MINIREVIEW - Eukaryotic Cell · Indrani Bose, 1Amy J. Reese, Jeramia J. Ory,1 Guilhem Janbon,2* and Tamara L. Doering1* Department of Molecular Microbiology, Washington University

EUKARYOTIC CELL, Aug. 2003, p. 655–663 Vol. 2, No. 41535-9778/03/$08.00�0 DOI: 10.1128/EC.2.4.655–663.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

A Yeast under Cover: the Capsule of Cryptococcus neoformansIndrani Bose,1 Amy J. Reese,1 Jeramia J. Ory,1 Guilhem Janbon,2*

and Tamara L. Doering1*Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110,1

and Unite de Mycologie Moleculaire, Institut Pasteur, 75724 Paris Cedex, France2

Few fungi are pathogenic to humans. Of these, Cryptococcusneoformans has emerged as an important cause of mortality inimmunocompromised patients, especially those with AIDS. Asa result, extensive research efforts have addressed the patho-genesis and virulence of this organism.

C. neoformans is a basidiomycetous fungus that is ubiquitousin the environment, where it is found in soil, in association withcertain trees, and in bird guano (16). Because of its ubiquity, ithas been suggested that most people are exposed to C. neo-formans early in life (41). The fungus is heterothallic, withmating types MATa and MAT�. Asexual reproduction takesplace either by budding or, in the case of MAT� cells, byhaploid fruiting in response to nutrient deprivation or expo-sure to the mating pheromone a factor (106). Sexual repro-duction occurs when cells of opposite mating types come to-gether to form a heterokaryon that ultimately leads to theproduction of basidia and basidiospores (64). Desiccated cellsand the spores formed by haploid fruiting or sexual reproduc-tion have all been suggested to serve as infective particles,which must be less than 2 �m in diameter to penetrate the lungparenchyma (44, 86). Infection occurs when the fungal parti-cles are inhaled and enter the alveolar space. In most immu-nocompetent individuals, this infection is either cleared orremains dormant until an immune imbalance leads to furtherdevelopment. In the setting of compromised immune function,however, the fungus disseminates, with particular tropism forthe central nervous system. In severe cases, cryptococcal infectionprogresses to a meningoencephalitis that is fatal if left untreated.

C. neoformans virulence is mediated predominantly by apolysaccharide capsule that surrounds its cell wall and hasmultiple effects on the host immune system. This structureprovides a physical barrier that interferes with normal phago-cytosis and clearance by the immune system. Capsule compo-nents inhibit the production of proinflammatory cytokines, de-plete complement components (by efficiently binding them),and reduce leukocyte migration to sites of inflammation (11).The capsule also constitutes the major diagnostic feature ofcryptococcosis, because its components can be detected in the

bloodstream and it can be visualized with light microscopy byusing India ink staining. The capsule excludes the ink particlesand forms characteristic halos (Fig. 1A) whose diameters areoften several times that of the cell. The elaborate structure ofthe capsule may also be appreciated by electron microscopy(Fig. 1B and C).

Given the importance of the capsule in cryptococcal disease,tremendous effort has been applied in recent years to under-standing its biology. This review focuses on the resulting ad-vances in our understanding of the structure and synthesis ofthe capsular components, the incorporation of these compo-nents into the existing capsular network, the association be-tween the capsule and the cell wall, and the regulation ofcapsule growth.

CAPSULE STRUCTURE

The capsule of C. neoformans is composed primarily of poly-saccharides. Studies of its structure have taken advantage ofthe fact that capsular material is shed copiously into the exter-nal milieu. This material, called the exopolysaccharide, caneasily be collected, purified, and used for structural studies.Historically, chemical degradation followed by paper, column,and/or gas-liquid chromatography and mass spectrometry wasused to determine the composition of the capsular material.These techniques identified two polysaccharide components:an abundant glucuronoxylomannan (GXM) and a minor ga-lactoxylomannan (GalXM). Their precise structures weresolved by the application of nuclear magnetic resonance tech-niques (27) and are shown in Fig. 2.

