MINIREVIEW
Covalently linked cell wall proteins of Candida albicans and their role
in ¢tness and virulence
Frans M. Klis, Grazyna J. Sosinska, Piet W.J. de Groot & Stanley Brul
Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
Correspondence: Frans M. Klis,
Swammerdam Institute for Life Sciences,
University of Amsterdam, Nieuwe
Achtergracht 166, 1018 WV Amsterdam, The
Netherlands. Tel.: 131 20 525 7834; fax: 131
20 525 7924; e-mail: [email protected]
Received 23 March 2009; revised 26 May 2009;
accepted 9 June 2009.
DOI:10.1111/j.1567-1364.2009.00541.x
Editor: Richard Calderone
Keywords
adhesion; amyloid; biofilm; cell wall model; GPI
proteins; proteomics.
Abstract
The cell wall of Candida albicans consists of an internal skeletal layer and an external
protein coat. This coat has a mosaic-like nature, containing c. 20 different protein
species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat
proteins vary widely in function. Many of them are involved in the primary
interactions between C. albicans and the host and mediate adhesive steps or invasion
of host cells. Others are involved in biofilm formation and cell–cell aggregation. They
further include iron acquisition proteins, superoxide dismutases, and yapsin-like
aspartic proteases. In addition, several covalently linked carbohydrate-active enzymes
are present, whose precise functions remain hitherto largely elusive. The expression
levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary
enormously. They depend on the mode of growth and the combined inputs of several
signaling pathways that sense environmental conditions. This is reflected in the
unusually long intergenic regions of most of these genes. Finally, the precise location
of several covalently linked CWPs is temporally and spatially regulated. We conclude
that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence
and that their expression is tightly controlled.
YEAST RESEARCH
Introduction
Candida albicans is widely found in birds and mammals. In
humans, it often resides on the skin and mucosal layers of
otherwise healthy individuals. In compromised hosts, however, C. albicans can cause serious diseases, ranging from
deep-seated mucosal infections to systemic infections,
which are frequently fatal. Candida albicans uses an arsenal
of molecular tools to overcome the body’s lines of defense.
An important role in the fitness and virulence of C. albicans
is reserved for those cell wall proteins (CWPs) that are
covalently linked to the skeletal cell wall polysaccharides.
They contribute to cell wall integrity, mask the b-glucan
layer, thus avoiding detection by dectin-1, promote biofilm
formation, mediate adherence to host cells and abiotic
medical devices, promote invasion of epithelial layers, offer
protection against the attacks of the innate immune system,
and are involved in iron acquisition. CWPs of C. albicans
have been extensively reviewed in recent years, reflecting the
growing realization of how important their role is in the
various stages of infection (Richard & Plaine, 2007; Chaffin,
2008; Hoyer et al., 2008; Nather & Munro, 2008; Gonzalez
et al., 2009). The field, however, is rapidly expanding. GelFEMS Yeast Res ]] (2009) 1–16
free proteomics of isolated walls has shown that at any time
the walls contain about 20 different types of covalently
linked CWPs and that CWP profiles can change dramatically depending on the environmental conditions. In addition, the presence of particular CWPs seems to be nichespecific and strongly correlated with either yeast or hyphal
growth of C. albicans. In this review, we will discuss the
identification and quantitation of covalently linked CWPs,
their characteristics and functions, and how they contribute
to fitness and virulence. We will also discuss some new
aspects that have come to light since earlier reviews concerning the regulation of CWP-encoding genes. Collectively,
these observations show that covalently linked CWPs play a
crucial role in the capability of C. albicans to survive and
grow in warm-blooded animals and to cope with the stress
conditions associated with host infection.
Cellular morphology and the molecular
organization of the cell wall
Candida albicans is a polymorphic fungus. The most
frequently observed growth forms are yeast cells, pseudohyphae, and hyphae. The hyphae are similar to the hyphae
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found in mycelial ascomycetes and possess an apical body
and septa with simple pores (Gow et al., 1980; Sudbery et al.,
2004). White-to-opaque switching, which manifests itself in
opaque colonies instead of the usual white colonies, also
occurs. ‘Opaque’ cells resemble normal yeast cells (‘white’
cells’), but they are considerably larger and more elongated,
and their walls are pimpled. In these pimples, cell walltraversing channels terminate that allow the transfer of small
vesicles over the wall. Opaque cells are better colonizers of
the skin than white cells and can shmoo and mate (Soll,
2004). Finally, like many other fungi, C. albicans can form
chlamydospores, thick-walled, large spherical cells that seem
to represent a resting stage. The available data indicate that
the walls of yeast cells, pseudohyphal cells, and hyphae
possess a similar molecular organization. However, the walls
of opaque cells and chlamydospores have been studied less
extensively.
Figure 1 shows a molecular representation of the cell wall.
The wall is bilayered with an external protein coat consisting
mainly of glycosylphosphatidylinositol (GPI) proteins. They
are often heavily mannosylated and phosphorylated, and,
hence, responsible for the binding of positively charged ions
and proteins by the cell wall (Horisberger & Clerc, 1988;
Cutler, 2001). Their sizes can differ hugely (compare, e.g.
Pga59 and Als4; Table 1 and Fig. 2). The protein coat
surrounds an inner polysaccharide layer consisting of
b-1,6-glucan, b-1,3-glucan, and small amounts of chitin
(Kapteyn et al., 2000; Chauhan et al., 2002; Ruiz-Herrera
et al., 2006; Nather & Munro, 2008). The GPI-CWPs in the
outer protein coat are covalently linked through a GPI
glycan (a truncated form of the original GPI-anchor) to
b-1,6-glucan molecules, which in turn are covalently linked
F.M. Klis et al.
to nonreducing ends of b-1,3-glucan molecules. b-1,6Glucans are highly branched, which prevents their alignment over extended regions, and lack a regular structure.
