificial lipid systems, in regard to the membrane lipid composition (membrane binding and membrane permeabilization with aegerolysin/ MACPF-protein complexes).
D.10 Educational activities
The project leader was appointed as a mentor of two completed PhD theses whose thematic is tightly connected to the thematic of the project J4-7162. Within her PhD thesis entitled "Interaction of fusion fluorescent protein ostreolysin A and lysophospholipids with lipid membranes and membrane domains" [COBISS.SI-ID 871031], dr. Maja Grundner evaluated the interactions of the fluorescent fusion protein ostreolysin A (OlyA-mCherry) with artificial and biological membranes. In order to better elucidate the interaction of OlyA-mCherry and lysophosphatidylcholine with membranes, its binding to artificial lipid membranes and membrane domains has been assessed. Using the microfluidic diffusion chamber, she has shown that the protein binds to the liquid ordered domains of phase separated giant unilamellar vesicles. Surface plasmon resonance experiments have shown that the OlyA-mCherry binding to lipid monolayer is lower compared to lipid bilayer. The interaction with lipid vesicles is reversible and the addition of lysophosphatidylcholine decreases the binding of OlyA-mCherry. Experiments using the Lagmuir balance were performed on lipid monolayers to demonstrate that the protein does not penetrate among lipid molecules. Further experiments with cholesterol oxidase showed that the availability of cholesterol plays an important role for OlyA-mCherry binding. In the second part of our study, the binding properties of OlyA-mCherry to membrane domains have been assessed. Tether pulling experiments on CHO cells were used to show the effect of the disruption of the cytoskeleton, the depletion of cholesterol and the incorporation of lysophosphatidylcholine into the plasma membrane on the tether force and on the protein binding. Within her PhD thesis entitled "Biological role and characterisation of aegerolysins and proteins with MACPF domain in filamentous fungus Aspergillus niger" [COBISS.SI-ID 285156096], dr. Maruša Novak performed a detailed investigation of the so-far cryptic biological role of aegerolysin proteins and proteins with MACPF domain in filamentous fungus A. niger. She has systematically studied the expression of the genes encoding these proteins in A. niger. She showed that the gene expression, and the consecutive protein production, depends on the growth and development of the fungus, and that the sporulation is crucial for the activation of the expression. Besides the sporulation, these genes can be activated also by the presence of other organisms, especially some soil bacteria, pointing to the involvement of aegerolysins and MACPF-proteins in defence mechanisms and/ or signalling. Maruša Novak has also showed that aegerolysins from A. niger specifically recognize and bind to ceramide phosphoethanolamine, a lipid present in membranes of invertebrate and some pathogenic bacteria. Fluorescenly tagged aegerolysins specifically interacted with insect cell membranes, but not with with mammalian cell membranes. These findings propose the use of aegerolysins as markers of membranes containing ceramide phosphoethanolamine, and consequently as biomarkers for detection of pathogenic microorganisms. In concert with their putative partnering protein, they could form cytolytic complexes that could be used for selective eradication of specific phytopathogens, especially insects.
D.09 Tutoring for postgraduate students
Dr. Maja Grundner was elected to participate at the international summer school "Cell Model systems" for doctoral students in Rome (Italy), where she presented the results of her doctoral dissertation (description is given above).
B.03 Paper at an international scientific conference
COBISS.SI-ID: 4110159