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Department of Chemical and Biological Engineering, University of Wisconsin – Madison, Madison, WI 53706, USA
Correspondence
Sean P. Palecek
palecek{at}engr.wisc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 1st St SW, Rochester MN 55905, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Jarvis, 1995; Pfaller et al., 1998; Pfaller & Diekema, 2007). C. albicans is also the most common fungus associated with biofilm formation on bioprosthetic materials (Lopez-Ribot, 2005). Cells in biofilms are much more resistant to antifungal agents, including amphotericin B and azoles (Al-Fattani & Douglas, 2004; Hawser & Douglas, 1995).
Adhesion between C. albicans cells and materials or host cells has been implicated as an early step in biofilm formation. Adhesion of C. albicans to mammalian tissues is also considered to be a very important determinant of pathogenesis (Calderone & Fonzi, 2001). C. albicans glycosylphosphatidylinositol-dependent cell wall proteins (GPI-CWP) are characterized by the presence of an N-terminal signal peptide and a C-terminal sequence signalling GPI anchor attachment (Dranginis et al., 2007). Many of these proteins are involved in mediating the adhesion of C. albicans cells to host materials and/or inert surfaces (Fu et al., 2002; Gaur & Klotz, 1997; Phan et al., 2007; Staab et al., 1999; Zhao et al., 2005, 2007). Previous studies suggest that the N-terminal domains of the adhesins that are members of the GPI-CWP family mediate binding to substrates and the GPI anchor is required for incorporating these proteins into the cell wall (Frieman et al., 2002; Loza et al., 2004). These adhesins, in many cases, also contain serine/threonine-rich tandem repeat domains essential for proper presentation of the N-terminal substrate-binding sites (Frieman et al., 2002; Loza et al., 2004). However, the conserved Als tandem repeats in Als5p facilitate adhesion to fibronectin and greatly increase cell–cell aggregation (Rauceo et al., 2006).
Previous studies have demonstrated that the C. albicans Eap1p is a member of the GPI-CWP family (Li et al., 2007). The C. albicans Eap1p adhesin was originally identified because of its ability to mediate adhesion to polystyrene when the EAP1 gene was expressed in a flocculin-deficient Saccharomyces cerevisiae strain. EAP1 expression enhanced attachment of S. cerevisiae to HEK293 kidney epithelial cells and played an additional role in mediating interactions between S. cerevisiae and C. albicans cells (Li & Palecek, 2003, 2005). Expression of EAP1 in a C. albicans efg1/efg1 mutant was able to restore its partially reduced adhesion to HEK293 epithelial cells (Li & Palecek, 2003). Deleting EAP1 in C. albicans reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. C. albicans eap1/eap1 mutant cells were also defective in cell–cell adhesion when those cells were grown in a parallel-plate flow chamber under shear flow (Li et al., 2007). The defect in Eap1p-mediated adhesion was also associated with a defect in C. albicans biofilm formation in an in vitro and an in vivo model (Li et al., 2007). In C. albicans, EAP1 is expressed at similar levels in both yeast and filamentous forms (Li et al., 2007); however, the relative expression of EAP1 compared to other adhesins and the localization of Eap1p remain unknown. Many insights into the functions of C. albicans adhesins have been derived in C. albicans (Fu et al., 2002; Li et al., 2007; Phan et al., 2007; Staab et al., 1999; Zhao et al., 2005, 2007). Heterologous expression of C. albicans adhesins in S. cerevisiae has also proven valuable in exploring C. albicans adhesive function (Loza et al., 2004; Rauceo et al., 2006). Eap1p mediates adhesion to many substrates, including synthetic materials, yeast cells and mammalian cells, when expressed in both S. cerevisiae and C. albicans, suggesting that S. cerevisiae may be used as a relevant heterologous system to study the adhesive function of Eap1p in C. albicans.