GXM makes up about 90% of the capsule mass. Its back-bone consists of mannose residues that are �-1,3 linked anddecorated with xylosyl and glucuronyl side groups (Fig. 2A).Roughly two of every three mannose residues are also 6-O-acetylated (8, 26, 61), with a preference for unbranched man-nose but with some acetylation of the mannose which is sub-stituted with glucuronic acid (56). GXM differs in the degree ofxylose addition and acetylation in various cryptoccocal strains.These variations, differentiated by specific antibody binding,contribute to the classification of C. neoformans into four se-rotypes, A through D (35, 108). A fifth serotype, AD, has beenproposed, as some strains exhibit characteristics of both the Aand D serotypes (50). The antiserum classification system,however, is not equipped to distinguish the subtle heterogene-ity in GXM structure that may correlate with the range ofvirulence seen among C. neoformans isolates. Therefore, it has

* Corresponding author. Mailing address for Guilhem Janbon:Unite de Mycologie Moleculaire, Institut Pasteur, 25 rue du Dr. Roux,75724 Paris Cedex, France. Phone: 33 145 688356. Fax: 33 145 688420.E-mail: [email protected]. Mailing address for Tamara L. Doering:Department of Molecular Microbiology, Washington UniversitySchool of Medicine, St. Louis, MO 63110. Phone: (314) 747-5597.Fax: (314) 362-1232. E-mail: [email protected].

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also been proposed that C. neoformans strains be classified onthe basis of the minimum GXM repeating unit, as determinedby proton nuclear magnetic resonance (27).

Analysis of GXM structure is complicated by variability onseveral levels. One instructive study involved isolates fromseparate episodes of cryptococcosis in individual patients (25).In several cases, the strains remained the same, as determinedby analysis of restriction fragment length polymorphisms (29)and electrophoretic karyotyping (78), but alterations in GXMstructure resulted in their assignment to different serotypes(25). Similar results have been observed after prolonged cul-tivation either in vitro or during cryptococcal infection in an-imal models (36). To further complicate the picture, all cells ina population may not bear identical GXM, as assessed by theirbinding of anticapsular monoclonal antibodies (F. Moyrandand G. Janbon, unpublished results).

GalXM constitutes about 7% of the capsular mass and has amore elaborate structure than GXM (96). It is built on an�-1,6-linked galactose polymer which bears side chains of dif-ferent lengths on alternate galactose residues (Fig. 2B). Aswith GXM, the side chain structures, which consist in thiscase of galactosyl, mannosyl, and xylosyl residues, vary be-

FIG. 1. Views of the cryptococcal capsule. (A) Differential inter-ference contrast micrograph of cells that were mixed with India inkafter induction of capsule formation by growth in low-iron medium(80). Scale bar, 3 �m. (B) Thin-section micrograph of a budding cellfixed in the presence of ruthenium red dye (80). Scale bar, 1 �m. (C)Quick-freeze, deep-etch image of the edge of a cell, with an arc of cellwall separating the cell interior (lower left) from the abundant cap-sule fibers emanating upwards. Scale bar, 0.15 �m. All images wereobtained as described by Pierini and Doering (80).

FIG. 2. Structures of the cryptococcal capsule components. (A)GXM of serotype B (27); (B) GalXM (96). Linkages between sugarsare printed next to the arrows connecting monosaccharides; arrowposition is not intended to represent linkage. Blue, mannose; gray,glucuronic acid; red, xylose; green, galactose. 6-O-acetylation of man-nose, which modifies the GXM backbone, is not shown.

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tween serotypes. The cap67 mutant strain has been partic-ularly useful in the elucidation of GalXM structure since itlacks GXM, thus simplifying analysis (55, 96, 97).

A third polysaccharide component identified in culture fil-trates of C. neoformans is mannoprotein, which forms a minorfraction of the exopolysaccharide (76, 83). These mannopro-teins have been the focus of immunologic studies, as theyappear to be involved in the induction of cell-mediated immu-nity (18, 28, 70, 75, 82) and cytokine production (48, 81), bothcritical functions for clearing the fungus. Several of these pro-teins have been purified from acapsular strains of Cryptococcusby concanavalin A affinity (49, 66), and one study suggests thatthey move through the cell wall and are released into theexternal environment (99). It is not clear whether the manno-proteins are capsule components. For this review, they are notconsidered part of the capsule structure and will not be dis-cussed further.

MUTANTS IN CAPSULE SYNTHESIS

The structures of the C. neoformans capsule polysaccharidesare complex (Fig. 2), and their biosynthesis requires many geneproducts (31). Both genetic and biochemical techniques havebeen applied to elucidate the steps in this pathway. One pro-ductive series of studies was based on analysis of acapsularcryptococcal strains (52). By complementing these mutants,Kwon-Chung and colleagues (19–22) identified four genes re-quired for capsule production. These CAP genes were shownto be essential for virulence in mouse models of cryptococcosis,emphasizing the importance of the capsule as a virulence fac-tor. Although proteins encoded by two of them have beenlocalized, Cap60p to the nuclear membrane (20) and Cap10pto the cytoplasm (21), the biochemical functions of the CAPgene products have yet to be determined.

