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Keywords:

  • adhesion;
  • amyloid;
  • biofilm;
  • cell wall model;
  • GPI proteins;
  • proteomics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 β-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. Gel-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 niche-specific 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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 wall-traversing 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 β-1,6-glucan, β-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 β-1,6-glucan molecules, which in turn are covalently linked to nonreducing ends of β-1,3-glucan molecules. β-1,6-Glucans 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 β-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).

image

Figure 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 GPI-CWPs 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 β-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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Table 1.   Experimentally validated covalently linked cell wall proteins of C. albicans
Protein5′-Intergenic region (bp)TATA boxNo. of AA and region of tryptic peptides*S/T-enriched regionFeatures and functions
GPI-CWPs
 Als14813+1260 79–311c. 330-GPIHigh levels of gene expression (Hoyer et al., 2008); N-terminal c. 300-residue Ig domain; found in band at the hyphal base (Fu et al., 2002); N-terminal sequence used as vaccine antigen (Spellberg et al., 2005); contributes to biofilm formation (Nobile et al., 2008a)
 Als32964+1155 77–311c. 330-GPIHigh levels of gene expression (Hoyer et al., 2008); N-terminal c. 300-residue Ig domain; uniformly distributed over the hyphal wall (Laforce-Nesbitt 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)
 Als43248+2100 77–91c. 330-GPIIntermediate 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 pH
 Als54353+1347 No datac. 330-GPICell 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
 Als641451366 No datac. 330-GPICell 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
 Als749321568 No datac. 330-GPICell wall location based on expression in S. cerevisiae (Sheppard et al., 2004); low gene expression (Hoyer et al., 2008); N-terminal c. 300-residue Ig domain
 Als92769+1889 No datac. 330-GPICell wall location based on expression in S. cerevisiae (Sheppard et al., 2004); intermediate levels of gene expression (Hoyer et al., 2008); N-terminal c. 300-residue Ig domain
 Cht23131583 22–299c. 300-GPIChitinase; N-terminal GH18 domain
 Crh112114+453 29–280c. 280-GPIN-terminal GH16 domain; transglycosylase (Pardini et al., 2006; Cabib et al., 2008)
 Csa19671018 No datac. 650-GPICell 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)
 Eap13451653 No datac. 140-GPICell 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)
 Ecm334430+423 139–341c. 350-GPIImportant for cell wall integrity (Martinez-Lopez et al., 2006); precise function unknown
 Hwp12081+634 No datac. 180-GPICovalently bound to cell wall β-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); N-terminal 14-mer peptide used as vaccine antigen (Xin et al., 2008)
 Hyr17223+919 143–286c. 350-GPIUnknown function; not found in mycelial fungi; the Hyr-like gene family is enriched in pathogenic Candida spp. (Butler et al., 2009)
 Ihd12368392 +c. 130-GPIUnknown function
 Pga4838451 42–385c. 370-GPITransglycosidase with an N-terminal GH72 domain; widespread among ascomycetes
 Pga10724+250 47–60c. 130-GPIN-terminal CFEM domain; loss of function results in fragile biofilms (Pérez et al., 2006); involved in heme-iron utilization (Weissman & Kornitzer, 2004)
 Pga302111277 123–149c. 150-GPIPga30, Pga31, and Rhd3 are homologous; function unknown; the Pga30-like gene family is enriched in pathogenic Candida spp. (Butler et al., 2009)
 Pga311619352 102–162c. 200-GPIN-terminus as presented in CGD possibly incorrect; Pga30, Pga31, and Rhd3 are homologous; function unknown; conserved in Candida spp.
 Pga452090+462 351–364c. 220-GPIFunction unknown; conserved in Candida spp.
 Pga598057+113 No dataAbsentCell 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
 Pga62806+213 No dataAbsentCell wall location confirmed by GFP tagging; contains two tandem repeats similar to the 3-cysteine domain of Pga59 (Moreno-Ruiz et al., 2009)
 Phr11016548 71–452c. 480-GPIN-terminal GH72 domain followed by an X8 domain (pfam07983) in the C-terminal half; transglycosylase involved in cell wall construction; incorporated at neutral/alkaline pH
 Phr21193544 23–269c. 480-GPIN-terminal GH72 domain followed by an X8 domain (pfam07983) in the C-terminal half; transglycosylase involved in cell wall construction; incorporated at acidic pH
 Rbt13415+721 262–271c. 270-GPIN-terminal Flo11 domain (pfam10182)
 Rbt51253+241 37–92c. 130-GPIN-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)
 Rhd3831+204 16–150AbsentPga30, Pga31, and Rhd3 are homologous; function unknown; conserved in Candida spp.
 Sap91555544 +c. 480-GPIYapsin-like protein mainly found in the PM; required for full cell wall integrity; also identified by immunogold labeling (Albrecht et al., 2006)
 Sap102000453 No datac. 390-GPICell wall location determined by immunogold labeling (Albrecht et al., 2006); yapsin-like protein mainly found in the wall; required for full cell wall integrity
 Sod41391+232 16–117c. 170-GPISuperoxide dismutase; contributes to clearance of ROS (Frohner et al., 2009)
 Sod53253+228 26–140c. 170-GPISuperoxide dismutase; plays a major role in the clearance of ROS (Frohner et al., 2009)
 Ssr13049234 23–79c. 80-GPIN-terminal CFEM domain; also identified by immunological means (Garcera et al., 2003)
 Utr24220470 75–310c. 370-GPIPutative transglycosylase (Pardini et al., 2006; Cabib et al., 2008) with a GH16 domain followed by C-terminal S/T-rich region; conserved in ascomycetes and basidiomycetes
 Ywp16033+533 93–349c. 160–410Strongly 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)
Non-GPI CWPs
 Mp651573378 128–371c. 60–110Putative transglycosylase with a C-terminal GH17 domain; found in fibrillar material; considered as a vaccine candidate (Pietrella et al., 2008)
 Pir15561346 19–346AbsentC-terminal conserved 4-Cys pattern; predicted to cross-link β-1,3-glucan chains (Ecker et al., 2006); protein levels increase during hypoxic conditions (Sosinska et al., 2008)
 Sim14601372 32–265c. 50–110C-terminal SUN domain; required for septation; synthetic lethality with SUN41 results in osmoremediable wall rupturing close to the septum (Firon et al., 2007)
 Tos14360468 288–370c. 140–230N-terminal DUF2403 domain and C-terminal DUF2401 domain
image