In the experiments described in this paper, we constructed deletion mutants of EAP1 and expressed wild-type Eap1p and Eap1p mutants in an adhesin-deficient S. cerevisiae strain to study the mechanisms by which Eap1p mediates adhesion and define the critical functions of the domains of Eap1p in the context of adhesion. We demonstrated that different regions of Eap1p mediated adhesion to distinct substrates.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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was used for general recombinant techniques according to protocols described by Sambrook et al. (1989). S. cerevisiae haploid strain SPY308 (
1278b MAT ura3-52 his3 : : hisG leu2 : : hisG flo8 : : kanr), diploid strain SPY311 (1278b MATa/ ura3-52/ura3-52 his3 : : hisG/his3 : : hisG leu2 : : hisG/LEU2 flo8 : : kanr/flo8 : : HIS3) and strain BJ5464 (BJ) (MAT ura3-52 trp1 leu2
1 his3200 pep4 : : HIS3 prb11.6R can1 GAL) were used as the hosts for all mutants. Yeast strains were routinely cultured in YPD medium (1 % yeast extract, 2 % peptone, 2 % glucose) at 30 °C. Synthetic complete media lacking specific nutrients and filamentous growth media have been described previously (Ahn et al., 1999; Kron et al., 1994). Synthetic low-ammonium (SLAD) medium contained 50 µM ammonium sulfate. Uracil was added to SLAD medium to a concentration of 0.2 mM to make SLAD+Ura. Galactose was added to media to replace glucose in order to express genes within plasmids containing the S. cerevisiae GAL1 promoter. Yeast cells were transformed using lithium acetate transformation (Gietz et al., 1992). Pseudohyphal growth assay and agar invasion assay were performed as previously described (Li & Palecek, 2003).
Plasmid construction.
Sequences of all oligonucleotides used in cloning are provided in Table 1. Plasmids designed to express GFP fusions to the N- and truncated C-termini of EAP1 were constructed in a similar manner to that described by Mao et al. (2003). A partial ORF encoding the N-terminal 42 amino acids of Eap1p was amplified from C. albicans SC5314 genomic DNA. This PCR product was digested with PacI and SpeI and ligated into pHwp1Sig.GFP.GPI (Mao et al., 2003) to yield pEap1Sig.GFP.Hwp1GPI. A partial ORF encoding the C-terminal 47 amino acids of Eap1p was amplified from C. albicans SC5314 genomic DNA. This PCR product was digested with BamHI and SmaI and ligated into pEap1Sig.GFP.Hwp1GPI to generate pEAP1.Sig.GFP.GPI. pEAP1.Sig.GFP.NOGPI is essentially identical to pEAP1.Sig.GFP.GPI except that 21 amino acid residues from the C-terminus of Eap1p encoding the GPI anchor signal were deleted. The sequences between the PacI and SmaI sites encoding Eap1p/GFP fusion proteins were also PCR-amplified from pEAP1.Sig.GFP.GPI and pEAP1.Sig.GFP.NOGPI then cloned into pCT302 (Boder et al., 2000) cut with EcoRI and XhoI to generate pCTEAP1.Sig.GFP.GPI and pCTEAP1.Sig.GFP.NOGPI, respectively.
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1N, HA-Eap1p2N, HA-Eap1p3N and HA-Eap1p4N, respectively. The PCR products produced by amplifying the genomic DNA of C. albicans strain SC5314 with oligonucleotides EAP1-1 and EAP1-1-Right, EAP1-2-Right, EAP1-3-Right, EAP1-4-Right, and EAP1-5-Right, respectively, were digested with SpeI and BamHI and ligated into pCTEap1.HA.Sig.GFP.GPI to yield HA-Eap1p.GPI, HA-Eap1p1C.GPI, HA-Eap1p2C.GPI, HA-Eap1p3C.GPI and HA-Eap1p4C.GPI, respectively. pCTEap1.HA.Sig.GFP.GPI was digested with SpeI and BamHI. The fragments amplified by PCR with oligonucleotides EAP1-2 and EAP1-4-Right and oligonucleotides EAP1-4 and EAP1-2-Right were cloned into this digested vector to generate HA-Eap1.Sig.TR1.GPI and HA-Eap1.Sig.TR2.GPI, respectively. For all constructs in which fragments were generated by PCR, the final constructs were verified by DNA sequence analysis.