To investigate whether the CAP genes are unique or mem-bers of larger gene families, we used their sequences to searchthe available C. neoformans genome. Because CAP59 andCAP60 are close homologs, they were treated as part of thesame gene family and only CAP10, CAP59, and CAP64 wereused to search sequence data from The Institute for Geno-mic Research (www.tigr.org). The protein sequence for eachgene was compared to the sequences of the assembled B3501contig database by using TBLASTN (4), and hits were consid-ered positive if a good match to at least half of the querysequence existed in the same contig (a good match was definedas a score greater than 50 bits, with an expect value below10�10). There are at least two homologs of CAP10 in thegenome, one of which is the recently identified CAP1 (65). Twoadditional homologs matched the first and last 50 amino acidsof Cap10p but had weak homology to the rest of the protein.Searches with the CAP59 sequence revealed two homologoussequences in addition to CAP60. CAP64 is also a member of agene family, with six homologs in C. neoformans.

To assess whether CAP genes were specific for Cryptococcusin the fungal world, we used the CAP10, CAP59, and CAP64sequences to search all fungi with publicly available genomesequences (Table 1). The only fungus with sequences homol-ogous to all three is another basidiomycete, the white rotfungus (Phanerochaete chrysosporium). Hybridization studiespreviously suggested that CAP59 is unique to the genus Cryp-

tococcus (79). However, the broader technique of sequenceanalysis indicates that many fungi, both pathogenic and benign,contain homologs of both CAP59 and CAP10. This suggeststhat these families of genes may play roles other than capsulesynthesis, as these other fungi lack capsules. Unfortunately, nogenome sequencing projects are under way for other encapsu-lated fungi. We also looked for CAP gene homologs beyondfungi (as assessed by BLink analysis of all genes and geneproducts in the nonredundant National Center for Biotechnol-ogy Information database). Interestingly, CAP10 has matchesin metazoans with sequences of unknown function in human,mouse, and Drosophila. CAP59 also matches a hypotheticalgene in the Leishmania major genome. CAP64, in contrast, hasno detectable homologs outside the Mycota.

A second set of genes that were identified genetically as hav-ing a role in capsule synthesis are the CAS genes. These werediscovered when one of our laboratories screened mutagenizedC. neoformans cells for those that were unable to bind, or weredefective in binding, monoclonal antibodies directed againstthe capsule (74). Analysis of these CAS mutants identified sixcomplementation groups, Cas1 through Cas6 (74), of whichseveral members have now been examined. The CAS1 gene(56) is one of a family of genes with orthologs in human,Drosophila, and plants. Analysis of the cas1 mutant capsuleshowed defects in O acetylation, and interestingly, cas1�strains were more virulent than the parental wild type in amurine model of cryptococcosis (56). The CAS2 gene (74) wasfound to correspond to the previously identified UXS1 gene.UXS1 encodes a UDP-glucuronate decarboxylase which formsUDP-xylose from UDP-glucose (5). This gene is necessary forboth capsule xylosylation and fungal virulence (74).

STEPS REQUIRED FOR CAPSULE SYNTHESIS

Genetic screens have clearly proven valuable in identifyinggenes whose products are required for capsule synthesis. Bio-chemical studies have tremendous power to complement thisapproach and help elucidate the functions of these gene prod-ucts. These experiments have generally focused on the specificevents in capsule biosynthesis that are outlined in Fig. 3. The

TABLE 1. Presence of CAP genes in the fungal worlda

Fungus CAP10 CAP59 CAP64

Cryptococcus neoformansb � � �Phanerochaete chrysosporiumc � � �Magnaporthe grisead � � �Neurospora crassae � � �Aspergillus fumigatusf � � �Histoplasma capsulatumg � � �Candida albicansh � � �Saccharomyces cerevisiaei � � �Schizosaccharomyces pombej � � �

a �, present; �, absent.b Data from http://www.tigr.org/tdb/e2k1/cna1/.c Data from http://www.jgi.doe.gov/programs/whiterot.htm.d Data from http://www-genome.wi.mit.edu/annotation/fungi/magnaporthe/.e Data from http://www-genome.wi.mit.edu/annotation/fungi/neurospora/.f Data from http://www.tigr.org/tdb/e2k1/afu1/.g Data from http://www.genome.wustl.edu/projects/hcapsulatum/.h Data from http://sequence-www.stanford.edu/group/candida/index.html.i Data from http://genome-www.stanford.edu/Saccharomyces/.j Data from http://www.sanger.ac.uk/Projects/S_pombe/.

