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Microbiol Mol Biol Rev, March 1998, p. 130-180, Vol. 62, No. 1
Department of Microbiology and
Immunology,
1092-2172/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cell Wall and Secreted Proteins of
Candida albicans: Identification, Function, and
Expression
SUMMARY
INTRODUCTION
CELL WALL COMPOSITION AND ORGANIZATION
Composition
Organization
Layering.
Fimbriae.
CELL WALL PROTEINS
Cell Wall versus Secreted Proteins
Extraction
Composition
Modification
Glycosylation.
Phosphorylation.
Ubiquitination.
Distribution and Expression
Enzymes with Cell Wall Function
Exo-
-(1,3)-glucanase.
-1,3-Glucan transferase.
Chitinases.
-N-Acetylglucosaminidase.
Glutaminyl-peptide-
-glutamylyl-transferase.
Hydrolytic Enzymes and Proteins with Extracellular
Targets
Acid proteinase.
Phospholipase.
Esterase.
Glucoamylase.
Hemolytic factor.
Acid phosphatase.
Miscellaneous.
Morphology-Associated Proteins
Epitope recognized by MAb 4C12.
Epitope recognized by MAb 3D9.
Hwp1p.
Hyr1p.
Immunomodulatory 65-kDa Mannoprotein (MP65)
Heat Shock Proteins
hsp90.
hsp70.
Heat shock or stress mannoproteins.
Glycolytic Enzymes
Enolase.
Phosphoglycerate kinase.
Glyceraldehyde-3-phosphate dehydrogenase.
Alcohol dehydrogenase.
Binding Proteins (Receptors) for Host Ligands
Serum proteins.
(i) Serum albumin and transferrin.
(ii) Fibrinogen.
(iii) Complement fragment C3d.
(iv) Complement fragment iC3b.
Extracellular matrix proteins.
(i) Laminin.
(ii) Fibronectin.
(iii) Entactin.
(iv) Vitronectin.
(v) Collagens.
Mannan adhesins and other binding proteins.
(i) Mannan adhesins.
(ii) Hydrophobic proteins.
(iii) Fimbriae.
(iv) Plastic binding proteins.
(v) Epithelial binding lectin-like protein.
(vi) Agglutinin-like proteins.
(vii) Adherence to Streptococcus spp. and other
bacteria.
(viii) Adherence to salivary proteins.
(ix) Miscellaneous.
WHERE ARE WE GOING? THE MYSTERIES AND CHALLENGES
FINAL COMMENT AND OUTLOOK
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis.
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.
Cell Wall and Morphology
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.
Cell Wall and Interactions with the Host
Two major aspects of the host-parasite interactions are the adhesion of C. albicans cells to host cells and tissues and the immunomodulation of the host immune response.
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.
Organization
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): Mannoprotein
GPI remnant
-1,6-glucan
-1,3-glucan
chitin. 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).
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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.
Celerin et al. (67, 68) reported that in the fungus Mycrobotryum violaceum, fimbriae are composed of a protein with strong similarity to collagen. The fimbrial protein from C. albicans discussed above does not appear to be related to this collagenous fimbrial protein (68). However, this type of collagenous fimbria appears to be conserved among fungal species since antiserum to the protein domain reacts with surface proteins of many fungi (67). The antiserum also reacts with 81- and 84-kDa surface moieties that may represent a second putative candidal fimbria (67). Although some fungi contain more than one type of fimbria (577), there is no additional evidence for multiple types of fimbriae in C. albicans. If such collagenous fimbriae are found in C. albicans, additional fimbria-mediated interactions of the microorganism with the host cells and tissues may be possible (see below).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.
Extraction
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).
Composition
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.
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There is a growing body of experimental evidence indicating that the
properties
expression, distribution, and chemical characteristics
of 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.
Modification
Posttranslational modifications of proteins include glycosylation, acetylation, prenylation, phosphorylation, ubiquitination and addition of a GPI moiety. Organisms use these modifications to confer structural options for proteins, to provide regulatory control of their functions and to target proteins to specific cellular locations. While not all of these modifications have been described in C. albicans, it is likely that the organism possesses the ability to modify its proteins by most, if not all, of the posttranslational modifications.
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).
-1,6- and
-1,3-glucan
moieties present in C. albicans cell wall mannoproteins may
be connected to a GPI anchor, which is known to be phosphodiester linked to the C-terminal amino acid of the mature protein (237, 238, 247, 268). This is in agreement with the hypothesis that proteins destined to be incorporated into the cell wall are linked to
-glucan through the glycan part of their GPI anchors
(104), as has been demonstrated for the S. cerevisiae
-agglutinin (306, 573). The GPI anchor
targets
-agglutinin to the cell wall (573). Pulse-chase
experiments indicate that a plasma membrane-bound form is released to
periplasmic space as an intermediate form that is then incorporated
into the cell wall (305, 306). Recent studies of the linkage
between mannoprotein and glucan suggest that the GPI remnant consists
of ethanolamine-phosphate-mannose5, with the terminal
mannose attached to the nonreducing end of
-1,6-glucan (268). A transglycosylation reaction is proposed to effect
the linkage.