They are water soluble and function as flexible linkers
between the coat proteins and the more rigid b-1,3-glucan
layer. In baker’s yeast, disulfide bridges in the external
protein coat restrict cell wall permeability (De Nobel et al.,
1990). This is probably also the case for C. albicans,
suggesting that some abundant CWPs are interconnected
by disulfide bridges. An attractive candidate for such protective, permeability-restricting coat proteins is Pga59, a
very short GPI-CWP with three cysteine residues (Fig. 2).
b-1,3-Glucan is the main structural polysaccharide and,
through its nonreducing ends, offers acceptor sites for
covalent attachment of b-1,6-glucan and chitin. Because
b-1,3-glucans are only moderately branched, they contain
multiple regions of contiguous, nonsubstituted glucose
residues. This allows them to form stable associations
mediated by interchain hydrogen bonds, resulting in a
continuous three-dimensional network around the cell. This
network is probably further strengthened by protein crosslinkages involving the repeats found in Pir-CWPs (Pir,
protein with internal repeats; Kapteyn et al., 2000; Ecker
et al., 2006). A dityrosine-containing polymer probably also
contributes to cell wall strength. First, at 42 1C, a condition
that causes cell wall stress, dityrosine-deficient cells grow
substantially slower, and second, dityrosine-deficient cells
are slightly more sensitive to caspofungin, which inhibits the
synthesis of b-1,3-glucan (Melo et al., 2008). The cell walls
of yeasts are highly elastic and are usually considerably
extended in living cells due to the turgor pressure exerted
on the walls. Consequently, cells visibly shrink when they die
Fig. 1. Cartoon of the cell wall of Candida albicans. The external protein coat, which is about 100 nm thick (Tokunaga et al., 1986; Bobichon et al.,
1994), has a mosaic-like nature and consists of both extended GPI-CWPs, including the Als family members (Hoyer et al., 2008), and much smaller GPICWPs such as Pga59 (Moreno-Ruiz et al., 2009). The protein coat is believed to be tightened by disulfide bonds. The internal skeletal layer of the mother
cell has a thickness of about 60–70 nm (Tokunaga et al., 1986; Bobichon et al., 1994). Pir-CWPs are proposed to cross-link the b-1,3-glucan network
through a glutamine-dependent transesterification that involves their repeats. The cell wall also contains a dityrosine-containing polymer (omitted for
clarity). Under some growth conditions, fimbriae are present, which can be released from the wall by reducing agents (reviewed in Tronchin et al., 1988;
Hazen & Hazen, 1992; Chauhan et al., 2002).
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Role of covalently linked CWPs of C. albicans
Table 1. Experimentally validated covalently linked cell wall proteins of C. albicans
Protein
5 0 -Intergenic
region (bp)
No. of AA and
region of tryptic
TATA box peptides
GPI-CWPs
Als1
4813
1
1260
79–311
c. 330-GPI
Als3
2964
1
1155
77–311
c. 330-GPI
Als4
3248
1
2100
77–91
c. 330-GPI
Als5
4353
1
1347
No data
c. 330-GPI
Als6
4145
1366
No data
c. 330-GPI
Als7
4932
1568
No data
c. 330-GPI
Als9
2769
1
1889
No data
c. 330-GPI
Cht2
3131
c. 300-GPI
Crh11
2114
1
Csa1
967
583
22–299
453
29–280
1018
No data
Eap1
3451
653
No data
c. 140-GPI
Ecm33
4430
1
c. 350-GPI
Hwp1
2081
1
423
139–341
634
No data
Hyr1
7223
1
c. 350-GPI
Ihd1
2368
Pga4
838
Pga10
724
1
919
143–286
392
1w
451
42–385
250
47–60
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S/T-enriched
region
c. 280-GPI
c. 650-GPI
c. 180-GPI
c. 130-GPI
c. 370-GPI
c. 130-GPI
Features and functions
High levels of gene expression (Hoyer et al., 2008); N-terminal c. 300residue Ig domain; found in band at the hyphal base (Fu et al., 2002); Nterminal sequence used as vaccine antigen (Spellberg et al., 2005);
contributes to biofilm formation (Nobile et al., 2008a)
High levels of gene expression (Hoyer et al., 2008); N-terminal c. 300residue Ig domain; uniformly distributed over the hyphal wall (LaforceNesbitt et al., 2008); N-terminal sequence used as vaccine antigen
(Spellberg et al., 2008); functions: adhesion, biofilm formation, invasion,
ferritin receptor (Zhao et al., 2006; Phan et al., 2007; Almeida et al.,
2008)
Intermediate levels of gene expression (Hoyer et al., 2008); N-terminal c.
300-residue Ig domain; pH 4-grown cells contain much more Als4 than
cells grown at neutral pHw
Cell wall location based on expression in S. cerevisiae (Sheppard et al.,
2004); low levels of gene expression (Hoyer et al., 2008); N-terminal c.
300-residue Ig domain
Cell wall location based on expression in S. cerevisiae (Sheppard et al.,
2004); low levels of gene expression (Hoyer et al., 2008); N-terminal c.
300-residue Ig domain
Cell wall location based on expression in S. cerevisiae (Sheppard et al.,
2004); low gene expression (Hoyer et al., 2008); N-terminal c. 300residue Ig domain
Cell wall location based on expression in S. cerevisiae (Sheppard et al.,
2004); intermediate levels of gene expression (Hoyer et al., 2008); Nterminal c. 300-residue Ig domain
Chitinase; N-terminal GH18z domain
N-terminal GH16z domain; transglycosylase (Pardini et al., 2006; Cabib
et al., 2008)
Cell wall location based on expression in S. cerevisiae (Lamarre et al.,
2000); four N-terminal CFEM domains; almost exclusively found in
growing buds; strongly expressed in and uniformly distributed over
hyphal walls (Lamarre et al., 2000); deletion results in fragile biofilms
(Pérez et al., 2006)
Cell wall location based on expression in S. cerevisiae and GPI-dependent
cell surface localization of HA-tagged Eap1 in C. albicans; deletant shows
reduced biofilm formation; N-terminal domain mediates yeast cell–cell
adhesion; S/T-rich regions mediate adhesion to polystyrene (Li & Palecek,
2003, 2008; Li et al., 2007)
Important for cell wall integrity (Martinez-Lopez et al., 2006); precise
function unknown
Covalently bound to cell wall b-glucan (Staab et al., 2004); N-terminal
domain serves as a substrate for epithelial trans-glutaminases (Staab
et al., 1999); crucial for biofilm formation (Nobile et al., 2008a); protein
levels increase upon oxygen and iron restriction (Sosinska et al., 2008); Nterminal 14-mer peptide used as vaccine antigen (Xin et al., 2008)
Unknown function; not found in mycelial fungi; the Hyr-like gene family
is enriched in pathogenic Candida spp. (Butler et al., 2009)
Unknown function
Transglycosidase with an N-terminal GH72z domain; widespread among
ascomycetes
N-terminal CFEM domain; loss of function results in fragile biofilms
(Pérez et al., 2006); involved in heme-iron utilization (Weissman &
Kornitzer, 2004)
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F.M. Klis et al.
Table 1. Continued.
