Departamento de Microbiología y Ecología, Facultad de Farmacia, Universidad de Valencia, Avda. Vicente Andrés Estellés s/n, 46100-Burjassot (Valencia), Spain
Correspondence
Eulogio Valentin
Eulogio.Valentin{at}uv.es
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ABSTRACT |
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A table showing putative GPI-proteins in Candida albicans can be found in Microbiology Online.
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INTRODUCTION |
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The cell wall of C. albicans is a complex biochemical entity composed mainly of three components, namely, glucans (1,3-- and 1,6-
-glucan), mannoproteins and chitin (Fleet, 1991
; Valentin et al., 2000
). The
-glucans are the main components, accounting for 5060 % by weight of the cell wall in C. albicans and other fungi. Chitin, a linear polymer of 1,4-
-linked N-acetylglucosamine units, is a relatively minor (110 %) but important constituent. In most fungi,
-glucans and chitin polymers account for the rigidity of the cell wall as well as its morphology (Sentandreu et al., 1994
). Mannoproteins represent 3040 % of the total cell wall and determine the surface properties, enabling C. albicans cells to interact and adhere to host tissues (Chaffin et al., 1998
).
Cell-wall mannoproteins can be divided into three groups according to the methods used for their extraction. One group can be solubilized by detergents, such as SDS, or chaotropic agents, such as urea, and is formed by mannoproteins loosely associated with other components of the cell wall (Elorza et al., 1985; Valentin et al., 1984
). The second group can be extracted by reducing agents, DTT or
-mercaptoethanol (Casanova et al., 1989
; Orlean et al., 1986
). The third group can be released from the cell wall only following enzymic degradation of
-glucans and chitin with
-glucanases or chitinase (Kapteyn et al., 1999
; Marcilla et al., 1991
; Montijn et al., 1994
; Valentin et al., 1984
; Van Rinsum et al., 1991
). Only the mannoproteins in this latter group are covalently bound to
-glucan and chitin. Two types of glucanase-extractable proteins have been reported: glycosylphosphatidylinositol (GPI)-dependent cell-wall proteins and proteins of the Pir family (proteins with internal repeats). The proteins of the first type have, as common characteristics, a high Ser/Thr content and a putative GPI-attachment site (Klis, 1994
; van der Vaart et al., 1995
). Proteins of the Pir family can also be extracted from the cell wall by a mild NaOH treatment (Kapteyn et al., 1999
; Mrsa et al., 1997
). The presence of a GPI anchor has been demonstrated in cell-wall proteins of different fungal species (Frieman et al., 2002
; Moukadiri et al., 1997
; Staab et al., 1999
; Wojciechowicz et al., 1993
). The presence of the GPI anchor seems to play an important role in the biology of C. albicans, as mutants are affected in morphogenesis, virulence and cell-wall composition (Richard et al., 2002
). The total number of glucanase-extractable mannoproteins identified in C. albicans so far is small, but it is likely that this number will increase in the very near future as a result of homology studies following BLAST searches in the genome database of this fungus.
In the present study, we have taken a sequence-dependent approach to identify cell-wall ORFs by screening the genome database of C. albicans for cell-wall proteins by an in silico analysis. From all the ORFs identified as potential cell-wall proteins, we selected IPF 3054 for further study. This ORF has a putative GPI-anchor sequence, is rich in Ser/Thr and has a high homology to the Saccharomyces cerevisiae Ssr1p cell-wall protein, which was cloned and studied by our group (Moukadiri et al., 1997).
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METHODS |
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Strains, growth conditions and transformations.
The C. albicans and Escherichia coli strains used in this study are listed in Table 1. C. albicans cells were grown routinely in YPD medium (1 % yeast extract, 2 % bactopeptone, 2 % glucose) (Sherman, 1991
) or SD medium (0·7 % yeast nitrogen base without amino acids, 2 % glucose) supplemented with the appropriate nutrients in amounts specified by Sherman (1991)
. YPD and SD media were solidified with 2 % agar. For germ tube induction, cells were cultured in modified Lee's medium as described previously (Elorza et al., 1988
).
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Nucleic acid manipulations and analysis.