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.
Distribution and Expression
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.
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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 compartments
cytoplasm 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.
Enzymes with Cell Wall Function
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.
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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)-glucanases has been reported mainly for S. cerevisiae, where at least two isoenzymes, arising by differential
glycosylation of a primary gene product, are secreted into the medium
(430). In C. albicans, exo-
-(1,3)-glucanase
activity was found to be secreted and exported mainly during germ tube
formation. Negligible enzyme was released into the medium when yeast
cells were grown (429). As with
-N-acetylglucosaminidase, this may be related to the more
porous nature of the germ tube cell wall (519). In contrast
to S. cerevisiae, only one exoglucanase has been detected in
C. albicans, and it accounts for most of the total glucanase activity present in the growth medium and cell extracts (307, 361,
428). However, there are some discrepancies between results reported from different groups. The enzyme purified from cell extracts
of C. albicans 1001 was reported to be a heterodimer of
subunits with molecular masses of 63 and 44 kDa (362).
Subsequently, the major exoglucanases secreted into the medium by
strains 1001 and 3153A were found to be identical, single
nonglycosylated polypeptides, with a molecular mass of about 38 kDa
(307). The peptides had significant chemical and
immunological similarity to the major exoglucanase secreted by S. cerevisiae. Cloning and sequencing of the gene EXG
(previously XOG1) coding for the exo-
-(1,3)-glucanase of
C. albicans (75, 308) revealed high identity to
the
-(1,3)-exoglucanase EXG1 gene cloned from S. cerevisiae (561). A single transcript was detected in
both yeast and hyphal forms, and the levels of expression appeared
proportional to the growth rate (74). Sequence analysis
indicated a signal peptide for secretion and a recognition by a
KEX2-like protease (75). A mature enzyme of 400 amino acid residues with no sites for N-linked glycosylation was
predicted. These results are consistent with the characteristics
(carbohydrate content and molecular mass) of the secreted enzyme
previously reported (307). Recombinant
exo-
-(1,3)-glucanase of C. albicans purified from
S. cerevisiae has been found to contain a number of short
blocks of sequence homology to several genes for cellulases of the
family A glucanases, including the conserved sequence site NEP, which
has previously been shown to be important in the catalytic function of
several cellulases (76). Glu-330 has been identified as the
catalytic nucleophile in the enzyme (308).
-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).
The C. albicans chitinase gene(s) was identified by use of degenerate PCR primers based on conserved fungal chitinase sequences (346). The deduced amino acid sequence encoded by the CHT2 open reading frame (ORF) predicted a polypeptide of 583 amino acids, and that encoded by CHT3 predicted a polypeptide of 567 amino acids. The deduced sequence from CHT1 consisted of 416 amino acids (347). The C. albicans chitinases were similar (36 to 38%) to that of S. cerevisiae (346). However, the similarity was 55 to 65% in the N-terminal region containing the putative catalytic domain. There are potential N-glycosylation sites in the sequences. Near the 3' end of the sequences of CHT2 and CHT3 is a sequence encoding a region that is rich in serine and threonine, which may be potential sites for O glycosylation. This region is not present in CHT1, which encodes a smaller putative protein (347). Expression of CHT2 and CHT3 was detected in both yeast cells and hyphae, although greater expression was associated with yeast growth (346). Expression of CHT1 was not detected under the various growth conditions examined. Preliminary results using a disruption of CHT2 suggested that the chitinase participated in cell separation as cells tended to form clumps or clusters.
-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).
-N-Acetylglucosaminidase from C. albicans is
specifically a chitobiase (519). It may act coordinately with chitinase, which is also present in this fungus (22),
in the hydrolysis of chitin to form GlcNAc. However, the role of these
enzymes in cell metabolism is not clear (223), but there is
no direct role in morphogenesis (383). In vivo, cleaving of GlcNAc from complex carbohydrates by C. albicans
-N-acetylglucosaminidase may provide a suitable carbon
source for the fungus. Alternatively, removal of GlcNAc residues from
glycoproteins of the fungal cell surface may cause conformational
changes that modify adhesion of C. albicans cells to host
tissues (55). Jenkinson and Shepherd (223)
reported that a C. albicans mutant defective in the
production of
-N-acetylglucosaminidase was less virulent
in an experimentally induced candidiasis mouse model than was the
wild-type strain, thus suggesting a possible role for this enzyme as a
candidal virulence factor. On the other hand, the phenotypic properties of the mutant suggest that the enzyme is not essential for the growth
of C. albicans cells (223). Hence, further work
is required to determine the role of this enzyme in pathogenicity. The
entire
-N-acetylglucosaminidase gene (HEX1)
from C. albicans has been cloned (55). The
organism appeared to use alternative transcription termination sites
depending upon growth conditions, since the HEX1 mRNA from
cells grown on GlcNAc was 200 nucleotides longer than the transcript
from cells grown on glucose. Plasmid-borne HEX1 also
responded to GlcNAc induction.
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.
Hydrolytic Enzymes and Proteins with Extracellular Targets
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 <