5 0 -Intergenic
region (bp)
No. of AA and
region of tryptic
TATA box peptides
S/T-enriched
region
Pga30
2111
277
123–149
c. 150-GPI
Pga31
1619
c. 200-GPI
Pga45
2090
1
Pga59
8057
1
352
102–162
462
351–364
113
No data
Pga62
806
1
213
No data
Absent
Phr1
1016
548
71–452
c. 480-GPI
Phr2
1193
544
23–269
c. 480-GPI
Rbt1
3415
1
c. 270-GPI
Rbt5
1253
1
721
262–271
241
37–92
Rhd3
831
1
Absent
Sap9
1555
Sap10
2000
204
16–150
544
1w
453
No data
Sod4
1391
1
c. 170-GPI
Sod5
3253
1
Ssr1
3049
Utr2
4220
232
16–117
228
26–140
234
23–79
470
75–310
Ywp1
6033
1
533
93–349
c. 160–410
Non-GPI CWPs
Mp65
1573
378
128–371
346
19–346
c. 60–110
Protein
Pir1
5561
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c. 220-GPI
Absent
c. 130-GPI
c. 480-GPI
c. 390-GPI
c. 170-GPI
c. 80-GPI
c. 370-GPI
Absent
Features and functions
Pga30, Pga31, and Rhd3 are homologous; function unknown; the
Pga30-like gene family is enriched in pathogenic Candida spp. (Butler
et al., 2009)
N-terminus as presented in CGD possibly incorrect; Pga30, Pga31, and
Rhd3 are homologous; function unknown; conserved in Candida spp.
Function unknown; conserved in Candida spp.
Cell wall location confirmed by GFP tagging (Moreno-Ruiz et al., 2009);
mature protein predicted to consist of 74 residues with three cysteine
residues and two potential N-glycosylation sites; N- and O-glycosylated;
proposed to be an abundant coat protein that cross-links CWPs by
disulfide bonding
Cell wall location confirmed by GFP tagging; contains two tandem
repeats similar to the 3-cysteine domain of Pga59 (Moreno-Ruiz et al.,
2009)
N-terminal GH72 domain followed by an X8 domain (pfam07983) in the
C-terminal half; transglycosylase involved in cell wall construction;
incorporated at neutral/alkaline pHw
N-terminal GH72 domain followed by an X8 domain (pfam07983) in the
C-terminal half; transglycosylase involved in cell wall construction;
incorporated at acidic pHw
N-terminal Flo11 domain (pfam10182)
N-terminal CFEM domain; loss of function results in fragile biofilms
(Pérez et al., 2006); involved in hemoglobin utilization (Weissman &
Kornitzer, 2004); protein levels increase upon oxygen and iron restriction
(Sosinska et al., 2008)
Pga30, Pga31, and Rhd3 are homologous; function unknown; conserved
in Candida spp.
Yapsin-like protein mainly found in the PM; required for full cell wall
integrity; also identified by immunogold labeling (Albrecht et al., 2006)
Cell wall location determined by immunogold labeling (Albrecht et al.,
2006); yapsin-like protein mainly found in the wall; required for full cell
wall integrity
Superoxide dismutase; contributes to clearance of ROS (Frohner et al.,
2009)
Superoxide dismutase; plays a major role in the clearance of ROS
(Frohner et al., 2009)
N-terminal CFEM domain; also identified by immunological means
(Garcera et al., 2003)
Putative transglycosylase (Pardini et al., 2006; Cabib et al., 2008) with a
GH16z domain followed by C-terminal S/T-rich region; conserved in
ascomycetes and basidiomycetes
Strongly expressed in exponentially growing yeast cells, but
downregulated in stationary phase and during filamentous growth;
adhesiveness and biofilm formation are inversely related with Ywp1p
levels; used as vaccine antigen (Granger et al., 2005)
Putative transglycosylase with a C-terminal GH17z domain; found in
fibrillar material; considered as a vaccine candidate (Pietrella et al., 2008)
C-terminal conserved 4-Cys pattern; predicted to cross-link b-1,3-glucan
chains (Ecker et al., 2006); protein levels increase during hypoxic
conditions (Sosinska et al., 2008)
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Role of covalently linked CWPs of C. albicans
Table 1. Continued.
5 0 -Intergenic
region (bp)
No. of AA and
region of tryptic
TATA box peptides
S/T-enriched
region
Sim1
4601
372
32–265
c. 50–110
Tos1
4360
468
288–370
c. 140–230
Protein
Features and functions
C-terminal SUN domain; required for septation; synthetic lethality with
SUN41 results in osmoremediable wall rupturing close to the septum
(Firon et al., 2007)
N-terminal DUF2403 domain and C-terminal DUF2401 domain
Based on MS data from De Groot et al. (2004); Pardini et al. (2006); Castillo et al. (2008); and Sosinska et al. (2008).
w
A. Sorgo, C. Heilmann, & G. Sosinska, pers. Observ.
GH#, glycoside hydrolase family # (CAZy database; Cantarel et al., 2009).
ROS, reactive oxygen species.
z
Fig. 2. Diagram of the GPI-CWP Pga59. Pga59 is both N- and Oglycosylated (Moreno-Ruiz et al., 2009). The three cysteine residues and
the two potential N-glycosylation sites are represented in bold. SP, signal
peptide; GPI, glycosylphosphatidylinositol addition signal.
(Arnold & Lacy, 1977). The elasticity of the wall reflects the
helical conformation of the b-1,3-glucan molecules. Such
helical molecules can assume various conformations varying
between a condensed and a fully extended state.
Cells in colonies and biofilms are surrounded by an
extracellular matrix (Joshi et al., 1973; Calderone et al.,
1984; Blankenship & Mitchell, 2006). This matrix consists of
proteins and polysaccharides, but its exact composition, its
architecture, and its precise relationship with the cell wall are
incompletely understood (Al-Fattani & Douglas, 2006).
Cell surface-associated cytosolic proteins
Many studies describe cell surface-associated cytosolic proteins that are extractable by various means from isolated
cells or cell wall preparations (reviewed in Nombela et al.,
2006; Chaffin, 2008). Cell surface-associated cytosolic proteins that are covalently linked to the cell wall have not been
found. Unfortunately, there are several factors that complicate the interpretation of these studies. (1) Incomplete
homogenization: because the copy numbers of individual
proteins range from a few hundred per cell to over a million
per cell, incomplete homogenization may result in the
FEMS Yeast Res ]] (2009) 1–16
presence of the most abundant cytosolic proteins, such as
glycolytic enzymes and elongation factors, in extracts from
incompletely disintegrated cell wall preparations. (2) Extraction of cells with 1% b-mercaptoethanol at 37 1C, an
often used extraction technique, results in leakiness of the
plasma membrane and a steady loss of cytosolic proteins
(Klis et al., 2007). (3) When cells die, they shrink due to the
loss of cell turgor. Conceivably, cytosolic proteins could then
become entangled in the inner layer of the cell wall.
