Download Molecular organization of the cell wall of Candida albicans

Medical Mycology 2001, 39, Supplement 1, 1±8
Molecular organization of the cell wall of Candida
albicans
F. M. KLIS, P. DE GROOT & K. HELLINGWERF
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam,
The Netherlands
We have recently presented a molecular model of the cell wall of Saccharomyces
cerevisiae. Here we discuss the evidence that a similar model is also valid for
Candida albicans. We further discuss how cell-wall proteins are linked to the skeletal
layer of the wall, and their potential functions. We emphasize that the composition
and structure of the cell wall depends on growth conditions. Finally, cell-wall
damage seems to activate a salvage mechanism resulting in restructuring of the cell
wall.
Keywords
cell-wall dynamics, GPI proteins, Pir proteins
Introduction
The fungal cell-wall functions as an exoskeleton that
determines the shape of the cell and helps to withstand
turgor pressure. As such it represents an excellent target
for antifungal drugs, the more so as several fungal cellwall components are absent in mammalian cells. The
macromolecules that are responsible for the mechanical
strength of the cell wall form a skeletal layer close to the
plasma membrane. This inner layer functions as a
scaffold for an external protein layer that limits the
permeability of the cell wall for large molecules, and
determines the antigenic properties and the hydrophobicity of the cell surface. The external protein layer may
also contain various adhesion proteins. In this review we
wish to present the current views of how the cell wall of
Candida albicans is organized at the molecular level. We
will focus on results from recent publications; for earlier
data we refer to two extensive reviews on this subject
[1,2].
(Table 1). Interestingly, the cell wall of C. albicans
contains considerably more b (1,6)-glucan, implying that
the b (1,6)-glucan molecules in C. albicans are either
more numerous or contain more glucose residues or both
[4,5]. C. albicans is a pleomorphic fungus that can grow
in the yeast form and pseudohyphally, but can also form
authentic parallel-sided hyphae. The data presented here
refer to the yeast form. The hyphal wall has a slightly
higher chitin content than the yeast form [1,6]. The
mannan side-chains of cell-wall proteins (CWPs) contain
numerous phosphodiester bridges [7]. As a result, C.
albicans cells have an isoelectric point of 2¢3 [8], and
their walls act as an ion exchanger capable of effectively
binding positively charged (in)organic ions and proteins
(see below). Interestingly, dityrosine has been identiŽ ed
Table 1 Cell-wall compositions of S. cerevisiae and C. albicans
grown in the yeast form
Wall dry weight (%)
Macromolecule
S. cerevisiae*
C. albicansy
Similarly to Saccharomyces cerevisiae, the cell wall of
C. albicans contains four classes of macromolecules
Mannoproteins
b (1,6)-Glucan
b (1,3)-Glucan
Chitin
50
5
40
1–3
35–40
20
40
1–2
Correspondence: Frans Klis, Swammerdam Institute for Life
Sciences, University of Amsterdam, Nieuwe Achtergracht 166,
1018 WV Amsterdam, The Netherlands. Tel.: ‡31 20 525 7834;
e-mail: [email protected].
These values tend to vary depending on medium composition and
environmental conditions.
*Ref. [3].
yRecalculated from [4].
ãã
Cell-wall composition
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ISHAM, Medical Mycology, 39, Supplement 1, 1±8
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Klis et al.
both in the lateral walls and the septa of hyphal forms
and in the walls of the yeast form [9]. Possibly, it crosslinks CWPs, thus contributing to virulence by offering
protection against proteolysis. Alternatively, it may
strengthen noncovalent adhesion to host cells by forming
cross-linkages between CWPs and surface proteins of
host cells.
Molecular organization of the cell wall
The cell wall of C. albicans has a layered structure. This
is beautifully illustrated by the work of Tokunaga and
co-workers [10]. Using cryoŽxation in combination with
scanning electron microscopy, they have shown that—
under the growth conditions used—the cell walls consist
of a homogeneous inner layer of about 100 nm and an
outer protein layer of about 180 nm, consisting of
densely packed Žbrillae organized perpendicular to the
cell surface. It is tempting to assume that the Žbrillar
structures visualized in this way are identical to the
Žmbriae isolated by shearing forces from intact cells (see
below) [11].
