INTRODUCTION

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Candida albicans is a serious agent of infection, particularly in immunocompromised patients. The delicate balance between the host and this otherwise normal commensal fungus may turn into a parasitic relationship, resulting in the development of infection, called candidiasis. The nature and extent of the impairment of normal host defense influence the manifestation and severity of infection. In general, superficial mucocutaneous candidiasis is frequent in patients with T-cell deficiencies, such as AIDS patients. The more serious, life-threatening, deep-seated or disseminated candidiasis is normally found in a spectrum of severely immunocompromised patients (29, 390). The fungus is not a mere passive participant in the infectious process, and a hypothetical set of virulence factors for C. albicans has been proposed and supported by various studies. These fungal attributes include the production of secreted hydrolytic enzymes, dimorphic transition (morphogenetic conversion from budding yeast to the filamentous growth form or hypha), antigenic variability, the ability to switch between different cell phenotypes, adhesion to inert and biological substrates, and immunomodulation of host defense mechanisms (for a review of these topics, see reference 96).

Initially, the cell wall was considered an almost inert structure that supplies rigidity and protection to the protoplast. Today, the cell wall is well established as being essential to almost every aspect of the biology and pathogenicity of C. albicans (64). The cell wall acts as a permeability barrier and is the structure that maintains the characteristic shape of the fungus. Also, as the most external part of the cell, the wall mediates the initial physical interaction between the microorganism and the environment, including the host. For these reasons, the cell wall of C. albicans is the focus of study by numerous research groups. Their objectives are the elucidation of both basic biological processes and functional mechanisms regulating the synthesis, organization, and environmental interactions of this complex macromolecular structure. Proteins have been implicated in most of the cell wall functions. Extensive reviews exist on different aspects of the cell wall of C. albicans (64, 478-482, 491, 493); however, recent reviews that focus on proteins have been limited primarily to the function of proteins as adhesins (49, 50, 110, 111, 151, 209, 406). This review focuses on both general characteristics of the cell wall and secreted proteins and specific aspects of individual proteins.

Although hydrolytic enzymes such as acid phosphatase were examined previously, studies on the identity and function of protein components began in the early to mid 1980s with studies in 1983 by Chaffin and Stocco (71), in 1985 by Elorza et al. (125) and Sundstrom and Kenny (524), and in 1986 by Pontón and Jones (414). In the first part of this decade, there has been an explosive growth in the number of studies of the cell wall proteins. These studies have been driven by presumed virulence functions of specific proteins and fueled by the realization that this is a complex, dynamic "organelle." Out of such impetus has come the identification of specific proteins not yet associated with specific pathogenic function and observations with more general import for the cell and cell wall proteins. This increasing knowledge of the protein component of the cell wall may result in a better understanding of the pathogenic mechanisms of the fungus and also may contribute to the design of innovative therapeutic regimens and diagnostic procedures (175, 333). At times, these studies have revealed several surprises and unexpected findings, which only add more attraction to the study of this fascinating microorganism. In writing this review, we have focused on the protein component with three objectives: (i) to summarize general aspects of proteins; (ii) to summarize studies on specific proteins or protein families; and (iii) to consider the implications, unanswered questions, and future research directions suggested by these studies.

Although the terms "dimorphism" and "dimorphic fungus", i.e., existing in two morphological forms, are well established and commonly accepted when referring to C. albicans, strictly speaking this fungus has the ability to adopt a spectrum of morphologies, and thus C. albicans could be considered a "polymorphic" or "pleomorphic" organism (244, 390). Since changes in the cell wall determine the shape of the whole fungal cell, the cell wall is the structure ultimately responsible for a given morphology. C. albicans can reproduce by budding, giving rise to the formation of yeast cells (also designated blastospores or blastoconidia). The production of germ tubes results in the conversion to a filamentous growth phase or hypha, also called the mycelial form. The formation of pseudohyphae occurs by polarized cell division when yeast cells growing by budding have elongated without detaching from adjacent cells. Under certain nonoptimal growing conditions, C. albicans can undergo the formation of chlamydospores, which are round, refractile spores with a thick cell wall. These morphological transitions often represent a response of the fungus to changing environmental conditions and may permit the fungus to adapt to different biological niches. The transition from a commensal to a pathogenic lifestyle may also involve changes in environmental conditions and dispersion within the human host. The ultrastructure, composition, and biological properties of the cell wall are affected by these morphological changes (64). Although progress has been achieved in the recent years, the molecular mechanisms governing these morphogenetic conversions are still not fully understood, partly due to the difficulty of genetic manipulations in this fungus (274, 275, 474). Recent reports that may herald rapid advances in this area have identified transcriptional regulatory genes, a general transcriptional repressor TUP1 (38), a putative transcriptional factor RBF1 (220), and a myc-like transcriptional factor EFG1 (516) that affect cellular morphology when their expression is altered. Most of the observations from these studies have been incorporated by Magee (310) into a model for the regulation of pseudohyphal growth.

Adhesion is a prerequisite for colonization and an essential step in the establishment of infection. C. albicans adheres to epithelial cells, endothelial cells, soluble factors, extracellular matrix, and inert materials implanted in the body of the host. Multiple adherence mechanisms appear to be used by C. albicans cells (49, 50, 110, 111, 151, 209, 252, 406). Physical interactions of this fungus with the host are mediated at the cell surface, and cell wall constituents implicated in binding have been designated adhesins (49). The large repertoire of adhesins displayed by this fungus may reflect the variety of host sites that it can invade (49, 50, 110, 111, 209). Specific characteristics of individual cell wall moieties participating in adhesion events are discussed later in this review.