Subsequent treatment with sodium dodecyl sulfate (SDS)
in combination with a reducing agent would unfold these
proteins, resulting in a random-coil conformation and
release from the walls. (4) As mentioned in the previous
section, the outer layer of the cell wall of C. albicans contains
many homogenously distributed, negatively charged sites
(Horisberger & Clerc, 1988), which are due to the presence
of phosphodiester bridges in the N-linked carbohydrate
side-chains of CWPs and to the carboxyl groups in wall
proteins (Cutler, 2001; Fradin et al., 2008). Consequently,
the cell wall acts as an ion exchanger and can bind many
proteins, depending on their isoelectric point and the pH of
extraction. (5) Intriguingly, electron microscopy of cells
grown on fatty acids or serum as the carbon source or
grown in vivo display so-called trans-cell wall structures
containing electron-dense (presumably proteinaceous) material penetrating the entire wall (Sheridan & Ratledge,
1996). Such structures were not observed in glucose-grown
cells. Unfortunately, the cells were not washed with an
isotonic solution, but with distilled water. This causes severe
cell wall stress by the rapid uptake of water and could result
in the formation of cracks in the wall. An alternative
explanation for the observations by Sheridan and Ratledge
is therefore that cells grown on poor carbon sources such as
encountered in vivo have much less robust walls than
glucose-grown cells and tend to form cracks in their walls
when subjected to mechanical stress.
To differentiate between cytosolic proteins that become
associated with the walls during the experiment and cytosolic proteins associated with the walls before the
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experiment, careful labeling of cell surface proteins by
enzymatic iodination or using a nonpermeant biotinylation
reagent is appropriate (Cappellaro et al., 1998; see, also, Klis
et al., 2007). In spite of the possibility of artifacts, the study
of cell surface-associated cytosolic proteins warrants close
attention. For the immune system of the host, it does not
matter whether cell surface-associated cytosolic proteins of
C. albicans result from fractures in the wall, cell walltraversing channels as observed in opaque cells, apoptosis,
or by an as yet not understood export mechanism. Indeed,
some glycoconjugate vaccines based on peptides from two
glycolytic enzymes (aldolase and enolase) and from methionine synthase (Met6) offer protection against systemic
candidosis in mice (Xin et al., 2008). Interestingly, it has
been shown by Murillo et al. (2005) that during the early
stages of biofilm formation, c. 30% of the cells stain with
phloxine B, but not with propidium iodide, indicating that
membrane permeability has changed, but that the cells are
still alive. For an extensive discussion of cytosolic proteins
that are potentially associated with the cell surface, the
reader is referred to the review of Chaffin (2008). In this
review, we focus on covalently linked CWPs.
Most covalently linked CWPs of C. albicans
are GPI-CWPs
There are two main classes of covalently linked CWPs: GPICWPs and non-GPI CWPs such as Pir1. The latter can be
released from the wall by mild alkali. A recent review
presents a list of 115 putative GPI proteins (Richard &
Plaine, 2007). GPI proteins possess two signal peptides
located at either end of the polypeptide chain. The
N-terminal signal peptide directs them to the endoplasmic
reticulum (ER). In the ER, the N-terminal signal is removed
and the C-terminal signal is replaced by a GPI anchor, a
preassembled lipid anchor that links them to the lumenal
leaflet of the membrane. Fungal GPI proteins follow the
secretory pathway until they reach the plasma membrane,
where some of them are retained (GPI-PMPs such as
Ecm331; PMP, plasma membrane proteins), whereas others
are released from the plasma membrane and incorporated
into the wall (GPI-CWPs such as the Als proteins and Hwp1)
(Dranginis et al., 2007; Mao et al., 2008). The distinction
between GPI-PMPs and GPI-CWPs is less clear-cut than this
terminology suggests. In Saccharomyces cerevisiae, a systematic search has shown that some mature GPI proteins are
indeed almost exclusively found in the wall and others almost
exclusively in the plasma membrane (Hamada et al., 1999).
Yet other CWPs are found in substantial amounts in both
locations. It seems likely that this is also the case in C.
albicans. The pre-GPI region of GPI-CWPs plays an important role in determining the final destination of fungal GPI
proteins (Dranginis et al., 2007; Mao et al., 2008).
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F.M. Klis et al.
Table 1 presents an overview of the CWPs in C. albicans,
which have been experimentally shown to be covalently
linked to the b-glucan–chitin network, by MS of cell walls
extracted with hot SDS combined with a reducing agent, by
enzymatic release from isolated walls, followed by immunological identification (Eap1, Hyr1, Hwp1; and in conjunction with green fluorescent protein tagging: Pga59 and 62),
by immunogold-labeling (Sap 9 and Sap10), or by expression cloning in S. cerevisiae and binding of the transformed
cells to antibody- or ligand-coated magnetic beads (the Als
protein family, Csa1; Lamarre et al., 2000; Sheppard et al.,
2004). This list is probably still incomplete because (1) the
levels of some CWPs may fall below the detection level
under the growth conditions tested; (2) suitable tryptic
peptides can be highly glycosylated (Hwp1 and Pga62),
complicating their identification; or (3) some CWPs lack
tryptic peptides that fall within the m/z range of the
instrument. Most of the identified CWPs are GPI-CWPs.
As GPI-CWPs are linked to the skeletal polysaccharides
through their C-terminal end, their N-terminal part is
facing outwards. As discussed above, the presence of a
particular GPI protein in the cell wall does not exclude the
possibility that a considerable number of copies or even the
majority are retained in the plasma membrane, and vice
versa.
Many GPI-CWPs possess a modular structure with their
functional domain located in the outwards-extending
N-terminal part of the protein. This N-terminal domain is
followed by one or more regions enriched in hydroxyamino
acids and often heavily O-glycosylated (Dranginis et al.,
2007; Hoyer et al., 2008). Consistent with this modular
organization, the MS-identified tryptic peptides of GPICWPs generally originate from the region that precedes the
serine- and threonine-rich part of the protein (Table 1).
Several GPI-CWPs show an internal tandem repeat region.
The corresponding intragenic tandem repeats tend to generate allelic and functional variability (Oh et al., 2005;
Verstrepen & Klis, 2006; Hoyer et al., 2008; Li & Palecek,
2008).
Location of CWPs
The transcript levels of many CWP-encoding genes in
S. cerevisiae are cell cycle-dependent, resulting in preferential incorporation of some CWPs either in small buds or in
large buds (reviewed in Klis et al., 2006; Smits et al., 2006).
Other CWPs are incorporated at specific locations such as
the bud neck, the birth scar, or the bud scar. The incorporation of CWPs into C. albicans is probably also temporally
and spatially regulated. For example, the GPI-CWP Csa1 of
C. albicans is only observed in growing buds of the yeast
form and not in mother cells; however, it is uniformly
distributed over hyphal walls (Lamarre et al., 2000). The
FEMS Yeast Res ]] (2009) 1–16
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Role of covalently linked CWPs of C. albicans
incorporation pattern of the GPI-CWP Utr2 is more complex (Pardini et al., 2006). In yeast cells, it is incorporated
into the wall at and around the presumptive bud site of the
mother cell and in small growing buds. Subsequently, it
forms a ring at the base of the bud neck. When yeast cells are
induced to switch to hyphal growth, Utr2 is incorporated at
the tip of the germ tube and in a ring at the septa. Other
CWPs seem to be preferentially expressed in yeast (Cht2,
Rhd3, and Ywp1; Granger et al., 2005) or hyphal cells (Als3,
Hyr1, Ihd1, Hwp1, and Sod5; Bailey et al., 1996; Staab et al.,
1999; Nantel et al., 2002; Laforce-Nesbitt et al., 2008).
