EUKARYOTIC CELL, Sept. 2009, p. 1373–1380 Vol. 8, No. 91535-9778/09/$08.00�0 doi:10.1128/EC.00044-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Lipophilic Dye Staining of Cryptococcus neoformans ExtracellularVesicles and Capsule�

Andre Moraes Nicola,1,2 Susana Frases,1 and Arturo Casadevall1*Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York,1 and University of Brasılia, Brasılia, Brazil2

Received 5 February 2009/Accepted 15 May 2009

Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressedindividuals. Recent studies have determined that this fungus produces vesicles that are released to theextracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes severalmolecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysac-charides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied ontransmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescentmembrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Twodialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles byfluorescence 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 dyesto stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. Afluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctatestructures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes providenew tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regionsin the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed ofpolysaccharide and nonpolysaccharide components.

Cryptococcus neoformans is an important cause of life-threat-ening systemic mycosis (5). It is believed that the fungus is ac-quired by inhalation and causes mild respiratory symptoms beforeestablishing a dormant state. In individuals with immune deficien-cies, such as seen with AIDS or cancer chemotherapy, latentinfections can reactivate and disseminate (5). This unicellularyeast is distinctive among other eukaryotic pathogens because it iscoated with a polysaccharide capsule, composed primarily by glu-curonoxylomannan (GXM), with galactoxylomannan and manno-proteins (3) as minor components. The capsule is considered itsmost important virulence attribute because it confers upon theyeast cell both defensive and offensive attributes in its interactionwith mammalian hosts. The capsule provides resistance to phago-cytosis and to phagocyte fungicidal reactive oxygen species (3).Capsular polysaccharides are also shed into host tissues, wherethey mediate a variety of immunomodulatory effects that un-dermine the capacity of the host to fight infection (10). Inaddition to the capsule, other major C. neoformans virulenceattributes include its ability to synthesize melanin, a cell wallpigment that augments resistance to oxidants and to antifun-gals, and several secreted enzymes, such as urease (9) andphospholipases (6, 8, 23).

GXM is synthesized inside the cell and subsequently ex-ported to the capsule (11, 12, 26). Because GXM fibers canhave molecular weights of more than a million (14), theirpassage through the cell wall, which is required for capsuleassembly, could present a formidable transport problem. Ro-drigues et al. recently proposed that trans-cell wall polysaccha-

ride export occurs by an extracellular vesicular system (19).These extracellular vesicles are formed in cytoplasmic multive-sicular bodies and cross the cell wall into the surroundingenvironment, where they presumably open to deliver theircontents (19). Vesicles purified from in vitro culture superna-tants contained GXM that could be recognized by specificantibodies and formed a capsule around acapsular mutants(19). These vesicles vary in size, some being up to 200 nm indiameter, and are heterogeneous in ultrastructural morphol-ogy, a hint that there might be different types of vesicles fordifferent types of cargo (18). In fact, further studies detectedlaccase, urease, and acid phosphatase enzymatic activities inthese vesicles, which along with detailed proteomic analysesdemonstrated that they carry a large number of proteins in-volved in virulence and form “virulence factor delivery bags”(18). Biochemical studies of vesicular composition revealedglucosylceramide, ergosterol, and phospholipids such as phos-phatidylcholine (PC), phosphatidylserine, and phosphatidyl-ethanolamine (1, 19). Genetic evidence for different vesiculartransport systems comes from the observation that C. neofor-mans sec6 mutants have defective extracellular laccase trans-port, despite having intact capsules (16).

The discovery that these vesicles are involved in the trans-port of several important virulence-associated components hasled to a surge in interest in their study. Extracellular vesicleshave been detected in the culture supernatants of Histoplasmacapsulatum, Candida albicans, Candida parapsilosis, Sporothrixschenckii, and Saccharomyces cerevisiae (1). Current studies offungal vesicles are hindered by the difficulties inherent to ob-servation of such small structures, which is possible only byusing time-intensive electron microscopy methods. We rea-soned that assays based on fluorescence, such as microscopy

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

� Published ahead of print on 22 May 2009.

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and flow cytometry, might be able to overcome this limitationand allow faster and more versatile observation of fungal ex-tracellular vesicles and their cargo. In this work we report theuse of fluorescent probes to visualize the extracellular vesiclesproduced by C. neoformans and provide insights about theircellular location and content.