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first to be considered here is the production of the activatedprecursors that serve as donors for capsule components. GXMsynthesis requires GDP-mannose, UDP-xylose, and UDP-glu-curonic acid as biosynthetic precursors; GalXM additionallyrequires UDP-galactose. The acetyl donor to GXM has notbeen identified. Since GXM is the major component of thecapsule and has a mannan backbone, biosynthesis of GDP-mannose is a critical early step. This requires the activity ofthree enzymes that are highly conserved in the fungal world,phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase (or GDP-mannose synthase) (Fig.3, element A). The gene encoding phosphomannose isomerasein C. neoformans (MAN1) has been identified and cloned(107). man1 mutants are defective in capsule production andGXM secretion, have morphological abnormalities, and areavirulent.

The nucleotide sugars UDP-glucuronic acid (which donatesa major component of GXM) and UDP-xylose (required forboth GXM and GalXM) can be made from UDP-glucose bythe sequential actions of two enzymes. UDP-glucose dehydro-genase leads to the formation of UDP-glucuronic acid (Fig. 3,element B), while UDP-glucuronate decarboxylase convertsUDP-glucuronic acid to UDP-xylose (Fig. 3, element C).These activities were detected in C. neoformans by Jacobsonand Payne (51, 53), and subsequent work in one of our labo-ratories has identified the genes encoding both of these en-zymes. One of these genes (UXS1), encoding UDP-glucur-onate decarboxylase, was found through its homology to abacterial gene that had been hypothesized to have a similarfunction (5). Although this decarboxylase is important for syn-thetic processes across biological kingdoms, the sequence forthis enzyme had not previously been obtained in any system.

Once biosynthetic precursors are made, the next probablestep in capsule synthesis is their localization to the site ofpolysaccharide construction. This is likely to require several

specific transporters (Fig. 3, element D), since the chargedprecursors are generally formed in the cytosol, while mosteukaryotic glycan synthesis occurs in membrane-bound com-partments. Several genes encoding putative nucleotide sugartransporters have been identified in C. neoformans by sequencehomology performed in our laboratories. Functional assays ofthese putative transporters should clarify their potential role incapsule formation and cryptococcal biology.

After appropriate precursors are transported into the com-partment of capsule biosynthesis, the monosaccharides mustbe assembled into polymers. As previously noted (31), thereare at least 11 linkages present in the various configurations ofGXM and GalXM. Each linkage probably reflects the functionof a different sugar transferase, because these enzymes areusually specific for the substrate, the moiety transferred, andthe bond formed. Constructing GXM and GalXM would there-fore require an array of sugar transferases (for examples, seeFig. 3, elements E to G) and an acetyltransferase (Fig. 3,element H).

Several distinct mannosyl transferase activities have beenidentified in C. neoformans by using crude membrane prepa-rations. Two of these, identified in efforts to isolate enzymesinvolved in capsule synthesis, are in fact �-1,2-mannosyltrans-ferases and therefore lack the requisite activity (32, 104). Athird, however, can transfer mannose from GDP-mannose to�-1,3-linked mannobiose and would therefore be capable ofbuilding the GXM backbone (32). This enzyme has beenpurified, and its peptide sequence has been used to obtainthe corresponding gene sequence. Inhibition of its expressionshows that it is required for normal capsule synthesis (U. Som-mer, H. Liu, and T. L. Doering, unpublished data). In addition,xylosyltransferase (Fig. 3, element G) and glucuronyltrans-ferase (Fig. 3, element F) activities have been detected in crudemembrane preparations of C. neoformans (103). Study of theseactivities led to the proposal that the order of addition of sidegroups to the mannan backbone is first, acetyl; second, glucu-ronyl; and third, xylosyl (103). However, subsequent investiga-tion of GXM structures in a mutant lacking xylose suggeststhat acetylation follows the addition of xylose (74).