However, the distinction between yeast- and hypha-specific
CWPs is sometimes less clear-cut than originally thought.
For example, when C. albicans is grown in a vaginasimulative medium at an acidic pH and transferred to
hypoxic conditions, Hwp1 levels in the wall increase considerably, although under these conditions hyphal growth
was not observed (Sosinska et al., 2008). Interestingly,
Hwp1p is also specifically incorporated into the wall of the
a/a portion of the bridge between mating partners (Daniels
et al., 2003). As already mentioned for Utr2, hyphal wall
incorporation also shows local variations. For example,
whereas Als3 and Hwp1 are uniformly distributed over the
hyphal tubes emerging from yeast cells that have switched to
hyphal growth, immunodetection of Als1 shows an intense
band at the hyphal base (Fu et al., 2002).
Most CWPs seem to be coat proteins, but Pir1 is an
exception to this rule. It is mainly found in the internal
skeletal layer, consistent with its proposed role as a b-1,3glucan cross-linking agent (Kapteyn et al., 2000; Ecker et al.,
2006). As Pir1 is not a GPI protein and can be released by
mild alkali from the walls, it seems possible that other alkalisensitive, non-GPI CWPs, such as Mp65, are also located in
the skeletal layer (De Groot et al., 2004, 2005).
General properties of covalently
linked CWPs
CWPs seem to be stable proteins once they are incorporated
into the wall (Ruiz-Herrera et al., 2002; E. Mol, pers.
observ.). Only a slow and limited release of wall-bound
CWPs into the medium takes place over time, corresponding to about 20% of the initial number of CWPs, probably as
a result of some cell wall remodeling in the mother cell
during each subsequent cell cycle (Hiller et al., 2007; E. Mol,
pers. observ.). The cell surface occupancy of covalently
linked CWPs in a yeast cell can be estimated as follows: an
unbudded yeast cell of C. albicans has an average cell length
of 5.67 mm and a width of 4.06 mm, which corresponds to a
volume of c. 49 mm3 and a surface area of 67 mm2 (KockováKratochvilová, 1990). Assuming a cell density of 1.12 g mL1
and a dry weight content of 34% (Kocková-Kratochvilová,
1990), a volume of 49 mm3 corresponds to c. 55 pg of wet
FEMS Yeast Res ]] (2009) 1–16
weight or c. 19 pg of dry weight per cell. In terms of dry
weight, the cell wall accounts for about 20% of the biomass.
As the polypeptide content of the wall is estimated at
2.4–3.3% (Pardini et al., 2006; Sosinska et al., 2008), the
wall of a single, unbudded cell contains 90–123 fg of polypeptide. Further assuming that the average molecular
mass of the polypeptide part of a CWP is 30 kDa, it can
be estimated that the wall surface is occupied by
c. 1.8–2.5 106 covalently bound CWP molecules or
c. 27–37 103 CWPs mm2. Similar estimates for the cell
surface occupancy of CWPs have been obtained for S.
cerevisiae (Dranginis et al., 2007; Yin et al., 2008).
The CWP coat determines two major collective cell surface properties: cell wall permeability and cell surface charge.
Restriction of cell wall permeability is due to the close
packing of CWPs, the presence of bulky N-linked protein
side-chains, and the formation of intermolecular disulfide
bridges (Zlotnik et al., 1984; De Nobel et al., 1989, 1990; Yin
et al., 2007). This protects the structural polysaccharides
against degradation by foreign glycanases and shields
b-glucan from detection by the mammalian b-glucan receptor dectin-1 (Zlotnik et al., 1984; Gantner et al., 2005;
Wheeler & Fink, 2006; Gow et al., 2007). Yeast cells and
filamentous forms differ in their ability to evade recognition
by dectin-1. Whereas filamentous forms of C. albicans are
completely shielded from detection, dectin-1 binds to birth
and bud scars of yeast cells, resulting in the activation of
dectin-1 (Gantner et al., 2005). Some small and abundant
GPI-CWPs seem to function primarily as coat-forming and
masking proteins. Possible candidates for such a function
are in S. cerevisiae Ccw12, which has a codon adaptation
index of 0.87, indicating that it is a very abundant protein,
and its counterpart in C. albicans Pga59. They are
N-glycosylated and, interestingly, contain three conserved
cysteine residues (Fig. 2). The second major cell surface
property is due to the presence of phosphodiester bridges in
N-linked carbohydrate side-chains of CWPs, which impart
negative charges to the cell wall (Horisberger & Clerc, 1988;
Cutler, 2001; Fradin et al., 2008).
Properties of CWP families and
individual CWPs
The Als, Hyr, and the Pga30 families of GPI-CWPs are
strongly enriched in the more common pathogenic Candida
species in comparison with nine species from the Saccharomyces clade (Butler et al., 2009). This strongly suggests that
these proteins play an important role in virulence and
adaptation to specific niches. However, the functions of
CWPs are manifold.
1. Coat-forming CWPs: as mentioned above, Pga59 is a
likely candidate for a coat-forming CWP that restricts the
permeability of the cell wall (Moreno-Ruiz et al., 2009).
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8
2. Hydrophobicity-conferring CWPs: Eap1 promotes adhesion to styrene, a hydrophobic polymer derived from
ethenylbenzene (Li & Palecek, 2003).
3. The Als adhesin family consists of eight GPI proteins,
seven of which have been experimentally validated as
covalently linked CWPs (Table 1). The mature Als proteins,
which have lost their N- and C-terminal signal peptide, are
multidomain proteins with four consecutive domains
(Ig–T–TR–stem; Ig, immunoglobulin; T, threonine-rich
domain of Als proteins; TR, tandem repeat domain of Als
proteins): the Ig domain of c. 300 amino acids, a region with
three tandem Ig-like sequences; the T-domain, a 127-residue
threonine-rich conserved domain; the TR domain, a region
consisting of a variable number of 36-residue, threoninerich, tandem repeats; and the stem domain, a highly
glycosylated Ser/Thr-rich domain of low structural complexity and variable length (Rauceo et al., 2006; Otoo et al.,
2008). Although it was originally thought that the Igdomain was solely responsible for the adhesive properties
of the Als proteins and the remainder of the protein was
functioning as a stalk domain, it is now clear that the
T-domain and the TR-domain have vital functions of their
own, particularly in cell–cell aggregation. In view of the
multiple functions assigned to Als proteins (Table 1), it
therefore becomes important to establish which domain is
directly responsible in each case.