MATERIALS AND METHODS

Fungal strains and media. C. neoformans isolates H99 (serotype A), 24067(serotype D), B3501 (serotype D), and CAP67 (a B3501-derived acapsular mu-tant) were used in this study. The cells were grown in either Sabouraud broth orminimal medium (15 mM glucose, 29 mM KH2PO4, 10 mM MgSO4, 13 mMglycine, and 3 �M thiamine [pH 5.5]). Capsule growth was induced by incubatingC. neoformans cells in 1:10 Sabouraud broth-MOPS (morpholinepropanesulfonicacid), pH 7.3, for 2 days at 37°C (27).

Probes. DiI (1,1�-dioctadecyl-3,3,3�,3�-tetramethylindocarbocyanine per-chlorate; Vybrant DiI cell labeling solution), DiD-DS (1,1�-dioctadecyl-3,3,3�,3�-tetramethylindodicarbocyanine-5,5�-disulfonic acid), BODIPY-PC[2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine], DiIC1(5) (1,1�,3,3,3�,3�-hexamethylin-dodicarbocyanine iodide), and SYTO RNASelect were purchased from Invitro-gen. Uvitex 2B, a probe that detects chitin in the fungal cell wall, was purchasedfrom Polysciences Inc., and AF488-18B7 was prepared by labeling the anti-GXMimmunoglobulin G1 18B7 with Alexa Fluor 488, according to the manufacturer’s(Invitrogen) instructions. Structures of the membrane probes used in this study areshown in Fig. 1.

Vesicle purification and staining. Extracellular vesicles were purified by themethod described before (19) with slight modifications. In brief, C. neoformanscells were removed from cultures in minimal medium (3 to 6 days, 30°C withshaking at 150 rpm) by centrifugation and filtration with a 0.4-�m filter, and thecell-free supernatant was concentrated by ultrafiltration on 100-kDa membranes.Vesicles were separated from this concentrate by ultracentrifugation at 100,000 � gfor 1 h. The vesicles were then washed twice with phosphate-buffered saline(PBS). The purified vesicles were then stained for 1 h in PBS containing anappropriate dye [5 �M DiI, 5 �M DiD-DS, 5 �M DiIC1(5), or 1 �M SYTORNASelect] and washed twice with PBS by centrifugation (1 h at 100,000 � g).The dye-stained pellet was then photographed with a Nikon D90 digital cameraand suspended in PBS for flow cytometry or in mounting medium (0.1 M propylgallate in PBS plus 50% glycerol) for fluorescence microscopy. A commercialpreparation of liposomes containing PC and ergosterol (Encapsula Nano Sci-ences) was used as a negative control.

Flow cytometry and microscopy. Dye-stained vesicles were analyzed with a BDFACSAria cell sorter equipped with four lasers emitting at 407, 488, 561, and 635nm. Data were analyzed offline with FlowJo software (www.treestar.com/flowjo).

Stained vesicles in mounting medium were imaged by epifluorescence micros-copy on a Zeiss Axioskop 200 inverted microscope equipped with a cooledcharge-coupled device using a 63�, 1.4-numerical-aperture (NA) objective. Im-ages were collected and deconvolved using a constrained iterative algorithm withAxioVision (Zeiss) software. Alternatively, a PerkinElmer UltraVIEW RS-3spinning disk laser confocal microscope equipped with a cooled charge-coupleddevice camera and a 100�, 1.4-NA objective was used. For experiments withwhole cells, C. neoformans cultures in Sabouraud broth were collected, washed,resuspended in PBS, and stained with the appropriated probes [5 �M DiI, 5 �MDiIC1(5), 100 �g ml�1 Uvitex 2B, 10 �g ml�1 AF488-18B7, 1 �g ml�1 BODIPY-PC] for 1 h. Spheroplasts were generated by overnight digestion with 40 mg ml�1

Trichoderma harzianum lysing enzymes (Sigma) in 1 M sorbitol and 0.1 M sodiumcitrate, pH 5.5, buffer and stained with 5 �M DiI. After washing and resuspen-sion in mounting medium, the cells were imaged on the Zeiss microscope de-scribed above or on a Leica SP2 laser scanning confocal microscope equippedwith a 63�, 1.4-NA objective. Deconvolved and confocal Z stacks were analyzedwith AxioVision or ImageJ (http://rsbweb.nih.gov/ij/) and tridimensionally recon-structed using VOXX (http://www.nephrology.iupui.edu/imaging/voxx/) soft-ware. Postcollection manipulation of the images involved only linear brightnessand contrast adjustments, which are indicated in the appropriate figure legends.

