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Eukaryotic Cell, September 2009, p. 1373-1380, Vol. 8, No. 9
1535-9778/09/$08.00+0     doi:10.1128/EC.00044-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Lipophilic Dye Staining of Cryptococcus neoformans Extracellular Vesicles and Capsule{triangledown}

André Moraes Nicola,1,2 Susana Frases,1 and Arturo Casadevall1*

Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York,1 University of Brasília, Brasília, Brazil2

Received 5 February 2009/ Accepted 15 May 2009

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 RNA-selective 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.

* Corresponding author. Mailing address: 1300 Morris Park Avenue, Forchheimer Building, Room 411, Bronx, NY 10461. Phone: (718) 430-3766. Fax: (718) 430-8701. E-mail: casadeva{at}aecom.yu.edu

{triangledown} Published ahead of print on 22 May 2009.

Eukaryotic Cell, September 2009, p. 1373-1380, Vol. 8, No. 9
1535-9778/09/$08.00+0     doi:10.1128/EC.00044-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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