Als proteins bind to diverse mammalian proteins. One of the
reasons is that the recognition of peptide ligands by the Als
proteins is degenerate and that their specificities overlap
each other only partially. This allows C. albicans to bind to a
large variety of host proteins (Klotz et al., 2004). Importantly, some members of the Als adhesin family have
amyloid properties (Rauceo et al., 2004; Fowler et al., 2007;
Otoo et al., 2008). This property probably contributes to
intercellular aggregation and biofilm cohesiveness (Nobile
et al., 2006a; Bastidas et al., 2009). Als proteins are also
involved in the formation of mixed aggregates consisting of
bacterial and fungal cells.
4. The adhesin Hwp1 represents a fascinating case of molecular mimicry. Its N-terminal domain is recognized as a
substrate by host transglutaminases at the epithelial surface.
As a result, C. albicans becomes covalently linked to epithelial
cells and cannot be washed away (Staab et al., 1999). As
discussed below, Hwp1 also plays a complementary role in
biofilm formation, together with Als1 and 3, possibly as a
result of a physical interaction between Als1 and 3 on the one
hand and Hwp1 on the other (Nobile et al., 2008a).
5. Pir1 is a cross-linking CWP. Consistent with its location
in the internal skeletal layer, Pir1 presumably cross-links
b-1,3-glucan chains (Kapteyn et al., 2000; Ecker et al., 2006)
(Fig. 3).
6. Carbohydrate-active enzymes: unexpectedly, a considerable number of CWPs have (predicted) glycosylase/transgly2009 Federation of European Microbiological Societies
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F.M. Klis et al.
Fig. 3. Proposed b-1,3-glucan cross-linking function of Pir1. The insert
shows the characteristic amino acid sequence in each repeat (R),
including the glutamine residue that is potentially involved in crosslinking. J, any hydrophobic amino acid.
cosylase activity (De Groot et al., 2004; Cantarel et al., 2009).
This raises the question of whether they are part of the
external protein coat and thus located far away from their
presumptive substrates or might be located elsewhere in the
wall. Alternatively, they could play a role in the formation of
the extracellular matrix of biofilms. For example, loss of the
putative cell wall (trans)glycosidase Sun41p, a non-GPI
CWP, results in strongly decreased biofilm formation on an
abiotic surface (Hiller et al., 2007; Norice et al., 2007).
7. Heme-iron acquisition: some CWPs (Als3 and Rbt5) are
involved in the acquisition of iron (Weissman & Kornitzer,
2004; Sosinska et al., 2008; Frohner et al., 2009).
8. Coping with oxidative stress: C. albicans incorporates the
GPI-modified superoxide dismutases Sod4 and 5 into its
wall, which help the cell cope with oxidative stress originating from innate immune cells (De Groot et al., 2004;
Martchenko et al., 2004; Fradin et al., 2005; Sosinska et al.,
2008; Frohner et al., 2009).
9. Invasion-related CWPs: other CWPs, such as Als3, have
been shown to act as an invasin, thereby facilitating endocytosis (Phan et al., 2007).
10. The yapsin-like proteins Sap 9 and 10 possess proteolytic activity, and in their absence, normal cell wall construction is affected, but their specific substrates are still largely
elusive (Albrecht et al., 2006; reviewed in Gagnon-Arsenault
et al., 2006).
Some simple tests to guide a more detailed evaluation of
the potential function of covalently bound CWPs (and other
proteins) are as follows: (1) an altered sensitivity to Calcofluor white, Congo red, SDS, or a cell wall-degrading
enzyme preparation (Ram & Klis, 2006); (2) slower growth
on agar plates containing 0.5 YPD to lower the osmotic
strength of the medium and hence to increase the turgor
pressure (Valdivia & Schekman, 2003) and kept at 37 or
42 1C; (3) cell surface hydrophobicity (Hazen & Hazen,
1992); (4) biofilm formation on an abiotic surface; (5)
invasive growth on agar plates; and (6) chlamydospore
formation (Jitsurong et al., 1993). More elaborate assays
measure (1) phenotypic changes resulting from conditional
gene overexpression/suppression (Fu et al., 2008); (2)
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9
Role of covalently linked CWPs of C. albicans
invasion of reconstituted human epithelia; (3) phagocytosis
(Fu et al., 2008; Frohner et al., 2009); (4) virulence when
C. albicans is supplied as a food source to the free-living
nematode Caenorhabditis elegans (Tampakakis et al., 2008)
or after injection into a Toll mutant of Drosophila melanogaster or larvae of the wax moth Galleria mellonella (Chamilos et al., 2006; Cowen et al., 2009); and (5) death rates
after systemic infection of mice. In case of large families,
heterologous expression in S. cerevisiae can help elucidate
the function of individual family members (Klotz et al.,
2004; Sheppard et al., 2004).
CWPs and biofilm formation
Candida albicans forms biofilms on both biotic and abiotic
surfaces such as tissues, dentures, and catheters. On abiotic
surfaces, they often show a bilayered organization, with a
basal layer of yeast cells, which seems to anchor the biofilm
to the surface, and an upper layer in which hyphae predominate (Chandra et al., 2001; Douglas, 2003). Biofilms
represent a complex, often hypoxic microenvironment.
Biofilm cells are generally surrounded by an extracellular
matrix that contributes to intercellular adhesion. Interestingly, the production of matrix material increases strongly
when the cells are subjected to a liquid flow (Al-Fattani &
Douglas, 2006). Knowledge of the molecular organization of
the extracellular matrix is limited. For example, it is unknown whether matrix polymers can become covalently
linked to each other. Biofilms have received ample attention
because biofilm formation on implanted medical devices is
responsible for many serious cases of candidosis (Ramage
et al., 2006). In addition, biofilm cells become much more
resistant to antifungal drugs, thus complicating their eradication.
A considerable number of covalently linked CWPs are
known to contribute to biofilm formation: the adhesins
Als1, Als3, and Hwp1; the CFEM domain-containing CWPs
Csa1, Pga10, and Rbt5; and Eap1 (Garcia-Sanchez et al.,
2004; Nobile et al., 2006b, 2008a; Pérez et al., 2006; Klotz
et al., 2007; Bastidas et al., 2009). Intriguingly, a strain
without Als1 and Als3 or a strain without Hwp1 can only
form rudimentary biofilms, but when the two strains are
mixed, extensive biofilm formation takes place. This demonstrates that Als1 and 3 and Hwp1 complement each
other in biofilm formation, and it also suggests that Als1 and
3 physically interact with Hwp1 (Nobile et al., 2008a). In
addition, the internal stretch of tandem repeats of the Als
proteins of C. albicans could, conceivably, promote biofilm
cohesion by self-association between opposing Als molecules (Klotz et al., 2007). The precise role of Csa1, Pga10,
and Rbt5 in biofilm formation is still unknown. However,
they share a CFEM domain (an c. 60-amino acid region with
eight conserved cysteine residues), raising the question of
FEMS Yeast Res ]] (2009) 1–16
whether this domain has a function in biofilm formation.