Another important aspect of interactions with the host, with direct implications for pathogenesis, is the potential of this fungus to modulate the immune response mounted by the host (64, 96, 107). The capacity of cell wall constituents, including glucan, chitin, and mannoproteins, to modulate (activate or depress) the immune response is well documented (11, 64). Mannans and mannoproteins display the most potent immunomodulatory activity, being able to regulate the action of virtually all arms of the immune system (natural killer cells, phagocytic cells, cell-mediated immunity, and humoral mechanisms) (64, 107, 402, 412). Although individual cell wall moieties with immunomodulatory properties are described below, readers are referred to excellent reviews on this topic (11, 64, 107).

CELL WALL COMPOSITION AND ORGANIZATION

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Composition

Approximately 80 to 90% of the cell wall of C. albicans is carbohydrate. Three basic constituents represent the major polysaccharides of the cell wall: (i) branched polymers of glucose containing -1,3 and -1,6 linkages (-glucans); (ii) unbranched polymers of N-acetyl-D-glucosamine (GlcNAc) containing -1,4 bonds (chitin); and (iii) polymers of mannose (mannan) covalently associated with proteins (glyco[manno]proteins). In addition, cell walls contain proteins (6 to 25%) and minor amounts of lipid (1 to 7%) (50, 64, 490, 493).

The microfibrillar polymers (-glucans and chitin) represent the structural components of the wall. They form a rigid skeleton that provides strong physical properties to the cell. From a quantitative point of view, -glucans are the main constituent, accounting for 47 to 60% by weight of the cell wall. Chitin is a minor (0.6 to 9%) but important component of the C. albicans wall, particularly of the septa between independent cell compartments, budding scars, and the ring around the constriction between mother cell and bud (126, 360).

On the other hand, mannose polymers (mannan), which do not exist as such but are found in covalent association with proteins (mannoproteins), represent about 40% of the total cell wall polysaccharide and are the main material of the cell wall matrix (50, 64, 480, 490, 493). The term "mannan" has been used also to refer to the main soluble immunodominant component present in the outer cell wall layer of C. albicans, called phosphomannoprotein or phosphopeptidomannan complex. This cell wall fraction contains homopolymers of D-mannose (as the main component), 3 to 5% protein, and 1 to 2% phosphate (436). The general features of cell wall mannoproteins in C. albicans are basically identical to those found for Saccharomyces cerevisiae, one of the most thoroughly investigated yeasts in this regard. Several studies have resulted in a detailed knowledge of the structure of this cell wall constituent in C. albicans (12, 262-265, 494-498). Thus, mannose polymers are linked to the protein moiety through asparagine (by N-glycosidic bonds through two GlcNAc [di-N-acetylchitobiose] residues) and threonine or serine (by O-glycosidic, alkali-labile linkages) residues. The N-glycosidically linked carbohydrate is composed of backbone chains of -1,6-linked mannopyranosyl residues to which oligosaccharide side chains are attached. The side chain mannopyranosyl residues contain -1,2, -1,3, -1,2, -1,6, and phosphodiester linkages as well as branches (-1,6) that are oversynthesized under acidic growth conditions (150, 152, 261-267, 494-498). The O-glycosidically-linked sugar component consists of single mannose residues and short, unbranched mannose oligosaccharides (412). Several studies raise the question of additional sugars present in cell wall constituents. These observations include the following: (i) not all proteinaceous moieties present in cell wall extracts from this fungus react with concanavalin A, a lectin recognizing -mannosylpyranose, or with polyclonal and monoclonal antibodies that recognize other mannan epitopes, such as factor 6, a mannooligosaccharide that confers serotype A specificity (57); (ii) differences in glycosylation and in sensitivity to neuraminidase have been detected in candidal receptors for complement (4, 563); and (iii) treatment with neuraminidase affects the electrostatic surface properties of C. albicans as detected with a fluorescent probe (227). As suggested in these studies, the observations raise the possibility that additional sugars are cell wall constituents. However, the observations could reflect the existence of contaminating proteases in the glycosidase preparation. Sugar residues other than mannose may define either additional functional or antigenic motifs or both in cell wall glycoproteins.

The percent composition of walls from yeast cells and filamentous forms are similar, although the relative amounts of -glucans, chitin, and mannan vary according to the C. albicans growth form considered (50, 480, 491). Hyphal cells contain at least three times as much chitin as yeast cells do (77, 127, 518). Chitin is the first polymer to appear in regenerating protoplasts (124, 375). Although the ratio of -1,3- to -1,6-glucan in the insoluble fraction is similar in yeast and hyphal cells, the insoluble glucan in the initial period of germ tube formation contains considerably more -1,3 linkages than that found in yeast and mature hyphal cells (518). The literature contains several reports on the identification of morphology-specific proteins and mannoproteins that are discussed later in this review.

The different cell wall components interact with each other to give rise to the overall architecture of the cell wall. Besides hydrogen and hydrophobic bonds, there is also experimental evidence for the presence of covalent linkages between different components (453, 482). Surarit et al. (527) reported the presence of glycosidic linkages between glucan and chitin in the nascent wall of C. albicans. Recent evidence indicates that mannoproteins may also establish covalent associations with -glucans (237, 238, 462, 463). It is suggested that -1,3- and -1,6-glucans are linked to proteins by phosphodiester linkages, a process that may involve the participation of a GPI (glycosyl phosphatidylinositol) anchor (238) (see below). Protein and mannoprotein species that are released only after digestion of the glucan cell wall network with -glucanases may play a key role in configuring the final cell wall structure characteristic of each growth form (yeast and mycelium) of C. albicans (453, 479-482). Interactions between glyco(manno)proteins and chitin also appear to exist in the wall of C. albicans cells as deduced from two lines of evidence: (i) chitinase treatment of isolated cell walls solubilizes protein moieties, and (ii) the kinetics of incorporation of protein and mannoprotein constituents into the walls of regenerating protoplasts is altered in the presence of nikkomycin, an antibiotic that blocks chitin synthesis (124, 319).

