The 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 60-80 nm 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 cryo-fixation 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 (~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 was 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 remodelling, which may also be the mechanism for release of extracellular vesicles.
Bibliographical noteNARG thanks The Wellcome Trust (080088, 086827, 075470, 099215 & 097377) and MRC Centre for Medical Mycology (MR/N006364/1) and acknowledges financial support from Gilead Sciences for a studentship and grant IX-EU-131-0262. Dr. Linda Soo Hoo and Tark Bunch of Gilead provided expert technical assistance in liposomal sample preparations and GF provided gold labelled test articles. JAM is funded in part from a research grant from Gilead Sciences Inc. ML was supported by the MRC (MR/J008230/1). AC was supported in part by 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733. We thank Debbie Wilkinson and Kevin McKenzie at the Imaging Core Facility at the University of Aberdeen for expert assistance with TEM.
- amphotericin B
- antifungal drug
- cell membrane
- fungal cell wall