The GPI-CWP Ssr1 also possesses a CFEM domain, but a
possible role for this protein in biofilm formation has not
been investigated. Eap1 promotes binding to polystyrene, a
highly hydrophobic substrate, and this might explain why
the loss of Eap1 results in reduced biofilm formation. On a
more speculative note, it is conceivable that some of the
wall-bound transglycosylases, together with soluble (trans)glycosylases, are responsible for forming covalent bonds
between cell wall and matrix macromolecules, or for assembling matrix macromolecules into supramolecular structures.
It is important to realize that naturally occurring biofilms
often house a mixture of species. For example, C. albicans
and Staphylococcus epidermidis form mixed biofilms and
have indeed been identified together in multimicrobial
infections (Al-Fattani & Douglas, 2006). Biofilms consisting
of C. albicans and oral streptococci have also been described
(reviewed in Ten Cate et al., 2009). In addition, C. albicans
and Pseudomonas aeruginosa interact in various body sites.
In vitro, they show an antagonistic interaction: C. albicans
hyphae are colonized by P. aeruginosa and eventually die
(Hogan & Kolter, 2002). However, P. aeruginosa does not
associate with or kill yeast cells. As yeast and hyphal cells
display different CWP profiles, a possible explanation for
this behavior is that P. aeruginosa specifically associates with
one or more hyphal CWPs.
Stress conditions that affect
CWP expression
Infection-associated stress conditions include hypoxia, as
found in biofilms and in the periodontal space, iron starvation as part of the normal host defense, low pH such as
found in the vagina or in the phagolysosomes of phagocytes,
carbon starvation after phagocytosis by macrophages or
neutrophils (Lorenz et al., 2004; Piekarska et al., 2006), and
nitrosative and oxidative stress as part of the defense
responses mounted by cells of the innate immune system
(Fig. 4). Antimicrobial peptides (AMPs) in the mucosal
secretions, such as the histatins in the saliva, represent
Fig. 4. Internal and external factors that affect the cell wall proteome.
2009 Federation of European Microbiological Societies
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10
another form of stress (reviewed in Kavanagh & Dowd,
2004). Similarly, the cells of the epithelial layers invaded by
C. albicans produce their own set of AMPs, such as the
a- and b-defensins. As C. albicans can survive temperatures
up to 45 1C, the human body temperature of 37 1C probably
does not represent a severe stress condition, if at all. Modern
medicine has introduced more tools to combat Candida
infections and thus represent novel stress conditions for
C. albicans. For example, the widely used azoles target Erg11
(lanosterol 14a-demethylase). This enzyme mediates a specific demethylation step in sterol biosynthesis. As a result,
ergosterol, the normal end product of this pathway, is
replaced by methylated sterols, which probably affect membrane properties such as membrane fluidity. In addition, the
methylated intermediates are likely suboptimal substrates
for the biosynthetic enzymes that operate after Erg11,
resulting in a general slowing down of sterol synthesis and
thus aggravating growth inhibition. The more recently
introduced echinocandins inhibit the synthesis of b-1,3glucan and thus weaken the cell wall (Liu et al., 2005).
Genomic transcript analysis and, to a much lesser extent,
cell wall proteome analysis have shown that the response of
C. albicans to these forms of stress often includes dramatic
changes in the transcript or protein levels of various
covalently linked CWPs (Niewerth et al., 2003; Bensen
et al., 2004; Lan et al., 2004; Copping et al., 2005; Hromatka
et al., 2005; Lee et al., 2005; Liu et al., 2005; Setiadi et al.,
2006; Vylkova et al., 2007; Castillo et al., 2008; Rauceo et al.,
2008; Sosinska et al., 2008). This strongly indicates that
covalently linked CWPs help the cell cope with stress
conditions (see also the next section).
Regulation and quantitation of
CWP expression
A full description of how the expression of CWP-encoding
genes is regulated would warrant a separate review. Importantly, the target genes of the signal transduction pathways
associated with sensing the environment and with morphogenesis, such as the cAMP-PKA pathway, the pH-sensing
Rim101 pathway, often include CWP-encoding genes. In
view of the role of the four known MAP kinase pathways of
C. albicans in mating, cell wall construction and cell wall
integrity, the yeast-to-hypha transition, invasive growth,
growth under embedded condition, adaptation to oxidative
stress, and the formation of chlamydospores, it seems likely
that they strongly affect the expression of CWP-encoding
genes as well. For a general overview of these pathways and
the corresponding positive and negative regulators, the
reader is referred to Monge et al. (2006); Arana et al.
(2007); and Biswas et al. (2007). Recently studied transcription factors that target CWP-encoding genes include the
pH-responsive transcription factor Rim101, which regulates
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F.M. Klis et al.
the expression of the GPI-CWP-encoding genes ALS1,
CSA1, HWP1, HYR1, IHD1, PHR1, PHR2, PGA10, RBT1,
and RBT5 (Bensen et al., 2004; Baek et al., 2006; Nobile et al.,
2008b); Flo8, which, together with Efg1, regulates the
expression of several hyphally expressed genes including the
GPI-CWP-encoding genes ALS1, ALS3, HWP1, IHD1, and
RBT5 (Cao et al., 2006); the filament-specific transcription
factor Ume6 (Banerjee et al., 2008; Carlisle et al., 2009); and
Cas5 and Sko1, both of which control the expression of
genes associated with cell wall integrity (Bruno et al., 2006;
Rauceo et al., 2008). In addition, the protein kinase Tor1
plays an important role in the negative regulation of several
covalently linked CWP-encoding genes, including ALS1,
ALS2, ALS3, CRH11, HWP1, RBT1, SAP10, and TOS1
(Bastidas et al., 2009).
Quantitative MS analysis of covalently linked CWPs is
just beginning to replace immunological means of quantitation (Yin et al., 2007, 2008). Fortunately, the available
evidence seems to indicate that under steady-state conditions, the transcript levels of CWP-encoding genes and the
corresponding protein levels are correlated (Klis et al., 2006;
G. Sosinska, unpublished data). Because covalently linked
CWPs are stable proteins and show only limited turnover,
this correlation does not hold during transitional states.
With this caveat in mind, transcript levels of CWP-encoding
genes as determined by genomic transcript analyses under
many environmental conditions can help predict in which
direction the corresponding protein levels in the walls will
move. Here, we present some general observations.
Although the eight members of the Als family have a
similar domain organization, the relative transcript levels of
the individual genes differ considerably. Some ALS genes
(ALS1, ALS2, and ALS3) show wide variations in transcript
levels, whereas ALS4 and ALS9 have intermediate levels and
ALS5, ALS6, and ALS7 transcript levels are generally low and
difficult to detect (reviewed in Hoyer et al., 2008) (Table 1).
