Using bioinformatic analyses, we identified the presence of the gene coding for aegerolysin and for a membrane attack complex/perforin (MACPF)-domain protein in the basidiomycete Lepista nuda, commonly known as the wood blewit. We named the aegerolysin nudolysin A (NudA) and the MACPF-domain protein nudolysin B (NudB). For our master's thesis, we expressed and obtained recombinant NudA and NudB proteins in bacteria Escherichia coli. We investigated the binding of NudA to membranes with specific lipid compositions, using the sedimentation test. We found that NudA binds to the ceramide phosphoethanolamine/cholesterol lipid mixtures and to the total lipid extract from the insect cell line Sf9. Furthermore, we also detected weak binding of NudA to sphingomyelin/cholesterol lipid mixture. Subsequently, we examined whether the combination of NudA and NudB forms cytolytic pore-forming complexes. Initially, we used a hemolysis test on bovine erythrocytes and found that the protein mixture NudA/NudB was hemolytic at pH 7.4 and that hemolytic activity is higher in acidic conditions (pH 6.0). Finally, we tested the cytolytic activity of the protein mixture NudA/NudB on the insect cell line Sf9 in vitro, where we found that the protein mixture exhibits cytolytic effects on these cells.
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