Nystatin belongs to a group of polyene macrolide antimicotics. Although it has a wide spectrum of activity towards pathogenic fungi, similar to amphotericin B, it is used mainly topically because of its nephrotoxicity when applied systemically. The biological target for polyene action is the plasma membrane of sensitive cells, where they can form membranespanning channels, permeable to different ions and even smaller molecules, resulting in the disturbance of the cellular electrochemical gradients, and ultimately cell death. The fungal cells whose principal membrane sterol is ergosterol are much more susceptible to polyenes than cholesterol-containing human cells, which is the basis of their clinical use. The consensus regarding the mechanism of the polyene membrane activity, especially the role of sterols, has not been reached yet. Therefore, the principal intention of the doctoral research has been to bring new insights about the mode of action of nystatin at the membrane level, which could improve the efficiency of its use in different infections as well as reduce its toxicity to human cells. In addition, the knowledge about the mechanism of the nystatin poreforming activity is essential for the development of the regulated delivery of drugs into cells. Firstly, we studied the membrane activity of nystatin in vesicles with dimensions comparable to the size of an average human cell (giant unilamellar vesicles – GUVs), which were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using the electroformation method. For this purpose GUVs filled with a sucrose solution were transferred using the micromanipulation system and micropipettes into an isomolar glucose solution with nystatin concentrations of 10 umol/L to 900 umol/L, and observed using the phase contrast microscopy. This novel methodological approach enabled us to study the osmotic phenomena and morphological responses of individual GUVs exposed to nystatin.
Three characteristic concentration-dependent responses of sterol-free GUVs were detected experimentally in three different, partially overlapping, nystatin concentration ranges. At low nystatin concentrations (150–250 umol/L) vesicle shape changes and various membrane formations with distinct temporal shape and length evolution patterns were observed. These patterns can be explained by the changes in the equilibrium leaflet-area difference (dA0), thereby revealing the steps of the nystatin membrane partition. The characteristic feature for the medium nystatin concentrations (250–400 umol/L) were transient tension pores associated with vesicle fading, which was intensified with increasing nystatin concentrations. At the highest nystatin concentrations (> 400 umol/L), two basic types of vesicle ruptures were encountered on the basis of the intensity of the osmotic process, i.e., the slow and fast ruptures (explosions). The occurrence of tension pores was attributed to osmotic processes leading to an increase of the lateral membrane tension as a consequence of the formaton of the transmembrane nystatin channels being more permeable to glucose compared to sucrose. As the surface density of nystatin channels increases, the tension pores become more durable (slow ruptures) and, eventually, explosions occur. Using a theoretical model, the dependency of the surface density of channels to the nystatin concentration was established and successfully correlated to different vesicle behavior. Therefore, we demonstrated that nystatin is able to form pores in the absence of sterols, which was debated untill now. Afterwards, we conducted experiments with GUVs containing cholesterol and ergosterol in different molar fractions. POPC-cholesterol GUVs namely enable the study of the toxic effects of nystatin on human cells, while POPC-ergosterol GUVs serve as model membranes for microbial fungal and trypanosomal kinetoplastid pathogens. It was shown that the nystatin partition and pore-forming activity are significantly increased in the ergosterol-containing membranes. This was demonstrated by the shift of the characteristic vesicle responses towards lower nystatin concentrations, while the basic concentrationdependent succession of phenomena was conserved. Furthermore, for POPC-ergosterol GUVs the long-lasting tension pores were characteristic and might represent an important mechanism also in in vivo conditions. In contrast, the addition of cholesterol into membranes in the molar fraction typical fot mammal cells (45 mol%) was found to be inhibitory for the nystatin action. It was also demonstrated that the nystatin activity is in a complex, nonlinear relation to the ergosterol membrane’s molar fraction. The comparison of the results to those of other studies in the field revealed that this could be the first confirmation of the existence of previously mathematically predicted sterol superlattices in the case of cell-sized vesicles. The theoretical modelling has shown that the sterol-induced changes of the osmotic phenomena
could not be explained adequately on the basis of the altered membarne mechanical characteristics, and were therefore interpreted mainly by the direct influences of the sterols on the membrane partition and the channel-formation process of nystatin. The basic toxicological research of the nystatin effects on the POPC-cholesterol GUVs were complemented by the study of the responses of Chinese hamster ovary (CHO) epithelial cells to the pore-forming agent nystatin, which were investigated using phase-contrast and fluorescence microscopy. As in the case of GUVs, the pore-forming activity of nystatin induced the osmotic processes resulting in the cell swelling and in tension pores. However, certain responses of CHO cells were different compared to GUVs. Firstly, no membrane protrusions were observed at lower nystatin concentration, which can be explained by the flattening of the invaginated membrane structures, such as caveolae, thereby compensating the increase of the surface of the outer cell membrane monolayer as a result of nystatin insertion. With increasing nystatin concentrations, the formation of spherically shaped blebs, gradually growing and coalescing into “cell-vesicles”, and finally cell ruptures, were observed. These osmotic phenomena in cells were compared to those in GUVs and interpreted using an extended theoretical model. By using the cell viability studies and the demonstration of the GFP-tagged actin cytoskleton degradation we were able to link them to the process of cell death.
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