Figure 2.  Diagram of the GPI-CWP Pga59. Pga59 is both N- and O-glycosylated (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.

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β-1,3-Glucan is the main structural polysaccharide and, through its nonreducing ends, offers acceptor sites for covalent attachment of β-1,6-glucan and chitin. Because β-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 cross-linkages 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 °C, 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 β-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 (Arnold & Lacy, 1977). The elasticity of the wall reflects the helical conformation of the β-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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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%β-mercaptoethanol at 37 °C, 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 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 wall-traversing 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

There are two main classes of covalently linked CWPs: GPI-CWPs 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).

Table 1 presents an overview of the CWPs in C. albicans, which have been experimentally shown to be covalently linked to the β-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 GPI-CWPs 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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 vagina-simulative 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 β-1,3-glucan 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 alkali-sensitive, 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 μm and a width of 4.06 μm, which corresponds to a volume of c. 49 μm3 and a surface area of 67 μm2 (Kocková-Kratochvilová, 1990). Assuming a cell density of 1.12 g mL−1 and a dry weight content of 34% (Kocková-Kratochvilová, 1990), a volume of 49 μm3 corresponds to c. 55 pg of wet 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 μm−2. 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 β-glucan from detection by the mammalian β-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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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).
  • 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, threonine-rich, 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 Ig-domain 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 β-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/transglycosylase 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).
image

Figure 3.  Proposed β-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 cross-linking. J, any hydrophobic amino acid.

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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 °C; (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) 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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 α- and β-defensins. As C. albicans can survive temperatures up to 45 °C, the human body temperature of 37 °C 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 14α-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 β-1,3-glucan and thus weaken the cell wall (Liu et al., 2005).

image

Figure 4.  Internal and external factors that affect the cell wall proteome.

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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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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; 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′-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′-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′-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′-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. pH-Conditional 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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 14-amino-acid peptide conjugated through a nonimmunogenic linker to a trisaccharide (β-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 pro-part 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References

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 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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cellular morphology and the molecular organization of the cell wall
  5. Cell surface-associated cytosolic proteins
  6. Most covalently linked CWPs of C. albicans are GPI-CWPs
  7. Location of CWPs
  8. General properties of covalently linked CWPs
  9. Properties of CWP families and individual CWPs
  10. CWPs and biofilm formation
  11. Stress conditions that affect CWP expression
  12. Regulation and quantitation of CWP expression
  13. Identification of vaccine antigens originating from covalently linked CWPs
  14. Outlook
  15. Acknowledgements
  16. References