Based on extensive research with S. cerevisiae [12–19]
and on comparative studies with C. albicans [20–22], the
molecular organization of the cell wall of C. albicans is
believed to be as follows (Fig. 1). The skeletal inner
layer is an elastic three-dimensional network of
branched b (1,3)-glucan molecules that are locally
aligned and is kept together by hydrogen bonding. This
network acts as a scaffold for the attachment of the other
macromolecules in the cell wall. Surarit et al. [23] have
presented evidence that in C. albicans b (1,6)-glucan
might be coupled to chitin through the C6 position of Nacetylglucosamine residues (b (1,6)-glucan!chitin). This
type of linkage has not been found in S. cerevisiae; in this
organism chitin is coupled covalently with its reducing
end to either a b (1,3)-glucan chain or to a short b (1,3)glucan side-chain of b (1,6)-glucan [17,18]. A comparably
detailed glycobiological analysis for C. albicans is still
lacking. Table 2 provides an overview of the postulated
basic structure of the polysaccharide–cell-wall protein
complexes in the cell wall of C. albicans. These will be
discussed in more detail in the next section.
Identi®cation and classi®cation of cell-wallassociated proteins
Molloy and co-workers have shown that preparations of
isolated cell walls of C. albicans may be heavily
contaminated with membrane proteins [25]. Biotinylation of cell surface proteins, using a membraneimpermeable reagent, is therefore an efŽcient alternative
means to identify authentic cell-surface proteins [26].
Although a few authentic cell-wall-associated proteins
are lost by extraction with hot sodium dodecyl sulfate
(SDS) such as Bgl2, a b (1,3)-glucan remodeling enzyme
[27], and the Žbrinogen-binding protein Pra1 [28], SDS-
Table 2 Putative polysaccharide–cell-wall protein (CWP) complexes in the cell wall of C. albicans
Fig. 1 Proposed molecular organization of the cell wall of
S. cerevisiae and C. albicans. The two main classes of CWPs are
glycosyl phosphatidylinositol (GPI-CWPs; GPI-anchor dependent
CWPs) and Pir-CWPs (CWPs with internal repeats). The b (1,6)glucan molecules are highly branched and thus water-soluble,
tethering GPI-CWPs to the b (1,3)-glucan network. GPI-CWPs
probably represent the major component of the external protein
layer. The Pir-CWPs are directly linked to the b (1,3)-glucan
network through an alkali-sensitive linkage and seem to be
distributed throughout the b (1,3)-glucan network. Some GPI-CWPs
may be double-linked through b (1,6)-glucan and through an alkalisensitive linkage (not shown here). The b (1,3)-glucan molecules are
moderately branched and form a three-dimensional, elastic network
that is kept together by hydrogen bonding between locally aligned
chains. The terminal nonreducing ends of the b (1,3)-glucan sidechains are believed to function as acceptor sites for b (1,6)-glucan
and chitin. This model is based on extensive research on S.
cerevisiae [12–19] and on comparative research on C. albicans [20–
22].
Polysaccharide–CWP complexes
References
b (1,3)-Glucan b (1,6)-glucan GPI-CWP
[20–22]
b (1,3)-Glucan$Pir-CWP
[22,24]
b (1,3)-Glucan$
[b (1,3)-glucan b (1,6)-glucan ] GPI-CWP
[16]
b (1,3)-Glucan$GPI-CWP
[16]
Chitin b (1,6)-glucan GPI-CWP*
[23]
For the sake of clarity, the potential attachment of chitin to b (1,3)-glucan in
the polysaccharide–CWP complexes is not shown. X!Y designates a
glycosidic linkage between X and Y in which X has provided the reducing
end and Y the nonreducing end. $ designates an alkali-sensitive linkage, the
nature of which is still speculative.
*In S. cerevisiae chitin may be linked to b (1,6)-glucan through a short b (1,3)glucan side-chain resulting in the polysaccharide–CWP complex: chitin!
b (1,6)-glucan GPI-CWP [17].
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2001 ISHAM, Medical Mycology, 39, Supplement 1, 1±8
Cell wall architecture
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extracted walls are strongly recommended for structural
studies. Various classes of cell-surface proteins have
been described.
1. Cell-surface proteins released by extraction from
intact cells with reducing agents [26]. These will include
CWPs that are linked through disulphide bridges to
other CWPs. They will also include soluble precursor
forms of covalently linked CWPs [26,29] or periplasmic
enzymes that are released because the cell wall has
become more permeable as a result of breaking
disulphide bridges [30]. For example, the secretory
protein Pra1 is released from the walls by reducing
agents [31]. Several authors have identiŽed glycolytic
enzymes at the cell surface of C. albicans [2,32,33,34].
