Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

Abstract

The three-dimensional ultrastructure of Cryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method. C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10(7) cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells of C. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure of C. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10-13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions by C. neoformans.

Antibody Interactions with the Capsule ofCryptococcus neoformans

“…This effect was most pronounced for MAb 2H1. The regional differences in MAb binding to the capsule may reflect structural differences in the architecture of the capsule as a function of distance from the cell wall or a higher epitope density in the inner capsule due to closer packing of polysaccharide fibrils near the cell wall (31). Alternatively, there may be a concentration gradient in polysaccharide molecules as a function of distance from the cell wall such that the capsule is less dense in the outer regions.…”

Effects of Voriconazole on Cryptococcus neoformans

“…Although the mechanism for this effect with voriconazole at 0.5 times the MIC is unknown, exposure of C. neoformans to subinhibitory concentrations of fluconazole has been shown to alter the organism's lipid profile (13). Given that capsule synthesis is associated with the accumulation of vesicles in the cell wall (34), it is possible that inhibition of ergosterol synthesis by voriconazole translates into membrane deficits that interfere with the vesicular trafficking required for capsule synthesis.…”

“…Whereas amphotericin B poorly penetrates into brain tissue (20) and fluconazole is generally fungistatic to C. neoformans, voriconazole penetrates into the cerebrospinal fluid (34) and can be cidal to C. neoformans. The present study shows that voriconazole is active against C. neoformans and that this effect is not affected by melanization provides another advantage of voriconazole compared to amphotericin B and caspofungin.…”

Human Antibodies against a Purified Glucosylceramide fromCryptococcus neoformansInhibit Cell Budding and Fungal Growth

“…This may happen due to the inability of GPI anchor cleavage by PI-PLC, a property of inositolacylated molecules found in C. neoformans (17), or to a more generalized process in which precursor molecules and enzymes are transferred to the cell wall in vesicles originating from the plasma membrane. Cell wall particles have been described in C. neoformans (47) presumably containing precursors of the capsule. Spherical invaginations which secrete vesicles outside the cell membrane have also been well documented by Takeo et al (53) in C. neoformans using the freeze-etching technique.…”