Delipidation of whole cells. Lipids were extracted from intact H99 cells grownin Sabouraud broth by sequential incubation in organic solvents. The cells werecollected, washed, and resuspended in PBS. Methanol and chloroform wereadded to a final proportion of 0.8:2:1 water-methanol-chloroform, followed by2:1 methanol-chloroform, pure chloroform, and pure methanol. Each extractionwas carried out with rotation at 37°C from 20 min to 2 h. The cells were thenwashed twice in PBS and stained and imaged by laser scanning confocal micros-copy as described above. Flow cytometric analysis was carried out in a BD LSRIIflow cytometer equipped with 488- and 633-nm lasers.

Purification and staining of capsular polysaccharide. Supernatant GXM waspurified from H99 culture supernatants by precipitation with cetyltrimethylam-monium bromide (CTAB) (7). Capsular polysaccharide was stripped from H99cells by treatment with dimethyl sulfoxide (DMSO) (4). To remove lipids, 1 mgof the DMSO-purified polysaccharide was diluted in PBS and extracted withethyl acetate at room temperature for 1 h with vortexing. All polysaccharidesamples were diluted in PBS at 1 mg/ml and centrifuged for 30 min at 20,000 � g toremove particulate matter. One milliliter of each solution and a control contain-ing only PBS were then stained with 5 �M DiI for 1 h at 37°C and centrifugedagain at 20,000 � g for 30 min.

RESULTS

Staining of purified vesicles. To assess the usefulness ofdialkylcarbocyanine dyes as probes for extracellular vesicles,we stained vesicular preparations with either DiI or DiD-DS

FIG. 1. Structure of the four membrane probes used in this study.

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and studied them by fluorescence microscopy. Purified vesiclesin buffer suspension stained brightly enough with the probes tobe readily observed by confocal microscopy as bright dots on adark background (Fig. 2A). Both probes are also useful forstaining the vesicles for flow cytometry (Fig. 2B). Althoughboth DiI and DiD-DS stained the vesicles, DiI staining wasbrighter than DID-DS staining, which resulted in better sepa-

ration between unstained and stained vesicles by flow cytom-etry. As controls, we also incubated the dialkylcarbocyaninedyes in buffer alone and stained purified vesicles with DiIC1(5),a dye similar to DiD-DS but without the two alkyl tails. Asexpected, no dye precipitation was observed in both cases,confirming that the probes are indeed staining purified vesiclesbased on their affinity for lipidic bilayers.

FIG. 2. Staining of purified vesicles with dialkylcarbocyanines. (A) Vesicles purified from C. neoformans supernatants after staining with DiI orDiD-DS and observation by confocal microscopy, showing small brightly fluorescent spots. Arrows indicate vesicles that are magnified in the insets. Theimage was adjusted after collection for brightness and contrast to enhance clarity. Bars, 5 �m (main parts of images) and 400 nm (insets). (B) Similarlystained vesicles were analyzed by flow cytometry, showing that individual vesicles can be observed, quantified, and analyzed. (C) Purified vesicles that werestained with DiD-DS and concentrated by centrifugation, resulting in visible staining of the 100,000 � g pellet (arrow). No staining of the pellet wasobserved with DiIC1(5), and no pellet was visible when DiD-DS in solution was centrifuged in the absence of vesicles.

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Having determined that these probes were useful for flowcytometry, we devised an experiment to explore the utility ofthe technique. As mammalian exosomes have been show tocontain RNA (25), we analyzed vesicles that were costainedwith DiI and the membrane-permeable RNA probe SYTORNASelect. The results revealed a subset of events thatstained positive with both dyes (Fig. 3A). To discard the pos-sibility of nonspecific binding, we used the same pair of dyes tostain commercial liposomes, which stained with DiI only. Un-stained vesicles from the same preparation were negative inboth channels. Formation of micelles, vesicles, or aggregatesby the probes themselves in solution was ruled out with acontrol consisting of DiI and SYTO RNASelect dissolved inPBS only (data not shown).