Cell wall architecture has been studied most extensively in S. cerevisiae and is likely to be a model for C. albicans since there are some similar observations, in particular sensitivity to enzymatic digestion, glucan-mannoprotein linkages, and candidate proteins, that fit the same model (237, 238, 246, 247, 268, 462, 463). In a very recent study, Kollár et al. (268) detected the presence of material containing all four major cell wall components, -1,3-glucan, -1,6-glucan, chitin, and mannoprotein. Their analysis indicated that -1,6-glucan has some -1,3-glucan branches that may be linked to the reducing end of chitin. The -1,6-glucan and mannoprotein are attached through a remnant of the mannoprotein GPI anchor. Reducing ends of -1,6-glucan may also be attached to the nonreducing end of -1,3-glucan. The proportion of cell wall polysaccharide involved in this type of structure is not clear. The following cell wall building block, where the linkages are indicated by the long dashes, is proposed (247, 268): MannoproteinGPI remnant-1,6-glucan-1,3-glucanchitin. The authors point out that these linkages are likely to be formed in the periplasmic space as a common end of the individual biosynthetic pathways. Chitin and -1,3-glucan are synthesized at the plasma membrane and extruded into the periplasm, mannoprotein is synthesized in the cytoplasm and transported through the secretory pathway, and -1,6-glucan synthesis may occur partially in the endoplasmic reticulum or Golgi complex (268). Not all components are necessarily present in a complex; therefore, the authors suggest that more chitin may be present in inner cell wall layers and more mannoprotein may be present in the outer layers.

Layering. Since polysaccharides are poorly reactive to the ordinary fixatives and stains used for transmission electron microscopy, only a few well-defined ultrastructural details are obtained by conventional protocols (Fig. 1A and B). However, transmission electron microscopy studies performed with more special techniques or with cytochemical stains and contrasting agents show several layers in the cell wall of C. albicans (Fig. 1C to F). The appearance of these layers is variable and seems to be related to the strain examined, growth conditions, morphology, and preparation of the specimens (50, 64, 422). Thus, there is no consensus about the number of layers present in the cell wall. Different authors have reported the presence of three to eight different layers (28, 64, 186, 421, 438). The outer cell wall layer appears as a dense network with a fibrillar or flocculent aspect (64, 480), whereas the inner wall layer appears contiguous with the plasmalemma with extensive membrane invaginations involved in anchoring of the cell wall to the membrane (192, 276). The microfibrillar polysaccharides glucan and chitin, the components that supply rigidity to the overall wall structure, appear to be more concentrated in the inner cell wall layer, adjacent to the plasma membrane. In contrast, proteins and mannoproteins appear to be dominant in the outermost cell wall layer (Fig. 1B), although they are also present through the entire wall and at the inner regions of the cell wall. Some of the latter proteins may be covalently associated with glucans. Evidence from several cytochemical and cytological studies indicate that the cell wall layering may be due to the distribution of mannoproteins at various levels within the wall structure (64). In any case, it seems clear that layering may be the result of quantitative differences in the proportions of the individual wall components (-glucans, chitin, and mannoproteins) in each layer rather than of qualitative differences (389).

View larger version (123K): [in this window] [in a new window]   FIG. 1.   Cell wall structure. (A) Transmission electron micrograph of a section of a C. albicans cell prepared by freeze-substitution, showing the cell wall as a thick, electron-dense, homogeneous structure. The presence of distinct layers was not evident in this preparation. Bar, 1 µm. Reprinted from reference 3 with permission of the publisher. (B) Thin sections of cells treated with gold-conjugated concanavalin A, showing an intense labeling with gold particles of the external wall surface. The surface exhibits a fibrillar appearance (arrows), suggesting that concanavalin A-reactive cell wall components, i.e., mannoproteins, are particularly abundant at the most external wall layers. The remaining wall structure also appeared as a homogenous structure in this transmission electron micrograph. Bar, 0.5 µm. Reprinted from reference 549 with permission of the American Society for Microbiology. (C) Other procedures for transmission electron microscopy examination of thin sections of C. albicans cells revealed more clearly the presence of an outer floccular layer (arrow) and showed that the remaining cell wall structure is not homogeneous and that some layering exists. Bar, 200 nm. Reprinted from reference 240 with permission of the publisher. (D to F) Complexity of the wall ultrastructure and presence of distinct layers in the cell wall of C. albicans as revealed by different scanning electron microscopy-based procedures such as cryo-scanning electron microscopy (D) and freeze-fracture, freeze-etch analysis (E and F). The presence of well-ordered, regularly arranged, radiating fibrils in the outer layer is particularly evident in the micrographs shown in panels E (hydrophilic cells) and F (hydrophobic cells). Bar, 0.3 µm in both panels. Panel D reprinted from reference 246 with permission of the publisher. Panels E and F reprinted from reference 191 with permission of the publisher.

Fimbriae. The outer cell wall layer that is composed mainly of mannoproteins appears as a dense network of radially projecting fibrils (28, 64), designated fimbriae (154, 583). These fibrils extend for 100 to 300 nm (190, 276) and are approximately 5 nm in diameter (28). Both filamentous forms and blastospores exhibit this characteristic feature (28). C. albicans fimbriae consist of many subunits assembled through noncovalent hydrophobic interactions (583). The major structural subunit of fimbriae is a glycoprotein with an apparent molecular mass of 66 kDa, while the unglycosylated protein has an approximate molecular mass of 8.64 kDa (583). In crude extracts, in addition to the 66-kDa moiety, components migrating with an electrophoretic mobility equivalent to proteins of 54, 47, and 39 kDa reacted with monoclonal antibodies (MAbs) raised against purified fimbriae, suggesting the presence of species with differing degrees of glycosylation (585). The hydrophobic status of the cells profoundly affects fimbrial structure. Hydrophilic cells have long, compact, evenly distributed fibrils, while hydrophobic cells have short, blunt fibrils (190) (Fig. 1E and F). The overall hydrophilic status may be due to masking of hydrophobic components by hydrophilic surface fibrils (190). Fimbrial components mediate the adherence of C. albicans to glycosphingolipid receptors on human epithelial cells (279, 583, 584, 586) as discussed later.

