Precise recognition of specific lipids and lipid domains on the plasma membrane is crucial for understanding cellular processes and for the development of novel diagnostic and therapeutic tools in cell biology and oncology. Aegerolysin family proteins, due to their unique ability to recognize specific lipids and lipid microdomains, offer exceptional biomedical potential, which has not yet been comprehensively evaluated in in vitro mammalian cell models.
The aim of this doctoral dissertation was to investigate the aegerolysins erylysin A (EryA), ostreolysin A6 (OlyA6), and its single-point mutant E69A, along with their membrane-attack complex/perforin (MACPF) domain protein partners pleurotolysin B (PlyB) and pulmolysin B (PulB). Our objective was to evaluate these recombinant proteins as innovative biomarkers for the detection of apoptosis and as therapeutic complexes for the selective destruction of urothelial cancer cells.
EryA is characterized by its specific affinity for cardiolipin (CL), standing out as the only known aegerolysin that does not bind to sphingomyelin. Using surface plasmon resonance, we demonstrated that the EryA-mCherry fusion protein binds specifically and irreversibly to CL in artificial membrane systems. Fluorescence microscopy revealed that EryA-mCherry labels CL on the plasma membrane and membrane blebs of apoptotic mammalian epithelial MDCK and RT4 cells. Thereby, it significantly overcame the limitations of annexin V-FITC, a standard apoptosis marker that proved to be non-specific in our study. Thus, we demonstrated that EryA-mCherry represents a highly specific and irreversible novel marker for apoptosis.
Furthermore, we investigated the diagnostic potential of the fluorescently labelled proteins OlyA6, which has an affinity for sphingomyelin/cholesterol lipid domains, and the E69A mutant, which has an affinity for both cholesterol-bound and free sphingomyelin, in urothelial cancer cell models. We found that the proteins selectively bind to the normal immortalized urothelial cell line (SV-HUC-1) and urothelial cancer cell lines (RT4, T24), while they do not bind to normal primary porcine urothelial (NPU) cells. These differences in binding directly coincide with the presence of uroplakins; the reduced or completely absent expression of uroplakins in cancer and normal cell lines allows aegerolysins access to target lipid domains on the membrane. In addition to structural accessibility, aegerolysin binding is significantly influenced by lipid composition of the plasma membrane, as cancer cells have an increased content of cholesterol and membrane rafts. Due to the introduced mutation, the E69A mutant exhibits a significantly higher affinity for membrane rafts, enabling its stronger binding to cells lacking uroplakins.
The therapeutic potential of cytolytic complexes of aegerolysins and MACPF domain-containing protein partners was evaluated by analysing their cytotoxic activity. The OlyA6/PlyB and E69A/PlyB complexes were highly effective in the selective elimination of cancer cells, while having no effect on normal NPU cells. The OlyA6/PlyB complex proved optimal for targeting non-invasive RT4 cancer cells, whereas the E69A/PlyB complex was more effective in eliminating invasive T24 cancer cells. Comparison of the MACPF domain-containing protein partners confirmed a distinct advantage of using the PlyB protein in cytolytic complexes compared to the PulB protein, as aegerolysin complexes with the PulB protein partner were less effective across all tested cell lines at equal concentrations. Multiparametric testing confirmed that the mechanism of cell death induced by the complexes is necrosis, occurring due to the formation of transmembrane pores, disruption of membrane potential, and loss of osmotic balance.
The obtained results demonstrate that fluorescently labelled aegerolysins represent highly selective diagnostic tools for the detection of apoptosis (EryA-mCherry) and the recognition of urothelial cancer cells (OlyA6-mCherry and E69A-mCherry). Furthermore, the combination of recombinant aegerolysins OlyA6 or E69A with the pore-forming partner PlyB, due to the specific recognition of membrane lipid composition, enables the necrotic lysis of urothelial cancer cells, thereby presenting a promising and innovative strategy for the targeted destruction of urothelial cancer cells.
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