This is consistent with the observation that several
proteins released by reducing agents do not react with
Concanavalin A, and thus do not seem to be glycosylated
[26]. It has been proposed that these proteins reach the
cell surface through a nonconventional export pathway,
bypassing the endoplasmic reticulum and the Golgi.
Alternatively, they may be abundant cytosolic proteins
from damaged cells that have become stuck in the wall or
are positively charged at the pH of the growth medium,
and bind therefore to the negatively charged wall [33].
As extraction with reducing agents is usually carried out
at a slightly alkaline pH, these proteins may subsequently become negatively charged, thereby forcing their
release from the walls. Their release from the cell wall
might also be facilitated because the wall becomes more
permeable.
2. CWPs that are linked through a GPI-remnant to
b (1,6)-glucan (GPI-CWPs). The b (1,6)-glucan moiety
may in turn be linked to b (1,3)-glucan or to chitin
(Table 2) [20–23]. The evidence for the presence of the
polysaccharide–CWP complex b (1,3)-glucan b (1,6)glucan GPI-CWP in C. albicans is based on comparative studies with S. cerevisiae. For this organism more
rigorous evidence exists (reviewed in [15]). The most
relevant observations in C. albicans are the following: (i)
CWPs released by endo-b (1,3)-glucanase digestion of
isolated walls and fractionated by SDS–PAGE run as
large, high-molecular-weight smears that react with both
b (1,3)-glucan and b (1,6)-glucan antisera [21,35]. On the
other hand, CWPs released by endo-b (1,6)-glucanase—
either as a recombinant enzyme or present as an
additional enzymatic activity in laminarinase—run as
relatively discrete bands [21,22]. The putative GPIproteins Als1 and Als3, proteins involved in cellular
adhesion, belong to this fraction [22]. (ii) The b (1,3)glucan and b (1,6)-glucan epitopes are both sensitive to
aqueous hydrouoric acid, which speciŽcally cleaves
phosphodiester bridges, suggesting that, similarly to S.
cerevisiae, a b (1,3)-glucan b (1,6)-glucan complex is
2001 ISHAM, Medical Mycology, 39, Supplement 1, 1±8
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linked through a GPI-remnant to the C-terminal end of
GPI-proteins [20,21].
Currently known putative GPI-CWPs in C. albicans
are Als1–Als9 [36], Csa1 [37], Hwp1 [38,39], Hyr1 [40],
Rbt1, Rbt5 and Wap1 [41]. In those cases in which the
entire sequence is publicly available they are predicted
to be GPI-proteins according to the algorithm developed
by Eisenberger and co-workers [42]. In several cases
their cell-wall location has been demonstrated immunologically [43,44]. Collectively, these data indicate that,
similarly to S. cerevisiae [45], the genome of C. albicans
may encode a large group of GPI-CWPs.
GPI-CWPs may be released enzymatically from the
cell wall by using aqueous hydrouoric acid [20,21],
which cleaves the phosphodiester bridge in the GPI
remnant of GPI-CWPs (–amino acidv –ethanolamine–
PO4–Mann–), b (1,6)-glucanase [22], b -1,3-glucanase
[22,26], or chitinase [22,46,47]. Extraction of isolated
walls with a b (1,6)-endoglucanase [48] releases at least
six proteins, with estimated molecular masses of 65, 100,
170, 220, 440 and 600 kDa [22]. The 440-kDa and 600kDa bands have been identiŽ ed as Als3 and Als1,
respectively, proteins involved in cellular adhesion. [22].
C. dubliniensis and C. tropicalis also contain a family of
ALS genes [49]. Furthermore, b (1,6)-glucanase digestion
of isolated cell walls released proteins that were crossreactive with an anti-Als serum, suggesting that
C. dubliniensis and C. tropicalis have similar cell-wall
architecture as C. albicans and S. cerevisiae. Finally, a
putative GPI-CWP, EPA1, encoding an adhesion protein, has been identiŽ ed in C. glabrata [50].
3. CWPs that are linked directly to b (1,3)-glucan
through an alkali-sensitive linkage, that is without an
interconnecting b (1,6)-glucan moiety [22,24]. Such proteins have been identiŽed initially in S. cerevisiae [14,51].
