Lipophilic Dye Staining ofCryptococcus neoformansExtracellular Vesicles and Capsule

Abstract

Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNAselective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components.

Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

“…Also of note is the high proportion of ribosomal and translation-related proteins, making up 20 to 30% of each sample. These, in conjunction with potential RNA cargo (30), are suggestive of a mechanism to influence host cell translation, particularly in the case of fungal cell internalization. Fungal small interfering RNA (siRNA) has recently been shown to interact with host cell RNA interference pathways (31), setting a precedent for involvement between molecular machinery of host and fungus cells.…”

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

“…Vesicle samples were stained with the lipophilic fluorophore dialkylcarbocyanine iodide (DiI) (catalog no. V22885; Invitrogen) as described previously (55). Briefly, vesicle suspensions were normalized to a sterol concentration corresponding to 2 g/ml and supplemented with a DiI solution (final concentration, 3 M).…”

Vesicle-associated melanization in Cryptococcus neoformans

“…Purified C. neoformans melanin ghosts were stained with the lipophilic probe DiI, which was used to stain C. neoformans extracellular vesicles (Nicola et al, 2009). Since the melanin purification protocol includes two extractions with chloroform, we also stained a different aliquot of the same melanin preparation that had not been subjected to the extraction steps.…”