Staining of C. neoformans cells. Having determined that thedialkylcarbocyanine dyes DiI and DiD-DS stained vesicles, weattempted to visualize and localize them in C. neoformans cellsusing fluorescence microscopy. Staining of intact or capsule-induced cells with DiI or DiD-DS revealed punctate structureslocated mostly on the outer edge of the capsule in encapsu-lated strains and on the surface of the cell wall in acapsularmutants (Fig. 4A and B). Some of these structures stained verybrightly, while others were much dimmer. Another stainingpattern, consisting of a uniform layer on top of the cell wall andstalklike attachments to the cell body (Fig. 4C), was observedmuch less frequently. No staining for the cytoplasmic mem-brane or any intracellular membrane structure was observed.Intracellular membranes in C. neoformans spheroplasts werelabeled (Fig. 4D), indicating that the probes were unable topenetrate the cell wall.

To test the stability of these hydrophobic regions, we ex-tracted the cells with methanol and chloroform and repeatedthe staining. Surprisingly, organic solvent extraction resulted incells that still stained brightly with DiI (Fig. 5A). Flow cytom-etry was used to quantify the amount of probe bound to eachcell, revealing that prior extraction with solvents increasedbinding of both dialkylcarbocyanine probes to the cells (Fig.5B). Staining of intact cells with DiIC1(5) also revealed similardots, confirming that in this case the binding was not depen-dent on the dialkylcarbocyanines’ affinity for lipid bilayers. Incontrast to DiI and DiD-DS, DiIC1(5) readily penetrated thecell and stained intracellular structures much more brightlythan the extracellular dots (Fig. 5D). To test whether the probewas binding directly to capsular polysaccharide, we incubateddifferent preparations of purified GXM with DiI, which re-sulted in formation of a stained precipitate (Fig. 5C). Flores-cence microscopic examination of the DiI-GXM precipitaterevealed large brightly fluorescent aggregates (data notshown).

FIG. 3. A population of vesicles stains with a probe for RNA.(A) Purified vesicles were simultaneously stained with DiI and theRNA-selective nucleic acid stain SYTO RNASelect. Double-positiveevents corresponding to RNA-loaded vesicles can be observed in theupper right quadrant. (B) Commercial liposomes made with PC andergosterol were stained using the same protocol; as expected, theystained only with DiI. (C) Control with unstained vesicles. All panelsare density plots, in which red-shifted colors code for increased densityof cells in an area.

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Unlike the experience with purified vesicles, the dialkylcar-bocyanines appeared not to be good probes to study extracel-lular vesicles in intact cells because of their propensity to staincapsule structures in delipidated cells. With this caveat inmind, we tested a second membrane tracer, the fluorescentphospholipid BODIPY-PC. The staining with BODIPY-PC

FIG. 5. Staining of delipidated cells and polysaccharide. C. neoformanscells were extracted with methanol and chloroform to remove lipids and thenstained with both DiI and DiD-DS. (A) Extracted and nonextracted cellswere stained with DiD-DS and imaged under the same conditions by laserscanning confocal microscopy. Extraction did not abrogate the binding of thedye. Staining with DiI produced the same result (not shown). Bar, 5 �m.(B) Native and extracted cells stained with DiI and DiD-DS and then ana-lyzed by flow cytometry to measure the amount of dye incorporated per cell.Bars represent the median fluorescence intensity of approximately 30,000cells in each condition. (C) Purified capsular polysaccharides were stainedwith DiI and then centrifuged. From left to right, the photograph shows thatthe dye does not precipitate by itself but forms aggregates with GXM purifiedfrom culture supernatants (CTAB-GXM) and from the capsule (DMSO-GXM), even after delipidation of polysaccharide samples with chloroform.(D) C. neoformans cells were stained with DiIC1(5), which resulted in brightstaining of intracellular structures and dim staining of extracellular dots. Thepanel on the right corresponds to the image on the left after linear brightnessand contrast adjustment to highlight the dimmer extracellular dots (arrow).

