Abstract
ABSTRACTThe fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles.
Highlights
The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield
Sample preparation techniques for transmission electron microscopy (TEM), such as high-pressure freezing followed by freeze-substitution (HPF-FS), have enabled us to visualize unprecedented architectural details such as the structure of the Candida mannoprotein fibrils [6, 7] and Cryptococcus capsule [1, 8] and the presence of membrane vesicles within the cell wall matrix [9, 10]
Several studies indicate that the outer mannoprotein layer of yeast determines wall porosity, and porosity assays based on measurement of cell lysis due to DEAE-dextran, poly-L-lysine, and glucanase suggest that molecules with a hydrodynamic radius up to 5.8 nm, equivalent to a molecular radius (Mr) of 400,000 Da, are able to permeate the wall [12, 13]
Summary
The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We report that the AmBisome liposome transits through the cell walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical cell wall porosity This implies that the cell wall has deformable, viscoelastic properties that are permissive to transwall vesicular traffic. Sample preparation techniques for transmission electron microscopy (TEM), such as high-pressure freezing followed by freeze-substitution (HPF-FS), have enabled us to visualize unprecedented architectural details such as the structure of the Candida mannoprotein fibrils [6, 7] and Cryptococcus capsule [1, 8] and the presence of membrane vesicles within the cell wall matrix [9, 10] The presence of such vesicles begs an explanation as to how such large vesicles transit from the membrane through the wall to the external fluid around a cell. It is known that the secretome, which contains many large glycoproteins such as invertase and other enzymes (up to 200,000 Da), is able to pass through the yeast cell wall [14]
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