CELL WALL PROTEINS

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Cell Wall versus Secreted Proteins

Should location or function determine the classification of a cell wall protein or secreted protein? Are proteins either one or the other? Proteins that are found in the in vitro growth medium are often called secreted or extracellular proteins. To reach this location, these proteins travel through the cell wall, where they coexist with cell wall-bound moieties and by location are proteins that contribute the total cell wall proteinaceous component. However, it seems reasonable to consider transiently associated proteins with an environmental destination as being secreted. On the other hand, how do we classify a protein when the association does not appear to be transient or when the postulated function of the protein is within the cell wall? There are proteins that are cell associated under one growth condition and secreted under another (519). Cytochemical detection of phospholipase activity shows a localized cell wall location in cells grown on yeast extract medium and a development of a more generalized cell wall, cell surface, and secreted localization in cells in contact with the chorioallantoic membrane (425, 427). There are enzymes recovered from culture supernatants whose functions are thought to be cell wall biosynthesis and remodeling (75). For most proteins, extracellular locations have not all been examined, so that some proteins that are reported as cell wall associated might also be found in culture fluids if examined. Several cell wall components that are not thought to be secreted have been detected in supernatants of C. albicans cell cultures (2, 178, 299, 536, 543). The relationship of some of these moieties with the cell wall structure is unclear. They may come from the outer wall layers. Alternatively, they may be released by lysed cells or as a consequence of the controlled degradation of the cell wall structure, required for wall expansion during growth. One of the criteria that has been used to demonstrate a cell surface location is binding of a ligand or antibody. When similar observations are made with extracellular proteins such as secreted acid proteinase (397) or phospholipase (425, 427), should this finding be differently interpreted, particularly when there may be a function for the cell associated protein (147, 433)? This discussion makes clear that for some proteins, classification as cell wall or secreted may be dependent upon the growth conditions of the organism, the conditions under which localization has been examined, and our view of the function of the protein. As the mechanisms of targeting proteins to subcellular locations are elucidated and protein functions more completely examined, these issues should be resolved. In this review, we have included both proteins whose function is thought to be in the cell wall and those whose role is thought be primarily extracellular.

In the next four sections, we consider general questions of cell wall protein extraction, protein composition of the extracts, protein modification, and distribution of proteins within the cell wall. In these studies, the emphasis has been on definition of cell wall protein by location, since the principal concern has been the removal of a cytoplasmic contribution to the extract. In the following sections, we review specific proteins that have been grouped by various functional and identity relationships.

Different techniques have been used to extract cell wall components of C. albicans. These include physical, chemical, and enzymatic methods and a combination of them. The choice of extracting reagents and techniques, the sequence of extraction methods, and the use of either intact cells or purified cell walls as the starting material may affect both qualitative and quantitative solubilization of cell wall components. In general, and due to the insolubility of both chitin and glucans, sequential alkali and acid treatments are required to effect their extraction (140, 141). In early studies, mannans were extracted from whole cells or isolated cell walls by alkali treatment and further precipitated with Fehling's solution as a copper complex (408). A milder procedure involved mannan extraction from cells resuspended in citrate buffer (pH 7) by autoclaving and further purification by precipitation with Fehling's solution (405) or with Cetavlon (66, 376, 392). This topic is covered more extensively in other reviews (141).

Proteinaceous components have been extracted or solubilized from the cell wall of C. albicans by a variety of techniques. Most of the studies have involved either detergents (such as sodium dodecyl sulfate or n-octylglucoside), reducing agents (such as dithiothreitol [DTT] and -mercaptoethanol [ME]), or hydrolases (such as proteases, Zymolyase, or other -glucanases, and chitinases) to release proteins from both isolated cell walls and intact cells (57, 59, 61, 71, 78, 122, 125, 319, 365, 414, 508, 524, 525). These reagents have been used alone or in combination. Although sulfhydryl compounds such as ME appear to be less efficient that hydrolases such as Zymolyase in releasing cell wall-bound proteins and glycoproteins (295), these chemical agents solubilize a complex array of proteinaceous components from the walls of intact C. albicans cells (57, 58). On the other hand, some -glucanases used to solubilize cell wall moieties actually are enzymatic complexes that may contain other unidentified or uncontrolled hydrolytic activities, which may alter the native characteristics of the released molecules.

Other extraction procedures have been less frequently reported and include chemical, enzymatic, and physical methods alone or in combination. -Elimination with NaOH has been used to release putative structural proteins (369). Ethylenediamine has also been used in structural studies to extract proteins (455). Salt (NaCl) was used to extract the surface determinant of a MAb (40) and a surface adhesin (225). Homogenization has been used to shear fimbriae (583), and -mannosidase treatment followed by sonication has been used to release wall antigens (195).