These observations suggest that the protein levels of ALS1,
ALS2, and ALS3 will vary strongly, depending on the
environmental conditions.
The transcript levels of several CWP-encoding genes
differ widely between yeast and hyphal cells (Kadosh &
Johnson, 2005). Using fetal calf serum to induce the switch
from yeast to hyphal growth, these authors found that after
1 h the transcript levels of ALS3, HWP1, HYR1, IHD1,
PHR1, and SOD5 had considerably increased, whereas the
transcript levels of PIR1, RHD3, and YWP1 had considerably
declined. This indicates that, in agreement with immunological observations, the CWP profiles of yeast and hyphal
cells will differ widely (Staab et al., 1999; Kapteyn et al.,
2000).
As already mentioned, various other infection-associated
stress conditions strongly affect CWP transcript levels
(Niewerth et al., 2003; Bensen et al., 2004; Lan et al., 2004;
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11
Role of covalently linked CWPs of C. albicans
Copping et al., 2005; Lee et al., 2005; Setiadi et al., 2006;
Vylkova et al., 2007; Nobile et al., 2008b; Sosinska et al.,
2008). These observations suggest that the regulatory control of CWP expression will be generally complex. This is
reflected in the unusually long 5 0 -intergenic regions of the
large majority of CWP-encoding genes (Table 1), suggesting
that they have long and complex promoters (Argimon et al.,
2007; Kim et al., 2007). In addition to the long 5 0 -intergenic
regions, c. 50% of the CWP-encoding genes contain a TATA
box (Table 1) as defined by Basehoar et al. (2004). In S.
cerevisiae, about 20% of the genes contain a TATA box
(consensus sequence: TATA[A/T]A[A/T][A/G]) in the 5 0 region ranging from 200 to 50. Importantly, TATA
box-containing genes are associated with stress and are
generally highly regulated. It is tempting to extrapolate these
data to C. albicans. Two elegant promoter dissection studies
of ALS3 and HWP1, respectively, illustrate the complexity of
the regulation of CWP-encoding genes (Argimon et al.,
2007; Kim et al., 2007). Both genes code for CWPs that are
preferentially expressed in hyphal cells and their promoter
regions integrate inputs from multiple activators and repressors. Both possess long 5 0 -intergenic regions (c. 3.0 and
2.1 kbp, respectively) and a TATA box. Both promoters also
contain two activation regions, one of which is essential for
activation, whereas the other more distal region serves to
amplify the response.
Why is it advantageous for the cell to be able to adjust the
individual CWP levels in newly formed walls? One possible
reason is that covalently linked CWPs are cell surface
proteins and thus subject to much greater variations in
environmental conditions than cytosolic enzymes. Individual CWP family members might be more suitable for
specific niches and extreme environmental conditions. pHConditional expression of CWPs is indeed well known
(Bensen et al., 2004; G. Sosinska, unpublished data). A
classical example is presented by PHR1 and PHR2, two
transglycosylases, which are expressed at neutral and acidic
pH, respectively (reviewed in Chauhan et al., 2002). In
addition, some CWPs might be useful for early stages of
infection such as adhesion, but might become superfluous
or even harmful in later stages of infection. For example, the
putative GPI-CWP Iff4 promotes mucosal infection, but its
presence during a systemic infection diminishes virulence
(Fu et al., 2008).
Identification of vaccine antigens
originating from covalently linked CWPs
For a general review of the development of a Candida
vaccine, the reader is referred to Mochon & Cutler (2005).
Recombinant forms of the N-terminal part of Als1 and Als3
(roughly corresponding to their first two domains, i.e. the
Ig- and the T-domain) have both been successfully used to
FEMS Yeast Res ]] (2009) 1–16
develop vaccines against various forms of candidosis in
mice. The Als3-based vaccine was shown to be more
effective against mucosal infections (Ibrahim et al., 2006;
Spellberg et al., 2006). Interestingly, the Als3-based vaccine
also offers protection against bacterial infection by Staphylococcus aureus (Spellberg et al., 2008). In addition, Mp65 is
being studied as a vaccine candidate (Gomez et al., 2000;
Pietrella et al., 2008). The GPI-CWP Ywp1 has also been
tested as a vaccine candidate, but with limited success,
probably because it seems to be yeast specific (Granger
et al., 2005). Recently, fully synthetic glycopeptides have
been described that offer protection against disseminated
candidosis in mice (Xin et al., 2008). They consist of a 14amino-acid peptide conjugated through a nonimmunogenic
linker to a trisaccharide (b-1,2-linked Man3). The peptides
originated from proteins found to be associated with the cell
wall during disseminated candidosis and were further
selected for their antigenicity. The trisaccharide sequence,
on the other hand, is a normal part of phospholipomannan
and of N-linked carbohydrate side-chains of cell surface
proteins (Nitz et al., 2002; Fradin et al., 2008). Both the
peptide and the carbohydrate epitope contributed to the
immunogenic response. Although all peptide–oligosaccharide combinations tested were immunogenic, they did not all
offer protection against infection. One of the more effective
combinations contained a peptide from the putative propart of the GPI-CWP Hwp1 (amino acids 28–41), whereas
others included peptides from Eno1 (enolase), Fba1
(fructose-bisphosphate aldolase), and Met6 (cobalamin-independent methionine synthase). Obviously, this approach
will allow testing of many more interesting peptides.
Outlook
The covalently linked CWPs in the protein coat of the wall
are the rank and file in the ongoing battle between
C. albicans and its host. They also play a major role in the
development of biofilms and in the interactions with other
microbial organisms. Nevertheless, our knowledge of their
precise function is in many cases still incomplete. This
argues for a continued and extensive functional analysis, as
initiated by Plaine et al. (2008). Their precise location and
their ‘visibility’ to the immune system are also often
unknown. Another related question is which CWPs possess
a carbohydrate-binding module and might therefore be
involved in cell wall construction, in the synthesis of the
extracellular matrix of biofilms, or even in the recognition of
host ligands (Montanier et al., 2009). We have further seen
that the protein composition of the wall can vary widely
during infection; this argues for determining the changes in
CWP profiles under infection-associated conditions. Such
responses are still relatively unexplored. The regulatory
mechanisms that are responsible for the composition of the
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12
protein coat are just beginning to emerge. Using a library of
transcription factor mutants (deletion and overexpression
strains) in conjunction with in silico and experimental
promoter analysis of CWPs will help one to identify the
transcription factors involved (Nobile & Mitchell, 2005).
Finally, we envision that relative and absolute quantitation
of CWPs will lay a solid foundation for a more quantitative
analysis of their regulation and expression and will assist in
identifying the best vaccine candidates.
Acknowledgements
We thank Clemens Heilmann, Leo de Koning, and Alice
Sorgo for comments and stimulating discussions. F.M.K
acknowledges the financial support by the EU Program
FP7-214004-2 FINSysB.
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