Fine Structure of Cryptococcus neoformans Grown In Vivo as Observed by Freeze-Etching

Abstract

Cryptococcus neoformans grown in the parasitic state was observed by the freeze-etching technique and was compared with that grown on culture media. Unlike other yeasts, this organism grown in vivo is very often devoid of the "ordinary" invaginations. The membrane of the cell grown in vivo was almost free from concavity and convexity except for many round depressions which represent the surface view of paramural bodies. Some of the paramural bodies were found to be multivesicular systems. Most were spherical invaginations containing a single vesicle or its ghost remaining after secretion of the vesicles. In clear contrast to the cell grown in vitro, the in vivo cell contained a great number of vesicles in the cytoplasm. These seemed to show high-secretion activity in C. neoformans grown in the parasitic state. On transfer from in vitro to in vivo, this organism enlarged the cell wall, capsule, and cell body. The appearance of a large vacuole, accumulation of storage organelles, and the existence of rodlike structures, seemingly lipid deposits, were also noted in the cytoplasm of the cell grown in vivo. The meaning of these results as well as the mode of capsular production are discussed.

A Eukaryotic Capsular Polysaccharide Is Synthesized Intracellularly and Secreted via Exocytosis

“…Early studies in C. neoformans suggested either intracellular or cell surface synthesis of capsule, but settling this question has remained elusive (Edwards et al, 1967;Takeo et al, 1973aTakeo et al, , 1973bSakaguchi et al, 1993;Feldmesser et al, 2001;Garcia-Rivera et al, 2004). The major conclusion from our study is that GXM is trafficked within secretory vesicles, supporting a model that capsular materials are synthesized in the Golgi and targeted to the plasma membrane for exocytosis.…”

“…The primary GXM-containing structures we observed were secretory vesicles. In early ultrastructural studies, Takeo et al (Takeo et al, 1973a, 1973b hypothesized that 30 -70-nm intracellular vesicles and/or ϳ50-nm vesicles in paramural bodies were involved in capsule synthesis. The typical intracellular vesicle size we observed was 100 nm, although it ranged from 60 to 130 nm (Figures 6 and 8).…”

“…A location for capsule synthesis was first hypothesized in 1967, when Edwards et al (1967) observed a halo between the cell wall and the capsule using conventional transmission electron microscopy (TEM) and suggested it was a "zone of capsule synthesis." Several years later, Takeo et al (1973aTakeo et al ( , 1973b demonstrated that this halo corresponded to a layer of 20-nm "granules," which they speculated were involved in polymerization of capsule precursors. These investigators used a freeze-etching technique to compare C. neoformans grown in vitro to cells grown in a mouse model of infection, which have more extensive capsule.…”

Fine Structure of Cryptococcus neoformans Grown In Vitro as Observed by Freeze-Etching

“…Interestingly, this organism also shows multivesicular bodies in the cytoplasm. This fact, in connection with the multivesicular paramural bodies found in an in vivo study (11), suggests the presence of multivesicular lomasomes, said to be distinctly absent in yeasts (6).…”

“…The wall particle is not attributable to artifact shrinkage of the capsular materials to the outermost part of the cell wall, since they are found deep enough from the wall surface. Furthermore, particles of the same size are also found in the inner part of the capsule (11).…”