There has been little comparison of the various methods used to extract the wall proteins. Casanova and Chaffin (57) compared five extracts for yeast cells and germ tubes: (i) ME extract of intact cells at alkaline pH; (ii) the Zymolyase extract of the treated cells; (iii) ME extract of isolated cell walls at alkaline pH; (iv) the Zymolyase extract of the treated cell walls; and (v) a sodium dodecyl sulfate (SDS)-ME extract of isolated cell walls. The extracts were examined by blotting with concanavalin A, two MAbs (MAb 4C12 to a high-molecular-weight component of germ tubes [59] and MAb 24.17 to a mannan epitope of a high-molecular-weight component [72]) and antiserum for factor 6. The authors concluded that the two sequential extracts obtained from intact cells were most satisfactory. In addition, although extraction with reducing agents is frequently thought to release medium to small components from the cell wall, this study showed that ME also released the high-molecular-weight components. These appeared to be larger than the same component present in Zymolyase extracts. ME and other reducing reagents are believed to solubilize mainly components associated with the outermost layers of the cell wall (50, 61, 295). These reagents also increase cell wall porosity and facilitate subsequent action of cell wall degrading enzymes (103, 589). The hydrolysis of glucan by Zymolyase or glucanases may release proteins enmeshed or covalently attached to the glucan. Proteins covalently attached to glucan are postulated to represent species contributing to cell wall structure (59, 122, 123, 125, 482). Covalent attachment of mannoprotein to glucan, perhaps through phosphodiester linkages, has been suggested, as noted below (237, 238, 462, 463).

A valid question is whether the proteins found in these extracts are genuine cell wall components. It has been suggested that treatment of intact C. albicans cells with reducing agents (DTT or ME) may release some intracellular macromolecular components (50). This question has been examined most thoroughly for extracts obtained with ME. As discussed later in this review, receptors or binding proteins for ligands that bind to the intact cell are found in such extracts. On the other hand, several proteins previously associated with a cytoplasmic function have also been found in the wall extracts (6, 8, 161, 303, 520). These observations led to additional experiments using different approaches to demonstrate that the proteins found in the extract were genuine wall components. Transmission electron microscopy demonstrated that each of the moieties was indeed present in the cell wall, including the cell wall interior (6, 8, 303). Chaffin and colleagues (6, 303) also used a more general method to identify genuine cell wall proteins. Intact cells were treated with a derivative of biotin that does not permeate the membrane and therefore does not label cytoplasmic proteins. The extracted biotinylated proteins included those previously thought to be confined to the cytoplasm. This demonstrated that the proteins were present in the cell wall prior to extraction and that their presence in the extract was not due to cytoplasmic contamination. Support for the validity of the extraction procedure was also obtained with parental and mutant strains of S. cerevisiae (294). Two members of the Ssa family of proteins (Ssa1p and Ssa2p) were detected in the cell wall and cytoplasm of the parental strain, whereas in the mutant strain missing these two proteins the remaining members of the family were detected in the cytoplasm but not in the cell wall. The failure to find Ssa proteins in the cell wall of the mutant strain demonstrated that the cell wall extract was not contaminated with the Ssa proteins of the cytoplasm. Hence, current evidence indicates that treatment with sulfhydryl compounds is a suitable method to release autochthonous cell wall protein and glycoprotein components without substantially altering their biological characteristics (6, 60, 61, 293, 295, 296, 300, 302, 377).

Analysis of the protein and glycoprotein constituents solubilized from isolated cell wall preparations and from intact cells of both candidal growth forms by different treatments has revealed both a complex array of protein-containing components and quantitative and qualitative differences in the protein composition of yeast and mycelial cell walls (57-59, 71, 125, 295, 319, 320, 324, 363, 414-416, 524) (Fig. 2). Some components have been characterized as high-molecular-weight mannoproteins (HMWM). The identity of these proteins may vary with the morphology of the organism. Several HMWM are released by treatment with -glucanases and may be covalently attached to structural polysaccharides. These HMWM may play an important role in modulating the organization of the different cell wall constituents to obtain the final supramolecular structure of the wall specific for each C. albicans morphology. In addition, HMWM contain large amounts of carbohydrate and consequently could be major elicitors of anticandidal host immunity (59, 62, 123, 159, 160, 297, 301, 326, 327, 415, 479, 523). In the medium- to low-molecular-weight range, from 20 to more than 40 polypeptide species (depending on the study considered) have been identified (61, 71, 125) (Fig. 2). As discussed above, the evidence suggests that these proteins are bona fide cell wall constituents.

View larger version (64K): [in this window] [in a new window]   FIG. 2.   Polypeptide composition of cell wall extracts and whole protoplast homogenates from C. albicans as revealed by different electrophoretic techniques (A and B). C. albicans cells from which samples were obtained were incubated in the presence of 14C-labelled protein hydrolysate and subsequently tagged with biotin. Double-labelled cell wall proteins and glycoproteins were extracted from intact blastoconidia (lanes 1 and 3) and germinated blastoconidia (lanes 2 and 4) by sequential treatment with ME (lanes 1 and 2) and digestion with Zymolyase 20T (lanes 3 and 4). Samples of protoplast homogenates from blastoconidia and germinated blastoconidia were run in lanes 5 and 6, respectively. Polypeptides were separated by SDS-PAGE and detected by fluorography (A) or transferred to nitrocellulose and detected with an avidin-peroxidase conjugate (B; lane S in this panel shows a mixture of prestained molecular weight standards run in parallel). Numbers and letters are used to identify and compare bands detected in the cell wall extracts by the different experimental procedures used. Although qualitative differences were observed (i.e., some polypeptides exhibited a strong radioactive label but were weakly biotinylated [band 6; star]), surface labelling of cells with biotin appeared to be a suitable technique to detect proteins in the wall of C. albicans. Thus, from the complex polypeptide pattern found in the protoplast homogenate samples as revealed by fluorography (A, lanes 5 and 6), only few species were labelled with biotin, indicating that most proteins released by ME and Zymolyase from intact cells are bona fide cell wall components. Brackets indicate a cluster of bands within a molecular weight range where many candidal moieties that represent receptors for host ligands have been identified (see the text). Reprinted from reference 61 with permission of the American Society for Microbiology. (C) The complexity of the polypeptide pattern of the cell wall extracts was clearly evidenced when analysis was performed by two-dimensional PAGE and silver staining (the polypeptide pattern shown corresponds to the ME extract from blastoconidia). Reprinted from reference 486 with permission of the American Society for Microbiology.