Microscopy.
To detect Eap1p-GFP fusion proteins and HA-tagged Eap1p mutant proteins, constructs were transformed into S. cerevisiae BJ5464 (BJ) cells and uracil prototrophs were selected on minimal glucose plates. Cells carrying these plasmids were cultured overnight at 30 °C in minimal medium containing galactose. The cells were then washed in PBS and immunofluorescence staining was performed as previously described (Li et al., 2007). To visualize yeast cell aggregates, cells were resuspended in PBS buffer and placed onto a microscope slide for photographing.
Western analysis.
Yeast cells expressing each Eap1p-GFP fusion construct were cultured as described above and harvested by centrifugation. To analyse the Eap1p-GFP fusion proteins in medium, the supernatant was concentrated using 10 kDa molecular mass cutoff Microcon centrifugal filters. Yeast cell walls were isolated according to the method of Mao et al. (2003). The proteins released into the supernatant by laminarinase digestion or from concentrated cell-free medium were subjected to SDS-PAGE and analysed by Western blotting using the ECL Western blot kit (Amersham) and an anti-GFP monoclonal antibody.
Polystyrene binding assay.
Yeast cells were cultured overnight at 30 °C in minimal medium containing galactose. After brief sonication to break cell clumps, equal number of cells was added to the wells of 24-well plates and incubated in sodium phosphate buffer (pH 6.0) for 2 h. Plates were washed a few times and the number of cells remaining attached was quantified by an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide]-reduction assay (Ramage et al., 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Mao et al. (2003) demonstrated that green fluorescent protein (GFP) fused to the N- and C-termini of the C. albicans GPI-anchored cell wall proteins Hwp1p, Als3p and Rbt5p was localized to the C. albicans cell wall. We used this approach to verify the cell wall localization of Eap1p in S. cerevisiae. A synthetic GFP (Cormack et al., 1997) was fused between 42 amino acid residues from the N-terminus and 47 amino acid residues from the C-terminus of Eap1p. The resulting fusion protein was expressed in S. cerevisiae under regulation of the GAL1 promoter. The pCTEAP1.Sig.GFP.GPI transformants expressing this fusion protein showed localized fluorescence at the cell surface (Fig. 1a), demonstrating that the EAP1-derived sequences were capable of targeting the fusion protein to the cell surface. We next used GFP fused to the 42 amino acids from the N-terminus but lacking the C-terminal sequence (pCTEAP1.Sig.GFP.NOGPI) to investigate if the GFP fusion was secured to the cell surface via a GPI anchor. This fusion did not localize to the cell wall, but was found inside the cell, appearing concentrated in extranuclear organelles (Fig. 1a). We tested the cell-free medium of the pCTEAP1.Sig.GFP.NOGPI transformants for secretion of the GFP fusion by Western blotting with an anti-GFP monoclonal antibody. The pCTEAP1.Sig.GFP.NOGPI transformants secreted a protein that was detected by the anti-GFP antibody and whose size corresponded to that of the fusion protein, whereas GFP was not detected in culture supernatants of the pCTEAP1.Sig.GFP.GPI transformants (Fig. 1a). To address whether the Eap1-GFP fusion proteins anchored to the plasma membrane or were incorporated in the cell wall, purified cell walls from S. cerevisiae strains transformed with GFP constructs were digested with β-glucanase. The GFP fusion protein was released from the cell walls of the pCTEAP1.Sig.GFP.GPI transformants and detected with anti-GFP antibodies (Fig. 1c). In control experiments, immunoreactive GFP proteins were not found in β-glucanase-treated cell walls of cells transformed with pCTEAP1.Sig.GFP.NOGPI nor in samples treated with buffer alone (Fig. 1c). The N- and C- termini of Eap1p were also required to localize Eap1p in C. albicans cell wall (Li et al., 2007). Based on these results, we concluded that Eap1p N-terminal and C-terminal sequences result in similar protein localization at the cell surface in S. cerevisiae and C. albicans.