CAPSULE ASSEMBLY AND DEGRADATION

Once individual capsule components are generated by thebiosynthetic pathways discussed above, they must be assem-bled into the elaborate meshwork of the mature capsule. Howare these new materials incorporated to generate and maintainthis crucial virulence factor? Capsule synthesis has been inves-tigated in two physiologically important situations: bud forma-tion, which occurs with concomitant encapsulation of thedaughter cell, and capsule synthesis on mature cells, which isinduced by altering growth conditions (80). In budding cells,new-capsule synthesis is directed into the bud, with no incor-poration of the maternal capsule into the nascent polysaccha-ride structure overlying the daughter cell (Fig. 4). In maturecells, new capsule material is incorporated just outside the cellwall, at the inner aspect of the existing capsule structure (80).This displaces the existing material outwards, although there isalso some mixing of old and new polysaccharide and an overallincrease in density of the polysaccharide structure with time.Material displaced by new synthesis could conceivably be shed

FIG. 3. Steps in GXM synthesis. Proteins with a role in biosynthesisare indicated by red letters and are discussed further in the text. (A)Phosphomannose isomerase; (B) UDP-glucose dehydrogenase; (C)UDP-glucuronic acid decarboxylase; (D) nucleotide sugar transportersspecific for GDP-mannose (D1), UDP-glucuronic acid (D2), and UDP-xylose (D3); (E) mannosyltransferase; (F) xylosyltransferase; (G) glu-curonosyltransferase; and (H) acetyltransferase. The color coding isthe same as described in the legend to Fig. 2, with the addition ofpurple to represent glucose.



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from the cell into the environment or it could remain associ-ated with the capsule, perhaps fixed in place by cross-linkingprocesses such as those suggested to occur during cell wallmaturation (67). To fully understand these processes, themechanism by which the capsule is initially associated with thecell must first be defined.

Early studies of how capsule material associates with the cellwere based on the observation that capsular exopolysaccharidecould bind to acapsular cells. Capsule material purified fromgrowth medium of wild-type cryptococcal cells was first shownto bind to acapsular mutants and to protect them from phago-cytosis (12, 13, 91). This interaction was specific for cryptococ-cal cells and cryptococcal capsule material (62) and was shownto be saturable and reversible (63). Chemical treatments ofacceptor cells and purified capsule material were subsequentlyinvestigated to test their effects on binding (89). These studiesled to a model in which polysaccharide capsule material bindsto a specific receptor on the surface of acapsular mutants.

To pursue the question of how capsule polysaccharides in-teract with the cell, an in vivo assay for the binding of capsule

to acapsular cells has been employed. In this assay, conditionedmedium from wild-type cells serves as a capsule source andindirect immunofluorescence with a fluorophore-tagged anti-capsule antibody is used to evaluate the transfer of polysac-charides to acapsular acceptor cells. Studies using this modelstrongly implicate cell wall glycan in capsule binding (A. J.Reese and T. L. Doering, submitted for publication).

The relationship of the capsule to the cell wall necessitatesan examination of C. neoformans cell wall composition. Whilemuch is known about the cell wall structures of Saccharomycescerevisiae and Candida albicans, considerably less is knownabout those of other fungal organisms (Table 2). Common cellwall structural building blocks include �-glucans and chitin (14,15). Other possible cell wall components in fungi include gly-coproteins (particularly mannoproteins), �-glucans (14), andmelanin (17). In S. cerevisiae (90) and Candida albicans (60),the main cell wall network is formed by branched �-1,3-linkedglucans, with chitin and several types of mannoproteins linkeddirectly to it. Other fungi, including C. neoformans, have a high�-1,3-glucan content, and their cell wall structures might beconsiderably different. Binding assays indicate that �-1,3-glu-can at the cryptococcal surface is required for acapsular cells tobind exogenous capsule material. Posttranscriptional silencingof a cryptococcal �-glucan synthase gene yields cells with poorability to assemble capsules, although the components are stillmade (Reese and Doering, submitted). It is interesting that inthe pathogenic fungi Blastomyces dermatitidis and Histoplasmacapsulatum, virulence is associated with �-1,3-glucan levels (47,59), although these fungi are not encapsulated.

While studies of capsule synthesis have progressed, capsuledegradation remains poorly understood. Although enzymes fromsoil microbes can degrade capsules (37), no cryptococcal or hu-man enzymes with this activity have been found. Nonetheless,capsule material must be degraded or remodeled by the cell toallow bud emergence, since buds are coated with newly syn-thesized capsule material (Fig. 4) (80). The mammalian hostmay also have mechanisms for capsule breakdown, but furtherstudies in this area are needed to clarify these events.