FIG. 4. Staining of whole cells with DiI. (A) Encapsulated C. neo-formans cells (B3501 isolate, capsule induced) were stained with DiI,Uvitex 2B, and the anticapsular antibody 18B7 conjugated to AlexaFluor 488. Fluorescence images are Z projections of deconvolvedstacks, which were also reconstructed in three dimensions. DiI-positivespots can be observed at the edge of the capsule. The images wereadjusted linearly for brightness and contrast. (B) Acapsular CAP67mutants that were stained with DiI and observed in a laser scanningconfocal microscope. The DiI-positive spots are directly on top of thecell wall. (C) Encapsulated isolate H99 was stained with DiD-DS andUvitex 2B and observed in a laser scanning confocal microscope. Alayer on top of the cell wall was uniformly labeled, as well as a pro-jection (arrow). (D) C. neoformans spheroplasts stained with DiI andimaged by epifluorescence microscopy followed by deconvolution. Thecells, which lost their shape and shrank due to the disruption of the cellwall, are stained. Bars (all panels), 5 �m.

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also revealed punctate structures on the outer edge of thecapsule, albeit with much lower brightness and with a lowerfrequency (Fig. 6A). Double staining with DiI (Fig. 6B) re-vealed that some DiI-positive structures also stained withBODIPY-PC but that the majority of the spots did not colo-calize. In contrast to DiI, BODIPY-PC readily penetrated thecell and stained intracellular membranes.

DISCUSSION

Dialkylcarbocyanines are amphiphilic molecules composedby a polar fluorescent head linked to two alkyl tails. The hy-drophobic alkyl chains insert into the lipophilic core of phos-pholipid bilayers, whereas the positively charged fluorophorerests close to the polar head groups, parallel to the membranesurface (2, 13). This binding mechanism makes them excellentprobes for lipidic membranes, a property that made dialkyl-carbocyanines attractive as fluorescent probes to detect extra-cellular vesicles.

Our results established that purified vesicles were efficientlylabeled with two different probes from this family: DiI, whichhas excitation (Ex) and emission (Em) maxima similar to thoseof rhodamine (Ex/Em, 549/565 nm) and the sulfonated

DiD-DS (Ex/Em, 644/665 nm). Epifluorescence and confocalmicroscopy was used to image the stained vesicles as brightdots. The fact that solid dots were observed instead of the ringthat would be expected from a vesicular structure can be ex-plained by the fact that the diameter of the vesicles is close toor even lower than the maximum theoretical resolutionachieved by optical microscopes.

The dyes are also suitable for staining vesicles for flow cy-tometry, a technique that permits fast quantitative analysis of alarge number of particles with different fluorophores. To testthe validity of this method, we costained purified extracellularvesicles with DiI and an RNA-selective nucleic acid probe,revealing a subpopulation of vesicles that may be associatedwith RNA. This finding could be explained by the presence ofrRNA, as ribosomal proteins have been detected in vesicleproteomics studies (18); alternatively, it could be an indicationthat these vesicles might carry mRNAs and microRNAs, re-sembling recently described exosomal shuttle RNAs, which areinvolved in mammalian cell-to-cell transmission of mRNAsand regulatory microRNAs (25).

In addition to staining purified vesicles, we attempted toprobe for lipophilic structures in the capsule of C. neoformansusing dialkylcarbocyanines. Although the capsule was previ-ously considered to be composed solely of polysaccharide, re-cent evidence indicates that it contains chitinlike material (17)and osmiophilic protuberances containing 3-OH oxylipins (21,22). The latter structures appear as distinct globules and pro-tuberances on the capsule of C. neoformans when stained withantibodies to 3-OH oxylipins. DiI staining revealed a variablenumber of discrete punctate structures, located mostly at thevery edge of the capsule on encapsulated strains or right on topof the cell wall in acapsular strains. A control experiment,however, revealed that both DiI and DiD-DS bound to thecapsule of cells that had been sequentially extracted with or-ganic solvents, which theoretically should be enough to strip offlipid membranes. We considered various explanations for theresistance of these lipophilic structures to organic solvents.One explanation is direct binding of the probes to the capsularpolysaccharide, forming aggregates via interaction between thepositively charged nitrogen atom at the head of the dialkylcar-bocyanines and negatively charged glucuronic acid residues incapsular polysaccharide. Consistent with this possibility is thefinding that staining intact cells with DiIC1(5) results in similardots on the edge of the capsule, as well as the aggregatesformed by purified GXM and DiI, even after the polysaccha-ride was delipidated by extraction with ethyl acetate. Giventhat these dyes are amphipathic, they may function like CTABin precipitating soluble GXM. Alternatively, the dye may bestaining the same type of osmiophilic protuberances in thecapsule, described as containing 3-OH oxylipins, which may beresistant to the organic solvents used. Consistent with thisinterpretation was the observation that some lipophilic struc-tures had stalklike attachments reminiscent of the protuber-ances described earlier (21, 22).