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). The N-terminal region contains a signal peptide and may also contain a substrate-binding domain based on the common structures among adhesins. Two alleles of EAP1 exist in C. albicans strain SC5314. The segment following the N-terminal region consists of 
90 and 17 copies of an imperfect peptide sequence, STPATE (Fig. 2, boxes with vertical lines), in the long and short allele, respectively. The long allele was used in this study. The region following this segment is homologous to the putative N-terminal adhesion domain (Fig. 2, grey boxes). The C-terminal tandem-repeat domain consists of 12 copies of an imperfect peptide sequence, TPAAPGTPVESQPVIPGTET (Fig. 2, hatched boxes). The C-terminal region contains a GPI anchor addition signal involved in cross-linking Eap1p to the yeast cell wall (Fig. 2). To investigate the function of each domain in Eap1p, we constructed a series of HA-tagged Eap1p deletion mutants by sequentially deleting one domain from the N-terminus or the C-terminus while leaving the signal peptide and the GPI anchor signal intact to ensure localization to the cell wall (Fig. 2). The HA epitope was engineered into the EAP1 coding sequence C-terminal to the 30th amino acid residue.
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1N, HA-Eap1p2N, HA-Eap1p3N (Fig. 3a), HA-Eap1p.GPI, HA-Eap1p1C.GPI, HA-Eap1p2C.GPI and HA-Eap1p3C.GPI (Fig. 3b) on the surface of yeast, but the antibody did not bind to yeast transformed with the plasmid encoding HA-Eap1p4N and HA-Eap1p4C.GPI (Fig. 3a, b). The HA epitope could also be detected by the anti-HA antibody when only the N-terminal or the C-terminal tandem repeat domain was expressed in the presence of the signal peptide and the GPI anchor addition signal (Fig. 3c). These results suggest that both the N-terminal and the C-terminal tandem repeat domains can project the N-terminus of the protein away from the cell and into the extracellular environment, where it is accessible to antibody binding. We also verified the surface expression of all the constructs using fluorescence-activated cell sorting (FACS) after sonication to break cell clumps, and a similar amount of each Eap1p mutant protein was found on the surface of yeast cells in which the construct was accessible to the anti-HA antibody (data not shown).
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1278b S. cerevisiae cells (Lambrechts et al., 1996; Liu et al., 1996; Lo & Dranginis, 1998). A S. cerevisiae haploid flo8 strain carrying an empty vector was unable to invade agar on synthetic medium lacking uracil, but wild-type EAP1 expression induced invasive growth in the flo8 strain (Fig. 4). The expression of any of the EAP1 mutants lacking the N-terminal domain was not able to restore invasiveness to the haploid flo8 strain, suggesting that the N-terminal domain of Eap1p was required for agar invasion (Fig. 4a). Expression of all C-terminal EAP1 mutants, except HA-Eap1p4C.GPI, was able to restore invasiveness to the haploid flo8 strain (Fig. 4b). Presumably the N-terminal domain of HA-Eap1p4C.GPI was not exposed to the extracellular environment; the HA tag on this protein was not accessible to an anti-HA antibody (Fig. 3b
). To further investigate the role of the N-terminal domain of Eap1p in haploid invasive growth, we expressed a construct encoding a chimera protein of the N-terminal domain of Eap1p and the C-terminal Ser/Thr-rich tandem repeat domain of Candida glabrata Epa1p. The C-terminal Ser/Thr-rich domain of Epa1p is able to project the N-terminal domain of Epa1p through the permeability barrier of cell wall and into the extracellular environment (Frieman et al., 2002). Expression of the C-terminal domain of Epa1p alone did not restore the ability of the haploid flo8 strain to invade agar, but the strain carrying the construct encoding the Eap1p-Epa1p chimera was able to invade agar (Fig. 4c), suggesting that the N-terminal domain of Eap1p is able to mediate invasive growth if projected beyond the cell surface.