FIG. 4. Cell wall and newly synthesized capsule overlying nascentbuds. Cell walls were tagged with fluorescein (green), and capsuleswere labeled with fluorophore-linked antibody to GXM (red). Cellswere then returned to culture to allow bud emergence. A comparisonof differential interference contrast (left) and fluorescence (right) im-ages shows that none of the original label extends beyond the mother-bud neck (arrow), indicating that there is no incorporation of maternalglycans in the new bud. Reprinted with permission from BlackwellPublishing (80).

TABLE 2. Major cell wall components of selected fungal organismsa

OrganismComponent or characteristic (reference)

�-Glucan �-Glucan Mannoproteins Chitin Able to melanize

Cryptococcus neoformans 15%b (mostly �-1,6; some�-1,3) (55)

35%b (mostly �-1,3;some �-1,4) (55)

Present (99) Present (16) Yes (102)

Saccharomyces cerevisiae 50%c �-1,3; 10%c �-1,6 (67) None (67) 40%c (67) 1–3%c (67) NoCandida albicans 40%c �-1,3; 20%c �-1,6 (60) None (60) 35–40%c (60) 1–2%c (60) NoSchizosaccharomyces pombe 55%c �-1,3; 6%c �-1,6 (46) 28%c �-1,3 (46) Likely present 0.5%c (46) ?Aspergillus fumigatus (mycelial form) 70%b �-1,3; 4%b �-1,6; 10%b

�-1,3/1,4 (7)Present (7) 3.5%c (7) Present (7) Yes (7)

Paracoccidioides brasiliensis Yeast form, 5%b �-1,3 (87);mycelial form, mostly�-1,3 (9)

Yeast form, 95%b

�-1,3 (87); mycelialform, little �-1,3 (9)

Likely present Present (yeast, my-celial form) (9)

Yes (42)

Blastomyces dermatitidis (yeast form) 5%b (58) 95%b (58) Likely present Present (10) Likelyd

Histoplasma capsulatum (yeast form) Present Present Likely present Likely Present Yes (77)

a The ratio of cell wall components is highly dependent on whether the organism is in hyphal or yeast form (15, 87, 90), so these are listed separately. Equivalentanalyses have not been performed for all organisms listed.

b Percentage of cell wall glucan.c Percentage of total cell wall mass.d J. D. Nosanchuk, personal communication.



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REGULATION OF THE CAPSULE

The C. neoformans capsule varies tremendously in size andmorphology under different growth conditions. As early as1958, Littman studied the effects of various nutrients on cap-sule formation and formulated a capsule-inducing mediumcontaining thiamine, sodium glutamate, mineral salts, maltose,and sucrose (68). Low-iron medium (98), alterations in pH,and a high ratio of CO2 to HCO3 (43) also induce capsuleformation, while other medium additives, such as 1 M sodiumchloride, suppress capsule formation (54). Suppression of cap-sule synthesis by sodium chloride is specific, as other hyper-tonic solutions do not have this effect. Capsule size also in-creases dramatically during infection (6, 40). The extent ofcapsule formation depends on the tissue location, with cells inthe brain having larger capsules than those in the lungs (84).This is probably due to differences in the CO2/HCO3 ratio orother capsule-modulating factors in those sites, again stressingthe highly regulated nature of this virulence factor.

Efforts to understand capsule regulation have focused atten-tion on second messengers, which mediate aspects of growthand development in eukaryotic cells. In particular, studies haveaddressed the second messenger cyclic AMP (cAMP), which isproduced in cells by the enzyme adenylyl cyclase. The activityof adenylyl cyclase in mammalian cells is regulated by hetero-trimeric G proteins (39), which are in turn activated by seventransmembrane receptors of the �-adrenergic receptor family.Binding of an appropriate ligand to the receptor leads theheterotrimeric G protein to exchange GDP for GTP, causingdissociation of the � subunit from the �� dimer (Fig. 5). De-pending on the organism, either one of these products mayactivate an adenylyl cyclase. The subsequent production ofcAMP has diverse effects, one of the best known of which is theactivation of cAMP-dependent protein kinase A (PKA). PKAin turn regulates downstream targets, including transcriptionfactors, leading to broad changes in cellular physiology (92).

cAMP has been shown to regulate capsule production inC. neoformans (2, 3). Implicated in the upstream pathway arethe GPA1 and CAC1 gene products, which have been shown tocontrol intracellular cAMP production. The GPA1 product ishomologous to the S. cerevisiae G� subunit Gpa2p (94), whilethe CAC1 product is the adenylyl cyclase (3). Both gpa1 and

cac1 mutants are unable to induce capsule production and arecorrespondingly avirulent, but overproduction of CAC1 cansuppress the gpa1 mutant defects (3). These defects can also becorrected by the addition of cAMP to the medium, therebybypassing the requirement for Gpa1p and Cac1p and support-ing the crucial role that signaling via cAMP plays in capsuleformation.