Genomic DNA from C. albicans was prepared using the method described by Fujimura & Sakuma (1993); total RNA isolation was done as described previously (Ramón et al., 1996
). Plasmid purification was performed using the FlexiPrep Kit commercial system (Amersham Biosciences). Standard DNA manipulation techniques were carried out using standard protocols (Sambrook et al., 1989
). DNA probes (amplicons IAE12 and IAE34) for Southern blot and Northern blot analysis were labelled by random primed incorporation of digoxigenin-labelled 2'-deoxyuridine 5'-triphosphate (DIG-labelled DNA) using the DIG DNA Labelling Kit (Roche) according to the manufacturer's instructions. Southern blot hybridization was performed as described by Ramón et al. (1996)
; Northern blot hybridization was done as described previously (Montero et al., 1998
). DNA and RNA concentrations were determined by measuring absorbance (A260) in a GeneQuant II RNADNA calculator spectrophotometer (Amersham Biosciences).
Plasmid construction for disruption of the CaSSR1 gene.
The CaSSR1 gene was disrupted by replacing part of the ORF (from amino acid 29 to 128) with a hisG : : URA3 : : hisG cassette (Fonzi & Irwin, 1993). The disruption cassette construction was achieved by using a two-step PCR amplification procedure with genomic DNA as template. In the first step, an amplicon of 607 bp was obtained from the genomic DNA using the sense primer IAE1 (5'-AGAAAAGCTTACGAAATAGGAGACGGGACC-3') and the antisense primer IAE2 (5'-AGCTCTGCAGAATAAACAAGCTGGTGGAGC-3') containing engineered HindIII and PstI restriction sites (underlined), respectively. The amplicon obtained (IAE12) was digested with HindIII and PstI, then subcloned into p5921 (Fonzi & Irwin, 1993
) containing the hisG : : URA3 : : hisG cassette; the resulting plasmid was named pIAE1-2 and contained the 5' region of the gene (positions -521 to +86 with respect to the start codon). In the second step, an amplicon of 444 bp containing the last 321 bp of the ORF plus the first 123 bp of the 3' downstream non-coding region was obtained by using the sense primer IAE3 (5'-TAAAGGATCCGAATCTAGTTCAGCCAAGGC-3') and the antisense primer IAE4 (5'-CATTGAGCTCTTATCAAAGAAAACATAACC-3') containing engineered restriction sites BamHI and SacI, respectively (underlined). The amplicon obtained (IAE34) was ligated into the BamHI and SacI sites of pIAE1-2 to create plasmid pI4 in which 297 bp (33 %) of the coding region were deleted.
Isolation of the CaSSR1 null mutant.
Disruption of CaSSR1 was achieved as described by Fonzi & Irwin (1993). CAI4 cells were transformed to Ura+ prototrophy with 10 µg of an HindIIISacI fragment from pI4. Transformed cells were selected as Ura+ in SD minimal medium lacking uridine and checked for integration of the cassette at the CaSSR1 locus by Southern blot analysis. One of the heterozygous disruptants recovered (designated C. albicans 30541-1) was used to select spontaneous Ura- derivatives in SD minimal medium containing 5-fluoro-orotic acid (Boeke et al., 1984
). These clones were analysed by Southern blot hybridization to identify those that had undergone intrachromosomal recombination between hisG repeats. One of these Ura- derivatives (termed C. albicans 30541-2) was used for replacement of the second CaSSR1 allele in a similar way using the HindIIISacI fragment from pI4. Transformed cells were selected as Ura+ and integration into the correct allele was verified by Southern blot analysis. One of the Ura+ transformants (designated C. albicans 30542-1) was used for 5-fluoro-orotic acid selection to Ura- auxotrophy. Ura- segregants were screened by Southern blot analysis using digestion with BglII to identify those carrying both disrupted CaSSR1 alleles. One of these CaSSR1 null mutants was designated C. albicans 30542-2.
Cell-wall purification.
Purified cell walls were obtained as described previously for S. cerevisiae (Pastor et al., 1984; Valentin et al., 1984
) except that intact cells were broken with glass beads [1·5 g (mg dry cells)-1] by shaking in a vortex mixer at room temperature for eight periods of 1 min each with intermediate periods of 1 min on ice. Using this method, breakage of the whole cell population was obtained, as monitored under the phase-contrast microscope. The purification procedure was continued by repeated washing (1200 g, 5 min) of the cell-wall pellet in cold PMSF (1 mM). The pellet was collected and operationally defined as the cell wall.