There is a growing body of experimental evidence indicating that the propertiesexpression, distribution, and chemical characteristicsof cell wall proteins and glycoproteins observed in vitro and in vivo are dependent on multiple factors. These include growth conditions, organism-related factors (such as growth state, morphology of the cells, strain and serotype, phenotypic switching, cell surface hydrophobic or hydrophilic status), and the nature of the biological specimens (intact cells or isolated wall preparations) that are subjected to analysis (5, 7, 24, 44, 57, 63, 106, 159, 190, 191, 195, 210, 284, 297, 301, 326, 416, 422, 477, 513, 529). Iron availability, which has been shown to be important for pathogens in establishing infection (46, 400, 566), affects the cell surface (529). There are quantitative but not qualitative changes in the profile of surface proteins associated with growth at different iron concentrations. Yeast cells of most strains grown in limiting or excess iron do not adhere as well to human buccal epithelial cells as do organisms grown at intermediate concentrations that support optimum growth. The effects of growth conditions on the expression of specific proteins are discussed for each protein in later sections. The cell wall may be thus envisaged as a highly dynamic "organelle." The fungus is capable of expressing differentially variable wall constituents that may be useful for switching between commensal and pathogenic lifestyles and for modulating and/or evading the immune host defense.

Glycosylation. Without any doubt, glycosylation is the most important modification of the proteins in the fungal cell wall. Attachment of sugar moieties to proteins results in the formation of the glycoproteins, which in the case of C. albicans are mainly mannoproteins. The general features of mannoproteins have been discussed above. However, the presence of nonglycosylated proteins has also been found in the cell wall of C. albicans (6, 8, 58, 61, 161). Mannosylated proteins can be broadly divided into two classes. The HMWM, most of which are larger than 200 kDa, are postulated to have structural functions within the cell wall. Some medium- to low-molecular-weight proteins also react with concanavalin A, indicating their mannan content. Within this broad division, the amount of carbohydrate attached to the same polypeptide may vary (59, 122, 364). Within the high-molecular-weight class, there is a difference in the size of the side chains associated with morphology. Oligosaccharides obtained from mannoproteins from yeast cells average 600 residues, and those from germ tubes average 300 residues (122). Cell wall proteins may also be O glycosylated with unbranched mannose chains containing one to a few residues (412). Elorza et al. (123) have suggested that some proteins are initially secreted as O-glycosylated proteins and become cross-linked with glucan and/or other N-glycosylated proteins only after incorporation into the cell wall structure.

Phosphorylation. Phosphorus is a minor component of the cell wall of C. albicans (77). It has been assumed that it is present in cell wall mannoproteins in phosphodiester linkages between mannose residues (17, 454). Bulk mannan from C. albicans can be fractionated into five fractions that differ in the amount of phosphate (393). Phosphomannoprotein complexes from cells of both C. albicans A and B serotypes have been characterized (261-263, 494-498). This material contained 0.9 to 1.6% phosphate depending on the morphology and strain considered, and the authors concluded that -(1,2) oligomannosaccharides were attached by phosphodiester linkage to other branching moieties. -(1,2) oligomannosidic epitopes were further observed on a C. albicans 14- to 18-kDa phospholipomannan moiety (545), a glycolipid with important immunologic properties (133-135, 228, 229, 424), whose mannose residues may be added differently from mannan (546). Not all the glycoproteins in the cell wall of C. albicans contain phosphate, and some proteins may contain phosphate but not carbohydrate (58).

Ubiquitination. Ubiquitin is a small (approximately 8,500-Da) polypeptide first isolated from bovine thymus (167). Sequence analysis of various ubiquitin genes has revealed striking evolutionary conservation among species (138). Ubiquitin plays important roles in protein modification, protein degradation, gene transcription, organization of chromatin structure, and stress resistance in higher eukaryotes (138, 139, 197). It is also associated with some cell surface protein and signaling functions (69, 368, 404, 504, 553). In yeast, a role for ubiquitination in endocytosis and/or turnover of plasma membrane protein receptors including -receptor has been demonstrated (198, 269, 442). The C terminus of ubiquitin is covalently attached to -amino groups of lysine in protein substrates by an enzymatic conjugation system. A large number of enzymes responsible for the formation and processing of ubiquitin-protein conjugates have been described (138), including in C. albicans (98). We have cloned a polyubiquitin gene (UBI1) of C. albicans that contains three tandem copies, head-to-tail spacerless repeats, of the sequence coding for the 76 amino acids of the ubiquitin protein (485). The gene has also been cloned from a different strain (15). Northern blot analysis revealed a single mRNA population of about 1 kb present in similar amounts in both yeast and mycelial cells (485). Indirect immunofluorescence demonstrated that ubiquitin determinants were located on the cell surface, and Western blot analysis of a ME extract demonstrated that several cell wall proteins contained ubiquitin-like epitopes. The cell wall species that are ubiquitinated are discussed below with the individual proteins. The role of ubiquitin in the cell wall whether in protein degradation, stress protection, or perhaps even modulation of activity of receptor-like molecules remains to be assessed.