Among the potential targets of cAMP, the C. neoformansPKA1 gene product has a clear role in capsule production (33),sinces a pka1 strain is acapsular and avirulent. In this case,capsule production is not restored by exogenous cAMP, sug-gesting that PKA acts downstream of cAMP in the signalingpathway (Fig. 5). Further investigations of PKA in C. neofor-mans are based on its subunit structure. Inactive PKA is atetramer of two catalytic subunits (encoded by PKA1) and tworegulatory subunits (encoded by PKR1). Binding of cAMP tothe regulatory subunits causes them to undergo a conforma-tional change and to dissociate from the catalytic subunits,releasing the latter from inhibition (Fig. 5). Loss-of-functionmutations of PKR1 should, therefore, lead to constitutive ac-tivation of PKA1. This is indeed the case, as pkr1 mutantsproduce larger capsules than wild-type cells do and are hyper-virulent in animal models. pkr1 mutants can also suppress thecapsule production defect of gpa1 cells (33). However, thecapsules produced by the pkr1 gpa1 double mutant are largerthan those produced by the pkr1 single mutant. This suggeststhat GPA1 may have additional targets, yet to be identified,that modulate capsule production (34). Once active, Pka1pkinase can cause changes in gene expression and physiology byphosphorylation of effector proteins. One candidate target ofPKA, which is involved in positive regulation of capsule pro-duction, is the transcription factor encoded by STE12, which isfound in two mating-type-specific forms (105, 110). This tran-scription factor is probably only one of several targets (Fig. 5),because loss of function of STE12� or STE12a leads only to adecrease in capsule size (23, 24, 110) and overexpression ofSTE12� does not overcome the capsule production defect of apka1 strain (33).

In many other systems, the Ras signaling protein plays a rolein the activation of adenylyl cyclase. However, RAS1 does notappear to be involved in capsule production in C. neoformans(1, 2). Other major signaling pathways in C. neoformans thathave been investigated include the mitogen-activated proteinkinase pathway and the signaling pathway of cyclophilins. Al-though both of these pathways have been shown to regulatevirulence and mating (100, 101), they are not clearly implicatedin capsule production.

OTHER CAPSULES

C. neoformans is unique in being the only fungus that is bothpathogenic to humans and in possession of a true polysaccha-ride capsule outside its cell wall. Tremella mesenterica, a closephylogenetic relative, possesses a similar capsule but does notcause disease (30). Several other fungi have capsule-like struc-tures, but they have not been well characterized. These fungiinclude Malassezia furfur (73), Rhinosporidium seeberi (93), Tri-chosporon beigelii (72), Blastocystis hominis (71), and Sporothrixschenckii (38).

Among other microbes, many gram-negative and gram-pos-

FIG. 5. cAMP signaling pathway in the regulation of the capsule.See text for explanation. Red symbols, Pkr1p; black circles, Pka1p;blue-gray circles, cAMP.



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itive bacteria also bear polysaccharide capsules. The numerousexamples among pathogens include Streptococcus pneumoniae,Neisseria meningitidis, and Pseudomonas aeruginosa (109). Cap-sules protect bacterial cells from desiccation, mediate theiradherence to host tissue, and assist in evasion of the host im-mune response (85). Some bacteria also shed exopolysacchar-ides into their surroundings as a method of avoiding immunesystem clearance. There is a tremendous variety of polysaccha-ride structures present in bacterial capsules (85, 109). All ofthem, however, are quite distinct from those of the C. neofor-mans capsule and, therefore, require an independent set of en-zymes for their synthesis and assembly.

The value of capsules has also been appreciated beyond themicrobial world with the use of microencapsulation techniquesin the food industry. In this application, alginate or other poly-saccharides are used to microencapsulate bacteria (57). Thisprocess makes use of the protective nature of polysaccharidecapsules to assist in the survival of probiotic bacteria.

FUTURE DIRECTIONS

The biogenesis of the capsule in Cryptococcus is a compli-cated process, and rapid advances in understanding its struc-ture, synthesis, and regulation have been seen in the past fewyears. However, there are vast areas that are still unexploredand numerous questions that remain unanswered. Filling thesegaps will require concerted effort in all fields of biology—biophysical, biochemical, genetic, and cell biological.