Solubilization of cell-wall proteins.
Conditions for solubilization of cell-wall proteins with SDS or zymolyase 20T have been described (Pastor et al., 1984; Valentín et al., 1984
); growth media and
-mercaptoethanol extracts were obtained as described previously (Casanova et al., 1989
; Elorza et al., 1988
).
SDS-PAGE and Western blot analysis.
Proteins were separated basically as described by Laemmli (1970) on SDS-10 % (w/v) polyacrylamide gels. For Western blot analysis, proteins were electrophoretically transferred from SDS-PAGE gels on to nitrocellulose filters (Hybond-C Extra; Amersham Biosciences) according to the method of Towbin et al. (1979)
. Filters were probed with rabbit antibodies against C. albicans yeast cells (PAbL) at a dilution of 1 : 1000, followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad). Antiserum binding was visualized by using the ECL (enhanced chemiluminescence) fluorescent labelling kit (Roche) following the manufacturer's instructions. Luminescence was recorded by exposing the filter to a radio-autographic X-Omat film (Kodak).
Subcloning of the CaSSR1 ORF into pADH.
The CaSSR1 ORF was obtained by PCR using the sense primer IAE5 (5'-CACAAGATCTATGGCTTCATTTTTAAAG-3') containing an engineered BglII (underlined) site and the antisense primer IAE6 (5'-CACACTCGAGTTACAATAAAACAGCACC-3') containing an engineered XhoI (underlined) site. A DNA polymerase with 3'5' proofreading activity (Ecotaq-Plus; Ecogen) was used to improve fidelity. We obtained an amplicon of 705 bp, from the ATG start codon to the TAA stop codon, which was subcloned into the commercial vector pGEM-T Easy (Promega). The amplicon was rescued by digestion with BglII/XhoI and ligated to BglII/XhoI-digested pADH (Bertram et al., 1996
) to give plasmid pADH-CaSSR1, which was used to transform the null mutant. The resulting transformed strain (C. albicans 30543) was tested for CaSSR1 overexpression.
Phenotypic analysis of mutants and the overexpressing strain.
Calcofluor white (CW) and Congo red (CR) sensitivities were tested by streaking cells on to plates containing different concentrations of CW or CR following the method described by van der Vaart et al. (1995). Aliquots (3 µl) of serial 1/10 dilutions of cells that had been grown overnight and adjusted to an OD600 value of 1 were deposited on to the surface of YPD or SD plates containing different concentrations of CW (0200 µg ml-1) or CR (025 µg ml-1); these samples were then grown at 28 °C and monitored for 3 days.
Sensitivity to zymolyase was also tested following the method described by van der Vaart et al. (1995). Exponentially growing cells were adjusted to an OD600 value of 0·5 (
4x106 cells ml-1) in 10 mM Tris/HCl, pH 7·5, containing 50 µg zymolyase 20T ml-1 and the decreases in the optical density were monitored over a 90 min period.
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RESULTS |
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From the ORFs analysed, we selected one (IPF 3054=CaSSR1) that presents a potential GPI region and whose product has high homology (62 %) with S. cerevisiae Ssr1p, an internal cell-wall protein (Moukadiri et al., 1997) (Fig. 1
A).
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Twelve of the resulting Ura+ transformants were analysed and 10 of them contained the desired insert at the CaSSR1 locus (data not shown). Southern blot analysis of a representative isolate, C. albicans 30541-1, after digestion with BglII, revealed that the cassette had integrated in the allele contained in a 3·08 kb BglII fragment originating a 7·08 kb fragment (Fig. 3); this is consistent with the replacement of one allele of CaSSR1 with the transforming DNA. The 3·08 kb BglII fragment corresponds to the other allele which was still present in the Ura+ transformants. Ura- segregants of C. albicans 30541-1 were selected on medium containing 5-fluoro-orotic acid (Boeke et al., 1984
) and examined by Southern blot analysis. Nine of the 12 independent segregants examined had undergone intrachromosomal recombination between the hisG repeats, resulting in the excision of the URA3 marker and one copy of hisG, whereas three of them had experienced an interchromosomal recombination event, reverting to the parental genotype (data not shown). Southern blot analysis of a representative intrachromosomal recombinant, C. albicans 30541-2, is shown in Fig. 3
. The 7·08 kb BglII fragment seen in C. albicans 30541-1 was absent, and a new band of 3·8 kb was present; this size corresponds to the correct intrachromosomal event.