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).
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; Li et al., 2007). S. cerevisiae haploid flo8 strains expressing the N-terminal and the C-terminal Eap1p mutants that lack one of the two Ser/Thr-rich tandem repeat domains (HA-Eap1p4N and HA-Eap1p4N.GPI) exhibited reduced adhesion to polystyrene compared to the wild-type and the Eap1p mutants that contain both tandem repeat domains (Fig. 6a, b; P<0.05). We also transformed the haploid flo8 strain with constructs encoding the N-terminal (HA-Eap1Sig.TR1.GPI) and C-terminal (HA-Eap1Sig.TR2.GPI) tandem repeat domains of Eap1p and found that the adhesion to polystyrene was restored by expressing these two tandem repeat domains bounded by the signal peptide and GPI anchor sequences of Eap1p (Fig. 6c; P<0.01). S. cerevisiae cells expressing the Eap1–Epa1 chimera did not exhibit enhanced adhesion to polystyrene as compared to cells expressing the C-terminal Ser/Thr-rich domain of C. glabrata Epa1p (Fig. 6c). These results suggest that the two Ser/Thr-rich tandem repeat domains are sufficient to mediate adhesion to polystyrene.
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; Li et al., 2007). To identify the domains of Eap1p involved in adhesion to mammalian cells, we expressed the N-terminal and C-terminal EAP1 mutants in a haploid flo8 strain and quantified the yeast adhesion to mammalian HEK293 cells. HA-Eap1p induced adhesion of the flo8 strain to HEK293 cells, but deletion of the Eap1p N-terminal tandem repeat domain dramatically reduced this adhesion (Fig. 7a, b; P<0.01). Interestingly, deletion of the N-terminal domain, which was required for adhesion to yeast cells, did not reduce adhesion to HEK293 cells (Fig. 7a). Likewise, expression of the chimera protein containing the N-terminal domain of Eap1p and the C-terminal domain of C. glabrata Epa1p had no effect on adhesion to HEK293 cells (Fig. 7c), indicating that the N-terminal domain of Eap1p is substrate specific. To test whether the N-terminal tandem repeat domain was sufficient to mediate adhesion to HEK293 cells, we expressed HA-Eap1Sig.TR1.GPI and found its expression significantly enhanced the adhesion to HEK293 cells (Fig. 7; P<0.01).
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/flo8 cells failed to form any filaments on low-nitrogen SLAD medium (Fig. 8), and EAP1 expression restored filamentous growth to a flo8/flo8 strain (Li & Palecek, 2003). The expression of both HA-Eap1p (wild-type) and HA-Eap1p1N restored the ability of a flo8/flo8 strain to form pseudohyphae, indicating that the N-terminal domain of Eap1p, which was involved in mediating yeast cell–cell adhesion, was not required for pseudohyphal growth (Fig. 8a). Expression of HA-Eap1p2N, HA-Eap1p3N and HA-Eap1p4N was not able to complement the pseudohyphal growth defect of the diploid flo8/flo8 strain (Fig. 8a), while expression of HA-Eap1p.GPI, HA-Eap1p1C.GPI, HA-Eap1p2C.GPI and HA-Eap1p3C did complement this pseudohyphal defect (Fig. 8b). To test whether the N-terminal tandem repeat domain alone is able to activate the pseudohyphal growth of the flo8/flo8 strain, we transformed the diploid flo8/flo8 strain with HA-Eap1Sig.TR1.GPI and found that pseudohyphal growth was restored (Fig. 8c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The availability of the C. albicans genome provides a strategy to predict ORFs that potentially encode GPI-CWP proteins based on their common sequence features. Thus far, 104 putative GPI-anchored proteins have been predicted and more than 65 % of these proteins have completely unknown functions to date (Richard & Plaine, 2007). Understanding whether these GPI-anchored proteins mediate adhesion to different materials, host cells, or other microbial cells should increase our ability to design strategies to prevent such adhesion and resulting infections.