The structure of the major capsular polysaccharides hasbeen established and suggests the synthetic precursors re-quired, but how is the synthesis of these polysaccharides initi-ated? Similarly, the question of whether the addition of newmaterial to the capsule occurs in units of single monosac-charides, short oligosaccharides, or extensive polysaccharidechains remains unanswered.

Our present understanding of the biochemical reactions incapsule synthesis is patchy. The participants in some of thepredicted reactions, such as GXM acetylation, have not yetbeen defined. Other capsular synthetic activities have been bio-chemically identified only in lysates and have yet to be studiedin detail or correlated with a corresponding gene. Only a fewhave been purified and cloned. Without pure enzyme prepa-rations or strains deficient in specific activities, it becomesextremely difficult to determine the order of the reactions thatadd side chain subunits to the polysaccharide backbone and,therefore, to delineate the entire biosynthetic pathway.

There are several approaches that can identify additionalgenes with potential roles in capsule synthesis. These includeidentifying gene families within Cryptococcus, finding crypto-coccal genes with homology to those encoding relevant pro-teins in other organisms, and examining mutants with alteredcapsules. Once important enzymes are identified and function-ally validated, their localization will be crucial in determiningthe sites and organelles involved in capsule synthesis. Deter-mination of the transport pathways that carry capsule compo-nents to the cell surface is another area that is important butlargely unexplored.

In the search for the molecular basis of capsule regulation, acAMP-signaling pathway that modulates capsule productionhas been identified. Genetics has been used to define many of

the members of this pathway. However, unanswered questionsremain. What is the nature of the signal that activates thepathway? What are the identities of the transmembrane recep-tor and of downstream effectors? A variety of nutrients andenvironmental conditions also affect capsule production, butwhat are the regulatory pathways by which they do so? Theregulation of capsule structure presents fascinating questionsin addition to those about how much capsule is made. Capsulestructure depends on environment (95), and studies with anti-capsule antibodies suggest that structure may vary among cellsin a population (Moyrand and Janbon, unpublished results),but the mechanisms controlling these differences are yet to bedefined.

The capsule polysaccharides that are shed into the externalmilieu enable C. neoformans to resist the host immune re-sponse. Little is known, however, about how this occurs. Arethe old parts of the capsule sloughed off as new material isadded, or is the release of capsular material a regulated pro-cess under specific cellular control? Also, how does degrada-tion of the capsule take place? The molecular mechanisms ofthese processes remain obscure.

Finally, although the structures of the main components ofthe capsule are now known, how do these components com-bine to form the three-dimensional structure of the capsule?Electron micrographs show an elaborate network of fibers thatextend out from the cell wall. However, these images do notaddress whether these strands are composed of one or multiplepolysaccharide components or how they group and branch. Abetter understanding of the biochemical and biophysical prop-erties of the capsule is needed to answer these questions.

For many years, the study of the cryptococcal capsule washindered by the absence of appropriate tools. This picture hasbeen completely redrawn by the recruitment and adaptation ofnew technologies to the cause of cryptococcal research, fromgenome sequencing (45, 88) and RNA interference (69) tostructural studies. These tools, coupled with the facts that genedisruptions are now routinely performed and animal modelsare in place to study in vivo effects, have considerably advancedour understanding of the Cryptococcus capsule. The comingyears will be an exciting time for studies of cryptococcal biol-ogy.

ACKNOWLEDGMENTS

We thank members of the Doering lab and Tom Kozel for helpfuldiscussions, Wandy Beatty for assistance in the preparation of Fig. 1,Andy Alspaugh and Chad Rappleye for comments on the manuscript,and John Heuser for quick-freeze deep-etch electron microscopy.

Studies of capsule synthesis in the Doering laboratory are supportedby NIH grants GM66303 and AI49173, a Burroughs Wellcome FundNew Investigator Award in Molecular Pathogenic Mycology, an awardfrom the Edward J. Mallinckrodt, Jr., Foundation, and a Craig FacultyFellowship. Studies of capsule synthesis in the Janbon laboratory aresupported by Ensemble contre le SIDA (grant AO13-3) and the Pas-teur Institute.

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MINIREVIEW - Eukaryotic Cell · Indrani Bose, 1Amy J. Reese, Jeramia J. Ory,1 Guilhem Janbon,2* and Tamara L. Doering1* Department of Molecular Microbiology, Washington University