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Characterization of CaSSR1 mutants
Proof that CaSSR1 encodes a cell-wall protein was obtained after the disruption of the two alleles of CaSSR1 and comparison of the zymolyase extracts from parental and null mutant strains cell walls by Western blot analysis with PAbL rabbit antibodies. As shown in Fig. 6, the antibody recognizes a 70 kDa species in the parental strain but not in the null mutant. Therefore, it was concluded that CaSSR1 encodes a cell-wall protein that is bound to
-glucan. No differences were observed in the protein patterns of the
-mercaptoethanol extract, SDS extract and spent medium (data not shown).
Phenotypic analyses of the mutants in comparison with their parental strain were performed. The specific growth rates of the yeast form of the parental strain C. albicans CAI4 and the Ura- strains 30541-2 and 30542-2 were similar and no differences in morphology were observed, as in S. cerevisiae ssr1 (Moukadiri et al., 1997). No differences in the kinetics of germ tube formation were observed between the mutants and the parental strain. Changes in the cell wall were studied by testing the sensitivities of the mutants to CW, CR and zymolyase, as described by van der Vaart et al. (1995)
for S. cerevisiae. Sensitivities to CW and CR did increase with respect to parental strain (Fig. 4
) and the sensitivity to zymolyase was also increased in the null mutant (Fig. 5
). These results suggest that the lack of CaSsr1p leads to a defective cell wall, as happens in S. cerevisiae ssr1 (Moukadiri et al., 1997
).
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DISCUSSION |
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The results obtained revealed that CaSSR1 encodes a cell-wall protein: (i) its disruption leads to the absence of a 70 kDa protein band in the material released from isolated cell walls by zymolyase (Fig. 6); (ii) overexpression of this gene leads to an increase in the amount of the 70 kDa protein band (Fig. 6
); and (iii) the deduced amino acid sequence presents the typical characteristics of a cell-wall protein, i.e. presence of a cleavable signal peptide, richness in Ser and Thr amino acids susceptible to O-glycosylation, as well as the probability of being modified by GPI addition.
The theoretical molecular mass of mature CaSsr1p is 20·2 kDa but in SDS-PAGE the protein appears with an apparent mobility of 70 kDa. This experimental mobility could be due to O-glycosylations and/or GPI-addition modifications. This anomalous electrophoretic behaviour has also been reported for other Ser- and Thr-rich cell-wall proteins (Lu et al., 1994; Moukadiri et al., 1997
; van der Vaart et al., 1995
). In S. cerevisiae, Ssr1p (Icwp) has an apparent mass of 140 kDa in SDS-PAGE, whereas in C. albicans its size is 70 kDa. This difference in size may be due to the fact that ScSsr1p has an N-glycosylation site (Moukadiri et al., 1997
), whereas this is lacking in CaSsr1p. As a consequence, ScSsr1p appears as a polydisperse band after extraction by zymolyase, whilst CaSsr1p does not. This polydispersity could be conferred by the size of the different mannan chains and by the presence of glucan side chains attached to the mannoprotein moiety of certain
-glucanase-extractable cell-wall proteins (Montijn et al., 1994
; van der Vaart et al., 1995
). CaSsr1p shares some common features with ScSsr1p, such as an abundance of Ser and Thr residues susceptible to O-glycosylation and a putative GPI-attachment site, which could play a role in the covalent linkage to the glucan network.