They are secretory proteins with an N-terminal signal
peptide, an internal repeat region, and a highly
conserved C-terminal region, but they lack a C-terminal
GPI addition signal. They have been designated as PirCWPs (CWPs with internal repeats). Later, similar
proteins have been identiŽ ed in C. albicans [22,24].
The main evidence for the presence of an alkali-sensitive
b (1,3)-glucan$Pir-CWP complex in C. albicans is as
follows: (i) immuno-gold labelling with ScPir2-antiserum
revealed the presence of a cross-reacting protein homogeneously distributed in the skeletal layer of the cell wall
similarly to S. cerevisiae [22]. (ii) When cell walls were
Žrst digested with b (1,6)-glucanase and subsequently
with a b (1,3)-glucanase, the second digestion freed a 235kDa protein that cross-reacted with ScPir2-antiserum
and with b (1,3)-glucan antiserum [22]. (iii) The b (1,3)glucan epitope was sensitive to mild alkali treatment
[22]. Extraction of cell walls obtained from biotinylated
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Klis et al.
cells with mild alkali released four protein bands,
including a 150-kDa protein band that reacted strongly
with the ScPir-antiserum [24]. (iv) Furthermore, using a
ScPIR2-derived probe, positive signals were obtained in
both Southern and northern analyses [24]. In agreement
with this, the genome of C. albicans contains at least one
open reading frame that shows signiŽcant similarity to
S. cerevisiae Pir-CWPs [22].
The observation that the linkage between Pir-CWPs
and b (1,3)-glucan is sensitive to alkali suggests that an
O-linked side-chain might be involved in this linkage. As
mentioned above, Pir-CWPs are predominantly distributed in the inner skeletal layer and not in the outer
protein layer [22]. A possible interpretation of these
results is that the reducing end of a b (1,3)-glucan chain
may be coupled to an O-linked side-chain of Pir-CWPs,
resulting in the following putative polysaccharide–CWP
complex: b (1,3)-glucan!Man1–7–O–CWP (Fig. 2).
4. The GPI-dependent cell-wall protein in S. cerevisiae, Cwp1, is a special case in that it was shown to be
linked both to the b (1,3)-glucan network as a normal
GPI-CWP, and as a Pir-CWP [16]. A similar cell-wall
protein, detected by Als1 antiserum, exists in C. albicans
(Piet de Groot, unpublished data).
Function of CWPs
The function of speciŽc CWPs may vary, but is in most
cases unknown. (i) Collectively, they limit the permeability of the wall, protecting the skeletal layer against
foreign, degrading enzymes, and the plasma membrane
against toxic compounds [52]. (ii) They may determine
the hydrophobicity [53] and antigenicity of the wall [2].
(iii) Other CWPs may be involved in cell-wall remodeling. (iv) Furthermore, they may have a role in cellular
adhesion and in virulence. In view of the natural habitat
of C. albicans, that is the mucosal surface of the human
Fig. 2 Linkage of Pir-CWPs to the b (1,3)-glucan network in
Candida albicans. Left panel: Pir-CWPs are linked to b (1,3)-glucan
through an alkali-sensitive linkage without an interconnecting
b (1,6)-glucan moiety [22,24]. Right panel: Hypothetical linkage
between the reducing end of a b (1,3)-glucan molecule and a sidechain (Man1 –7 ) O-linked to a serine or threonine residue in a PirCWP. Note that O-glycosidic linkages are sensitive to alkaline
conditions. Man: mannose. |: polypeptide backbone of Pir-CWP.
body, one may expect to Žnd a wide variety of adhesion
proteins in its outer cell wall [54]. For example, Alonso
and co-workers have identiŽed a lectin-like adhesion
protein that speciŽcally recognizes the N-terminal
domain of type IV collagen [55]. Furthermore, fucosespeciŽc adhesins have been identiŽ ed on germ tubes [56].
Interestingly, a large family of GPI-CWPs, designated
the ALS (agglutinin-like sequence) family, has been
identiŽed that all have a similar tripartite structure [57]
(Fig. 3), and that are predicted to be involved in
adhesion to mammalian cells [36]. This family has nine
known members, each possessing an N-terminal domain
with clear sequence similarity to the N-terminal half of
Sag1, the sexual agglutinin of mating type a cells of
S. cerevisiae. Interestingly, this region of Sag1 is
responsible for sexual agglutination and is believed to
contain three immunoglobulin variable-like domains
[58]. It has further been shown that expression of
ALS1 and ALA1/ALS5 in S. cerevisiae makes them
adhesive towards mammalian cells, supporting the idea
that ALS proteins are genuine adhesion proteins [59,60].