As described above, transmission electron microscopy studies of the C. albicans cell wall show the existence of several layers. The structural appearance of these layers is variable and seems to be related to the strain examined, the growth conditions, the morphology (yeast cells or germ tubes) exhibited by the microorganism, and the sample preparation protocol (50, 64, 422) (Fig. 1). After total removal of proteins and mannoproteins by treatment with strong alkali or heating, the cell wall appeared to be significantly thinner with loss of any appreciable layering. These structural changes were paralleled by the absence of electron-dense components detectable with ordinary electron microscopy dyes, concanavalin A binding sites, and positive staining with reagents specific for mannoproteins (periodate-silver). Thus, the cell wall layering appears attributable to the distribution of mannoproteins at various levels within the wall structure (64). Treatment of cells with sulfhydryl agents and hydrolytic enzymes, coupled with specific cytochemical staining, has consistently shown that mannoprotein constituents are preferentially located at the outermost layer of the wall of C. albicans cells. The surface has a fibrillar or flocculent aspect with thin, delicate filaments or fimbriae (Fig. 1; see below). This material is present mostly in virulent strains, and it is also more abundant in isolates exhibiting increased adherence to host tissues (64). Proteins whose biological function is at the cell surface or the extracellular environment may nevertheless be found at the innermost layer of the wall and through the wall as they travel from the plasma membrane and periplasmic space to their destination (478). Thus, some proteins destined for the extracellular environment may also be obtained in cell wall extracts. Evidence for this mannoprotein traffic in C. albicans has been reported (423). Immunoelectron microscopy shows that proteins on the cell surface visualized by indirect immunofluorescence are also detected at the cell surface as well as near the plasma membrane with some protein distributed through the wall (6, 303). On the other hand, a protein that is not detected at the cell surface by indirect immunofluorescence but is present in cell wall extracts is located only in the interior of the wall (8). Using a panel of MAbs to localize proteins, Pontón et al. (416) demonstrated various distribution classes of cell wall proteins: (i) expressed only on the germ tube surface, (ii) expressed on the germ tube surface and within the yeast cell wall, (iii) expressed on both yeast cell and germ tube surfaces, and (iv) expressed within the wall of both germ tubes and yeast cells. A fifth category, expressed only on yeast surfaces, is also reported (296). Proteins that are associated with -glucans should be concentrated near the plasma membrane with the structural polysaccharide. In any case, since asymmetry in mannoprotein distribution is evident in C. albicans walls (64), layering is more likely to be the result of quantitative differences in the proportions of the individual wall components (-glucans, chitin, and mannoproteins) in each layer rather than qualitative differences (389).

Differences in the distribution of proteins and glycoproteins at the cell surface are also noted. This asymmetry may be related to the physiologic role played by each particular moiety. High-molecular-weight mannoproteins that may play an important and active morphogenetic role in modulating the organization of the cell wall (59, 62, 123, 160, 326, 327, 480) are homogeneously distributed on the cell surface (59, 62, 159). Some proteins and mannoprotein moieties that are receptors for different host ligands exhibit clustering or asymmetric cell surface distribution (60, 296, 328). The distribution of common or morphology-associated, homogeneously or heterogeneously distributed cell surface antigens of C. albicans as revealed by immunofluorescence microscopy is shown in Fig. 3.

View larger version (135K): [in this window] [in a new window]   FIG. 3.   Surface expression of cell wall proteins. Phase-contrast (A and C) and immunofluorescence (B, D, and E to I) of C. albicans blastoconidia (B) and mycelial filaments (M) reacted with different polyclonal and monoclonal antibody preparations raised to protein and glycoprotein cell wall constituents. Some antibodies recognized antigens that appeared to be specific for or preferentially expressed in germ tubes (A and D) or blastoconidia (E and F). Arrows in panels A to D point to the location of mother blastoconidia (A and C) that exhibited no fluorescence (B and D). Some antigens appeared to be homogenously distributed on the surface of mycelial filaments (B and D) or blastoconidia (G). However, patches of greater fluorescence intensity were observed with other antisera preparations (H and I), suggesting that antigens recognized by such antisera were heterogenously and randomly distributed within the cell wall structure. The pictures in panels B and D to G are from standard immunofluorescence microscopy observations. Panels H and I show single-focal-plane sections of different cells obtained by confocal fluorescence microscopy and associated software. Bar, 10 µm (except for panel G, which is 1.25 µm). Panels A to D reprinted from reference 59 with permission of the American Society for Microbiology. Panels E and F reprinted from reference 24 with permission of the American Society for Microbiology. Panel G reprinted from reference 72 with permission of the American Society for Microbiology. Panels H and I reprinted from reference 328 with permission of the American Society for Microbiology.

At least three cell moieties appear to have posttranslational regulation of their localization in the cell wall. A MAb that recognized a hyphal surface protein detected a smaller protein in the plasma membrane of yeast cells (394). The C3d binding protein was detected as a 60-kDa moiety in germ tube cell walls, while a 50-kDa component was found in yeast cell membranes (563). The third example is a 30-kDa protein found in the cell wall of germ tubes but not yeast cells (5). However, the gene is expressed in yeast cells and presumably translated, although the cellular location of the protein is not known. Another group of proteins to be discussed below, i.e., enolase, hsp70, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), well-known cytoplasmic proteins, have recently been described as also present in the cell wall. They may represent proteins with regulation of partition between two cellular compartmentscytoplasm and cell wall.

Other proteins, such as hydrolytic enzymes [discussed below], are altered in their expression by environmental substrates. Another example of an apparently environmentally regulated protein is the 58-kDa fibrinogen binding protein that is expressed by cells growing on Lee medium but not by cells growing on yeast extract-peptone-glucose medium (5). The foregoing discussion makes clear that the expression and localization of proteins in the cell wall are complex and dynamic processes. The presence and location of proteins and glycoproteins in the cell wall are likely to be affected by several mechanisms including gene expression, posttranslational regulation, subcellular partitioning, and targeting of destination within the cell wall.

As discussed above, a number of hydrolytic enzymes have been recovered from both cell-associated locations (cell wall and periplasm) and culture medium whose function is postulated to be within the cell wall (Table 1). These enzymes are thought to be involved in cell wall biosynthesis or the remodeling that accompanies growth and division of cells.

Exo--(1,3)-glucanase. Secretory exo--glucan hydrolases (-glucanases or -glucosidases) are widely occurring enzymes in many yeast and fungal species. Although the exact physiological roles of these enzymes are unknown (141, 386), they participate in the metabolism of -glucan, which is the main structural microfibrillar polymer of the cell wall in C. albicans (64). The most widely accepted biological role of glucanases is limited hydrolysis of cell wall glucan during morphogenetic events (141, 386). -Glucanases have been described to be associated with the C. albicans cell wall (387, 428).