Adherence activity of the GPI-anchored proteins has commonly been ascribed to the N-terminal domains of these proteins because deletions in these regions usually abrogate binding activity (Frieman et al., 2002; Loza et al., 2004; Sheppard et al., 2004; Staab & Sundstrom, 1998; Wojciechowicz et al., 1993). N-terminal globular domains bind peptide or sugar ligands, with millimolar to nanomolar affinities. These affinities and the location of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell–cell associations (Dranginis et al., 2007). Here, we demonstrated that the N-terminal domain of Eap1p contains a ligand-binding domain mediating adhesion to yeast cells. This domain is also capable of rescuing the invasive defect of a haploid flo8 S. cerevisiae strain. The increase in invasion upon expression of Eap1p domains may be the result of the increased cell–cell adhesion. Numerous mutations that increase cell–cell adhesion via FLO11-dependent or FLO11-independent mechanisms in S. cerevisiae also enhance invasive growth (Guo et al., 2000; Palecek et al., 2000; Svarovsky & Palecek, 2005; Vyas et al., 2003).
The tandem repeats in Eap1p appear to have no direct role in yeast–yeast binding, but instead may project the N-terminal ligand-binding domains away from the body of the cell and into the extracellular environment. Previous studies also suggested that tandem-repeat-containing domains are essential for stabilizing the correct conformation of the N-terminal ligand-binding domain and contributing positively to the adherence activity mediated by members of the Als family of adhesins (Loza et al., 2004; Rauceo et al., 2006). Our data also demonstrated that the Eap1p Ser/Thr-rich domain containing tandem repeats was able to mediate adhesion to polystyrene and to mammalian epithelial cells directly, in addition to its function of projecting the N-terminal domain into the extracellular environment. Further, the N-terminal tandem repeat domain of Eap1p was able to promote the pseudohyphal growth of S. cerevisiae, suggesting that this tandem repeat domain was also able to induce morphological change of this fungus. The mechanism by which Eap1p induces pseudohyphal formation in S. cerevisiae is not known, but may be related to its adhesive properties, as the endogenous S. cerevisiae adhesin Flo11p and endochitinase/endoglucanse regulation of mother–daughter separation influence filamentous differentiation (King & Butler, 1998; Lo & Dranginis, 1998; Pan & Heitman, 2000). However, EAP1 expression does not appear to strongly influence morphology in C. albicans (Li et al., 2007). Our results indicate that tandem repeats of GPI-anchored proteins possess multiple functions, including modulating protein structure, mediating adhesion to certain substrates, and signalling morphological changes.
The tandem repeats are thought to trigger frequent recombination events in genes or between genes and pseudogenes, causing expansion and contraction of the gene size in S. cerevisiae, and the size variation creates quantitative alterations in phenotypes (Verstrepen et al., 2005). Different clinical isolates of C. albicans have differing numbers of repeats for the same ALS gene (Hoyer, 2001). The two alleles of EAP1 in C. albicans strain SC5314 also contain differing numbers of repeats in the N-terminal tandem repeat domain. Our results suggest these tandem repeat domains can directly mediate adhesion in addition to their roles in generating cell-surface diversity through genetic recombination and epigenetic regulation.
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ACKNOWLEDGEMENTS |
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Edited by: J. Pla
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Received 1 October 2007;
revised 22 December 2007;
accepted 10 January 2008.
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) at its C-terminus (amino acids 1075–1121). The open boxes on the left denote amino acids 31–140, the putative yeast–yeast adhesion domain. Boxes with vertical lines denote amino acids 141–675, the N-terminal serine/threonine-rich domain with repeats. Grey boxes denote amino acids 676–748, the domain that is homologous to the putative yeast–yeast adhesion domain. Hatched boxes denote amino acids 749–997, the C-terminal Ser/Thr-rich domain with repeats. The box with horizontal lines denotes amino acids 998–1074, the C-terminal region. The diagram is drawn to scale. SP, signal peptide; HA, haemagglutinin epitope of influenza virus