Because the binding of DiI to the capsule hindered its use-fulness for observing vesicles in intact cells, we used a secondprobe for lipid bilayers. This probe, a PC molecule labeled inone of its alkyl tails with a green fluorescent hydrophobicfluorophore, labeled similar punctate structures in the edge ofthe capsule. Double staining with DiI revealed some DiI-pos-

FIG. 6. Double staining of intact cells with DiI and fluorescent PC.(A) C. neoformans cells that were stained with a green fluorescentphospholipid probe, which resulted in a punctate staining on the edgeof the capsule. (B) C. neoformans cells (B3501 isolate, capsule in-duced) were stained simultaneously with BODIPY-PC and DiI. Thedouble staining reveals limited colocalization of PC and DiI. Images inboth panels were linearly adjusted for brightness and contrast.

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itive spots that were also stained with fluorescent PC, butwithout complete colocalization. The location of the stainedstructures matches previous ultrastructural experiments, whichhave occasionally located vesicles (19) and 3-OH oxylipin glob-ules (21, 22) near the edge of the capsule in encapsulated C.neoformans cells. The punctate pattern we observed withBODIPY-PC also resembles the one reported after stainingwith human antibodies specific for 3-OH oxylipins (21, 22).Although these results do not allow us to conclusively link thepunctate BODIPY-PC-stained structures with either vesiclesor lipid-containing protuberances (21, 22), the results are con-sistent with both possibilities and indicate the existence oflipophilic regions in the capsule.

There were significant differences in the staining of C. neo-formans with DiI, DiD-DS, and BODIPY-PC. Both DiI andDiD-DS were useful for studying purified vesicle preparations.However, when used to study whole cells, they bound to cap-sular polysaccharides and cell wall structures. In contrast,BODIPY-PC appears to be the most specific for cell-associ-ated lipids, with the caveat that its staining is not as bright orstable as that of the other dyes. Hence, dialkylcarbocyaninedyes are more useful with purified vesicle preparations whileBODIPY-PC is more suitable for studying whole cells. Stainingof the cell wall with the dialkylcarbocyanines, but not withBODIPY-PC, was observed occasionally. Rodrigues et al. (20)reported the presence of glucosylceramide in the cryptococcalwall, which could provide the stained sites we observed. Li-pophilic dye staining of the cell wall is both consistent with andsupportive of the existence of lipids in the cell wall. The findingof lipids in the cell wall is relevant to models by which glyco-sylphosphatidylinositol-containing cell wall proteins are an-chored in cell wall structures, with the important caveat thatthese dyes could also bind to cell wall polysaccharides given theaffinity of DiI for isolated GXM.

Extracellular vesicles are increasingly recognized as a newsecretion system in fungi that is likely to have a critically im-portant role in fungal pathogenesis (15). In addition to theoriginal discovery in C. neoformans, extracellular vesicles havebeen found in Histoplasma capsulatum and several other asco-mycetes (1) and also in the parasite Trypanosoma cruzi (24),making them attractive for studies on pathogenesis and even-tual development of novel therapeutic tools. The finding ofhydrophobic regions on the polysaccharide capsule raises thequestion as to their physiological function and/or role in cap-sule structure. The lipophilic regions visualized with fluores-cent probes on the capsule edge bear a tantalizing resemblanceto vesicles and the lipophilic protuberances described by others(21, 22). Although the detailed answer to what these structuresare must await future studies, we lean toward the interpreta-tion that the lipophilic regions are related to the previouslydescribed 3-OH oxylipid-rich protuberances (21, 22) and/ortranscapsular vesicles (18, 19).