-1,3-Glucan transferase. Many yeasts, including C. albicans and S. cerevisiae, contain a highly conserved protein with a size ranging from 31.5 to 34 kDa depending on the species (196). It is a major cell wall mannoprotein in S. cerevisiae (466). In C. albicans, a protein of 34 kDa is secreted by protoplasts and observed as an aggregate in gel filtration (122). When released by Zymolyase, it eluted as a low-molecular-weight species. The protein appeared to have a single N-linked oligosaccharide of 2.5 kDa and a residual protein moiety of 31.5 kDa. A cell wall protein with an approximate molecular mass of 34 kDa was isolated as a by-product during purification of an endo-(1,3)--glucanase from the material secreted to the medium by C. albicans (178). The enzyme displayed a unique glucanosyl transferase activity and did not contain any exo- or endo--glucanase activity. The authors suggested that this 34-kDa protein is a glucan-branching enzyme responsible for the transformation of the initial linear -(1,3)-glucan into the branched -(1,3)--(1,6)-glucan that is found in the cell wall of the fungus. The C. albicans BGL2 gene encoding the -1,3 glucan transferase has been cloned (471) and is similar to the S. cerevisiae BGL2 gene (245). The S. cerevisiae enzyme has been described as an exoglucanase (245) and as an endoglucanase (373). However, more recently, the S. cerevisiae enzyme has been shown to be homologous to the sequence of the C. albicans enzyme and corresponds to Bgl2p (165). More sensitive assays revealed that at low concentrations of glucose oligosaccharides, glucanase activity was observed, while at higher concentrations, glucosyltransferase activity predominated. The enzyme transfers -1,3 glucan oligosaccharides from a donor -1,3 glucan to an acceptor -1,3 glucan, forming a linear polymer joined by a -1,6 linkage (587). This activity would permit the enzyme to participate in cross-linking or repair of glucan within the cell wall.

Chitinases. Chitinases are produced by many organisms including chitin-containing organisms that produce both chitin synthases and chitinases. In C. albicans, the most likely role for these enzymes, like glucanases, is limited hydrolysis of cell wall chitin during morphogenetic events (141). C. albicans contains three chitinase genes, CHT1, CHT2, and CHT3 (346). More than half the chitinase activity detectable in whole-cell extracts is associated with secreted enzyme (periplasmic and cell wall) (170). Reactivity is detected in supernatants of washed cells and with intact cells by using a substrate that does not enter the cell (346). Chitinase production increases during exponential growth and is greater in cells grown on yeast extract-peptone-dextrose (YEPD) medium than on a minimal medium. One of the roles of chitinase in S. cerevisiae, where chitin is confined primarily to the septum, is mother-daughter separation. Disruption of S. cerevisiae CTS1 results in clumps of cells due to failure of the cells to separate (273). Treatment of C. albicans yeast cells with a chitinase inhibitor also leads to inhibition of cell separation and clumps of cells (170).

-N-Acetylglucosaminidase. Production of -N-acetylglucosaminidase by C. albicans cells is induced by the presence of GlcNAc in the medium (519). GlcNAc, the -N-acetylglucosaminidase reaction product, also induces the synthesis of other enzymes required for the metabolism of this amino sugar (171, 492, 519). The enzyme may function as a chitobiase that, in conjunction with chitinase, completes the hydrolysis of chitin to provide both nitrogen and carbon sources from chitin. The enzyme, for which two molecular forms with different glycosylation levels appear to exist (364), is secreted and deposited into different regions of the cell envelope of both the yeast and mycelial forms (365, 426, 519). The enzyme is also released to the culture medium of both growth forms but to a greater extent (at least fourfold greater) during hyphal formation (519). It is suggested that the germ tube wall is more porous than that of yeast cells and, consequently, that release of extracellular enzymes to the medium is facilitated by the hyphal formation process. -N-Acetylglucosaminidase acts on a number of natural and synthetic substrates including diacetylchitobiose and triacetylchitotriose. In its native form, the enzyme appears to be a monomer with a molecular mass of 66 kDa (519).

Glutaminyl-peptide--glutamylyl-transferase. The activity of the enzyme glutaminyl-peptide--glutamylyl-transferase (transglutaminase) has been detected in cell extracts obtained from both growth phases of C. albicans. The activity was associated mostly with the cell wall fraction, whereas the cytosol contained almost negligible amounts of enzyme activity (456). This distribution suggested an extracellular (cell wall-bound) location for transglutaminase in intact cells. Although the wall component displaying this enzymatic activity has not been characterized, the postulated transglutaminase activity could be involved in the formation of covalent bonds between different wall proteinaceous moieties (456). In this context, it has been suggested that formation of such covalent linkages could play a role in maintaining the spatial organization of the cell wall during the biogenesis and assembly of this structure.

The enzymes in the previous section are postulated to find substrates and to have their primary function within the cell wall. This section considers enzymes whose substrates are not associated with the cell wall but are found in the environment (Table 1). The action of these enzymes may provide access to nutrients for the organism. When hydrolysis of these substrates or action of extracellular proteins affects the function and viability of the host, the enzymes may be considered virulence factors that contribute to the establishment of infection. These proteins are at least transiently associated with the cell wall during their translocation across the cell wall to the external environment. However, in some cases, the association is less transient. The distribution of some of these enzymes is variable and, under some growth conditions, may be primarily cell associated in the periplasm and cell wall. Some of these proteins have also been localized to the cell surface by the same criteria that have established a cell surface location for adhesins whose function is at the cell surface. One of these extracellular proteins, Sap, also found at the cell surface, may contribute to adherence (209, 406). We have also included in ^<