None of the currently known GPI-CWPs is essential, but
deletion of HWP1 or RBT1 results in signiŽcantly
reduced virulence in animal models [39,41].
Adhesion has been shown to be a two-step process,
involving an initial step mediated by adhesion proteins,
followed by a stabilizing step that involves an epithelial
transglutaminase that uses the GPI-CWP Hwp1 as its
substrate. As a result, C. albicans cells become covalently linked to host cells [39,61]. Furthermore, one may
speculate that the capability of C. albicans to form
dityrosine bridges [9] may also contribute to this
stabilization step.
The Žmbriae discussed above are the obvious location
for adhesion proteins. Isolated Žmbriae have been
puriŽed and analysed, which resulted in the identiŽcation
of an abundant protein with a molecular weight of
66 kDa [11]. This seems to rule out a role for ALS
proteins, because they all have higher apparent molecular weights. However, the fractionation range of the
gel system used in this study did not allow the detection
Fig. 3 ALS genes encode putative GPI proteins that consist of
three domains. SP, signal peptide; the conserved N-terminal domain
of the mature protein is probably involved in cellular adhesion
[36,57]; the central domain consists of a variable number of 36amino-acid repeats; the C-terminal domain is rich in serine and
threonine; GPI, GPI anchor addition signal. As the C-terminus is
predicted to connect the protein through b (1,6)-glucan to the
b (1,3)-glucan network, the adhesion domain is well positioned for
cell–cell interactions.
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2001 ISHAM, Medical Mycology, 39, Supplement 1, 1±8
Cell wall architecture
of high molecular weight proteins. Therefore the
possibility that Žmbriae also contain ALS proteins
or other high molecular weight adhesins cannot be
excluded formally.
Cell-wall dynamics
Yeast and hyphal modes of growth
C. albicans is a pleomorphic fungus and can grow in the
yeast, pseudohyphal and in the hyphal form. Yeast
growth is characterized by temporal control of two
modes of cell-wall expansion. Using polylysine-coated
beads to tag the cell surface, Staebell & Soll observed
that during the Žrst two-thirds of bud growth, the bud
grew largely apically, with some general extension [62].
Subsequently, cell-wall growth in the apical zone shuts
down and cell-wall growth required for the remaining
bud growth is due to general expansion. During hyphal
growth, however, apical cell-wall growth predominates
and general expansion is minimal. In other words,
general wall extension, which probably requires cell-wall
remodeling to allow the insertion of new wall material,
has a much more important role in the yeast form than in
the hyphal form of growth.
The timing of chitin deposition in the lateral walls
seems to differ between yeast and Žlamentous forms.
When yeast cells were treated with Calcouor, which
preferentially binds to chitin, the lateral walls of the
mother cell uoresce strongly, in contrast to the walls of
the growing bud (Els Mol, personal communication),
suggesting that, similarly to S. cerevisiae [63], chitin
Table 3
deposition in the lateral walls is delayed until after
cytokinesis. On the other hand, hyphae labelled with
tritiated N-acetylglucosamine incorporate label preferentially at the growing apex [6], indicating that during
hyphal growth chitin deposition in the lateral walls is not
delayed.
Because of these observations, one expects the cell
walls of the yeast and hyphal form to be different.
Indeed, Mormeneo and co-workers have shown that
walls from yeast cells contain considerably more alkaliextractable proteins than hyphal cells, suggesting that
yeast cells may have more Pir-CWPs [64]. The protein
compositions of yeast and hyphal cell walls also differ
[22,26,43,65]. Several putative GPI-CWPs (Als3, Als8,
Hwp1, Hyr1, Rbt1 and Wap1) are hyphal-speciŽ c.
Interestingly, four of the corresponding genes (ALS3,
ALS8, HWP1, and HYR1) contain multiple so-called Eboxes (consensus sequence, 50 -CANNTG-30 ) in their
promoter regions, which are recognized by the bHLH
(basic helix–loop–helix) transcription factor Efg1 [66].
Responses to cell-wall stress
The yeast cell wall is a highly dynamic entity. In
S. cerevisiae, an estimated 1200 genes directly or
indirectly affect cell-wall structure and organization
[67]. Table 3 shows that in C. albicans also, growth
conditions affect cell-wall composition and structure.