In summary, we report the use of two dialkylcarbocyaninesand a fluorescent phospholipid to study C. neoformans extra-cellular vesicles and cells. The results establish the feasibilityand versatility of fluorescent lipid probes for the study of fun-gal vesicles. They also suggest that nucleic acids could beadded to the list of possible vesicular cargo. The availability oflipophilic dyes that are suitable for light microscopy and flow

cytometry significantly expands the range of available method-ologies for vesicular and capsular studies.

ACKNOWLEDGMENTS

We thank Erin McClelland for the Alexa Fluor 488 18B7 antibodyand Peter Williamson and Marcio Rodrigues for helpful discussion ofthe data.

This work was supported by National Institutes of Health grantsAI33774, HL59842, AI33142, and AI52733 to A.C.

REFERENCES

1. Albuquerque, P. C., E. S. Nakayasu, M. L. Rodrigues, S. Frases, A. Casade-vall, R. M. Zancope-Oliveira, I. C. Almeida, and J. D. Nosanchuk. 2008.Vesicular transport in Histoplasma capsulatum: an effective mechanism fortrans-cell wall transfer of proteins and lipids in ascomycetes. Cell. Microbiol.10:1695–1710.

2. Axelrod, D. 1979. Carbocyanine dye orientation in red cell membrane stud-ied by microscopic fluorescence polarization. Biophys. J. 26:557–573.

3. Bose, I., A. J. Reese, J. J. Ory, G. Janbon, and T. L. Doering. 2003. A yeastunder cover: the capsule of Cryptococcus neoformans. Eukaryot. Cell 2:655–663.

4. Bryan, R. A., O. Zaragoza, T. Zhang, G. Ortiz, A. Casadevall, and E. Da-dachova. 2005. Radiological studies reveal radial differences in the architec-ture of the polysaccharide capsule of Cryptococcus neoformans. Eukaryot.Cell 4:465–475.

5. Casadevall, A., and J. R. Perfect. 1998. Cryptococcus neoformans. ASMPress, Washington, DC.

6. Chayakulkeeree, M., T. C. Sorrell, A. R. Siafakas, C. F. Wilson, N. Pantarat,K. J. Gerik, R. Boadle, and J. T. Djordjevic. 2008. Role and mechanism ofphosphatidylinositol-specific phospholipase C in survival and virulence ofCryptococcus neoformans. Mol. Microbiol. 69:809–826.

7. Cherniak, R., H. Valafar, L. C. Morris, and F. Valafar. 1998. Cryptococcusneoformans chemotyping by quantitative analysis of 1H nuclear magneticresonance spectra of glucuronoxylomannans with a computer-simulated ar-tificial neural network. Clin. Diagn. Lab. Immunol. 5:146–159.

8. Cox, G. M., H. C. McDade, S. C. Chen, S. C. Tucker, M. Gottfredsson, L. C.Wright, T. C. Sorrell, S. D. Leidich, A. Casadevall, M. A. Ghannoum, andJ. R. Perfect. 2001. Extracellular phospholipase activity is a virulence factorfor Cryptococcus neoformans. Mol. Microbiol. 39:166–175.

9. Cox, G. M., J. Mukherjee, G. T. Cole, A. Casadevall, and J. R. Perfect. 2000.Urease as a virulence factor in experimental cryptococcosis. Infect. Immun.68:443–448.

10. Ellerbroek, P. M., A. M. Walenkamp, A. I. Hoepelman, and F. E. Coenjaerts.2004. Effects of the capsular polysaccharides of Cryptococcus neoformans onphagocyte migration and inflammatory mediators. Curr. Med. Chem. 11:253–266.

11. Feldmesser, M., Y. Kress, and A. Casadevall. 2001. Dynamic changes in themorphology of Cryptococcus neoformans during murine pulmonary infection.Microbiology 147:2355–2365.

12. Garcia-Rivera, J., Y. C. Chang, K. J. Kwon-Chung, and A. Casadevall. 2004.Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellulartrafficking of capsular glucuronoxylomannan. Eukaryot. Cell 3:385–392.

13. Gullapalli, R. R., M. C. Demirel, and P. J. Butler. 2008. Molecular dynamicssimulations of DiI-C18(3) in a DPPC lipid bilayer. Phys. Chem. Chem. Phys.10:3548–3560.

14. McFadden, D. C., M. De Jesus, and A. Casadevall. 2006. The physicalproperties of the capsular polysaccharides from Cryptococcus neoformanssuggest features for capsule construction. J. Biol. Chem. 281:1868–1875.

15. Nosanchuk, J. D., L. Nimrichter, A. Casadevall, and M. L. Rodrigues. 2008.A role for vesicular transport of macromolecules across cell walls in fungalpathogenesis. Commun. Integr. Biol. 1:37–39.

16. Panepinto, J., K. Komperda, S. Frases, Y. D. Park, J. T. Djordjevic, A.Casadevall, and P. R. Williamson. 2009. Sec6-dependent sorting of fungalextracellular exosomes and laccase of Cryptococcus neoformans. Mol. Micro-biol. 71:1165–1176.

17. Rodrigues, M. L., M. Alvarez, F. L. Fonseca, and A. Casadevall. 2008.Binding of the wheat germ lectin to Cryptococcus neoformans suggests anassociation of chitinlike structures with yeast budding and capsular glucu-ronoxylomannan. Eukaryot. Cell 7:602–609.

18. Rodrigues, M. L., E. S. Nakayasu, D. L. Oliveira, L. Nimrichter, J. D.Nosanchuk, I. C. Almeida, and A. Casadevall. 2008. Extracellular vesiclesproduced by Cryptococcus neoformans contain protein components associ-ated with virulence. Eukaryot. Cell 7:58–67.

19. Rodrigues, M. L., L. Nimrichter, D. L. Oliveira, S. Frases, K. Miranda, O.Zaragoza, M. Alvarez, A. Nakouzi, M. Feldmesser, and A. Casadevall. 2007.Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryoticsolution to the problem of fungal trans-cell wall transport. Eukaryot. Cell6:48–59.

20. Rodrigues, M. L., L. R. Travassos, K. R. Miranda, A. J. Franzen, S. Rozen-

VOL. 8, 2009 OBSERVING FUNGAL VESICLES WITH FLUORESCENT PROBES 1379

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tal, W. de Souza, C. S. Alviano, and E. Barreto-Bergter. 2000. Humanantibodies against a purified glucosylceramide from Cryptococcus neofor-mans inhibit cell budding and fungal growth. Infect. Immun. 68:7049–7060.

21. Sebolai, O. M., C. H. Pohl, P. J. Botes, C. J. Strauss, P. W. van Wyk, A.Botha, and J. L. Kock. 2007. 3-Hydroxy fatty acids found in capsules ofCryptococcus neoformans. Can. J. Microbiol. 53:809–812.

22. Sebolai, O. M., C. H. Pohl, P. J. Botes, P. W. van Wyk, and J. L. Kock. 2008.The influence of acetylsalicylic acid on oxylipin migration in Cryptococcusneoformans var. neoformans UOFS Y-1378. Can. J. Microbiol. 54:91–96.

23. Shea, J. M., T. B. Kechichian, C. Luberto, and M. Del Poeta. 2006. Thecryptococcal enzyme inositol phosphosphingolipid-phospholipase C confersresistance to the antifungal effects of macrophages and promotes fungaldissemination to the central nervous system. Infect. Immun. 74:5977–5988.

24. Trocoli Torrecilhas, A. C., R. R. Tonelli, W. R. Pavanelli, J. S. da Silva, R. I.Schumacher, W. de Souza, N. C. E Silva, I. de Almeida Abrahamsohn, W.Colli, and M. J. Manso Alves. 2009. Trypanosoma cruzi: parasite shed vesiclesincrease heart parasitism and generate an intense inflammatory response.Microbes Infect. 11:29–39.

25. Valadi, H., K. Ekstrom, A. Bossios, M. Sjostrand, J. J. Lee, and J. O. Lotvall.2007. Exosome-mediated transfer of mRNAs and microRNAs is a novelmechanism of genetic exchange between cells. Nat. Cell Biol. 9:654–659.

26. Yoneda, A., and T. L. Doering. 2006. A eukaryotic capsular polysaccharide issynthesized intracellularly and secreted via exocytosis. Mol. Biol. Cell 17:5131–5140.

27. Zaragoza, O., and A. Casadevall. 2004. Experimental modulation of capsulesize in Cryptococcus neoformans. Biol. Proced. Online 6:10–15.

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