Furthermore, just as in S. cerevisiae [19], a salvage
mechanism seems to be activated in response to various
forms of cell-wall stress, resulting in increased levels of
chitin in the lateral walls, probably to reinforce the cell-
Growth and stress conditions known to affect cell-wall composition and structure in Candidas albicans
Conditions
Medium
Composition
Low pH
Temperature
Cell-wall stress
mnn9: truncated N-chains
pmt1: defective O-glycosylation
bgl2 and phr1: defective
remodeling of b (1,3)-glucan
inhibition of b (1,3)-glucan
synthesis by papulacandin
Azole-induced membrane stress
Defective signaling
mkc1
hog1
Cell-wall related phenotypes
References
Altered CWP composition
Increased thickness of the mannan layer
Increased transcript levels of CaHSP150/PIR2 at 37 o C compared to 24 o C
[22,26]
[68]
[24]
Increased sensitivity to b (1,3)-glucanase
Higher levels of chitin, a chitin–b (1,6)-glucan–CWP complex, and Pir-CWPs
Higher levels of chitin, a chitin–b (1,6)-glucan–CWP complex, and Pir-CWPs
Increased chitin levels and increased sensitivity to nikkomycin
[22,69]
Increased sensitivity to nikkomycin
[70]
Increased chitin levels and increased sensitivity to nikkomycin
[72,73]
Increased sensitivity to cell-wall lytic enzymes, nikkomycin, SDS and caffeine.
These phenotypes are osmotic-remediable.
Increased resistance to nikkomycin
[74,75]
[22]
[27,71]
[76]
Nikkomycin is an inhibitor of the synthesis of chitin. Hypersensitivity to SDS and caffeine indicates weakened cell walls [67]. MKC1
encodes the MAP kinase of the cell integrity pathway, and HOG1 encodes the MAP kinase of the osmostress pathway.
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2001 ISHAM, Medical Mycology, 39, Supplement 1, 1±8
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Klis et al.
wall (see reviews by Popolo et al. and Navarro-Garc´õ a
et al., this issue). This is accompanied by increased
sensitivity to nikkomycin, which inhibits chitin synthesis.
When Candida cells are challenged with azoles, which
interfere with sterol synthesis, similar phenotypes are
observed as in the case of cell-wall stress [70,73].
Presumably, the lack of ergosterol in the plasma
membrane affects plasma membrane-associated cell-wall
polymer synthases, thus resulting indirectly in cell-wall
stress. Similarly to S. cerevisiae, deletion of MKC1,
encoding the MAP kinase of the cell integrity pathway,
leads to weakened cell walls, and this phenotype can be
suppressed by osmotically stabilizing the medium with
sorbitol. Interestingly, hog1 cells are more resistant to
nikkomycin and the cell wall perturbing compound
Congo Red, suggesting that the high-osmolarity glycerol
pathway also has a role in regulating cell-wall composition and structure. For an extensive discussion of the
topics mentioned in this section, the reader is referred to
the contribution by Popolo et al. and Navarro-Garcõ´a et
al., this issue.
Perspectives
Several fascinating questions remain to be investigated.
For example, how does the molecular organization of the
hyphal wall differ from the yeast cell wall and which
differences are related to the yeast and hyphal modes of
growth? Is the cell-wall model presented here valid for
other Candida species? The few available data suggest
that this might be the case, but signiŽcant differences
cannot be excluded. Cell-wall remodeling and assembly
enzymes are promising targets for new antifungal
compounds; however, most of them have not yet been
characterized or even identiŽ ed. There is clear evidence
that the cell wall is a highly dynamic entity, but the
various changes in cell-wall composition and structure
have not yet been studied in detail or categorized, and
we do not understand why cells change their cell wall in
speciŽc ways in response to various stress conditions.
The signalling pathways that are involved in regulating
the construction of the cell wall are only known in
outline. Another unanswered question is why Candida
species apparently uses so many adhesion proteins. What
are their ligands and might they be tissue- or organspeciŽc? A comparative study of the postulated recognition domains of the ALS family might be helpful in this.
Genomic studies will certainly help to answer these
questions but, for a complete picture, a more glycobiological approach is also urgently needed.
Acknowledgements
We thank Els Mol for help and Lois Hoyer for
collaboration. This work has received Žnancial support
from the Dutch Foundation for Technical Sciences,
GLAXO, and the European Union (Framework Program V).
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