To obtain some information about the possible function of CaSsr1p, we analysed the phenotype of Cassr1 mutants and the overexpression of CaSsr1p. Both the null mutant and the overexpressing strain behaved similarly to the parental strain with respect to growth rates, morphology and kinetics of germ tube formation, indicating that CaSsr1p is not absolutely necessary for viability in cells growing under the experimental conditions and for the dimorphic transition. CW and CR are compounds that interfere with the assembly of polymers in the cell wall (Elorza et al., 1983
; Kopecka & Gabriel, 1992
) and they have been used for detecting S. cerevisiae cell-wall mutants (Ram et al., 1994
; van der Vaart et al., 1995
); when both the Cassr1
null mutant and an overexpressing strain were grown in media containing either CW or CR, an increased sensitivity was observed when compared to the parental strain. This result suggests that in a normal cell-wall structure CaSsr1p must be present in an adequate concentration with respect to the other cell-wall components, and that any deviation from this concentration causes a change in the architecture of that structure as in S. cerevisiae (Moukadiri et al., 1997
).
Sensitivity to zymolyase has also been used to find cell-wall defects (van der Vaart et al., 1995). In the present work, depletion of CaSsr1p caused an increase in sensitivity to zymolyase, as happens in S. cerevisiae (Moukadiri et al., 1997
); however, overabundance of Ssr1p in S. cerevisiae does not induce changes in sensitivity when compared with the parental strain, whereas in C. albicans the overexpression of CaSSR1 renders the cells as sensitive to zymolyase as the null mutant. These results suggest that this increased sensitivity should be related to changes in the structure of the glucan network rather than a decrease in the thickness of the outer layer of the cell wall, as suggested by van der Vaart et al. (1995)
for Cwp2p in S. cerevisiae.
We do not know whether CaSsr1p forms disulphide bridges with other cell-wall proteins, but if that is the case, the protein is also bound to -glucan because it is not released by reducing agents (i.e.
-mercaptoethanol).
Further studies with modified versions of CaSSR1 should provide insights into the organization and structure of the C. albicans cell wall.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Bertram, G., Swoboda, R. K., Gooday, G. W., Gow, N. A. R. & Brown, A. J. P. (1996). Structure and regulation of the Candida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase. Yeast 12, 115127.[CrossRef][Medline]
Boeke, J. D., LaCroute, F. & Fink, G. R. (1984). A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197, 345346.[Medline]
Calderone, R. A. & Braun, P. C. (1991). Adherence and receptor relationships of Candida albicans. Microbiol Rev 55, 120.[Medline]
Caras, I. W. & Weddell, G. N. (1989). Signal peptide for protein secretion directing glycophospholipid membrane anchor attachment. Science 243, 11961198.[Medline]
Caras, I. W., Weddell, G. N., Davidz, M. A., Nussenzweig, V. & Martin, D. W., Jr (1987). Signal for attachment of a phospholipids membrane anchor in decay accelerating factor. Science 238, 12801283.[Medline]
Casanova, M., Gil, M. L., Cardeñoso, L., Martinez, J. P. & Sentandreu, R. (1989). Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans. Infect Immun 57, 262271.[Medline]
Chaffin, W. L., Lopez-Ribot, J. L., Casanova, M., Gozalbo, D. & Martinez, J. P. (1998). Cell wall and secreted proteins of Candida albicans: identity, function, and expression. Microbiol Mol Biol Rev 62, 130180.
Elorza, M. V., Rico, H. & Sentandreu, R. (1983). Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. J Gen Microbiol 129, 15771582.[Medline]
Elorza, M. V., Murgui, A. & Sentandreu, R. (1985). Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cells. J Gen Microbiol 131, 22092216.[Medline]
Elorza, M. V., Marcilla, A. & Sentandreu, R. (1988). Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties. J Gen Microbiol 134, 23932403.[Medline]
Fleet, G. H. (1991). Cell walls. In The Yeasts, vol. 4, pp. 199277. Edited by A. H. Rose & J. S. Harrison. New York: Academic Press.
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.
Fox, J. L. (1993). Fungal infection rates are increasing. ASM News 59, 515518.
Frieman, M. B., McCaffery, J. M. & Cormack, B. P. (2002). Modular domain structure in the Candida glabrata adhesion Epa1p, a 1,6-glucan-cross-linked cell wall protein. Mol Microbiol 46, 479492.[CrossRef][Medline]
Fujimura, H. & Sakuma, Y. (1993). Simplified isolation of chromosomal and plasmid DNA from yeasts. Biotechniques 14, 538540.[Medline]
Gattiker, A., Gasteiger, E. & Bairoch, A. (2002). SCANPROSITE: a reference implementation of a PROSITE scanning tool. Appl Bioinform 1, 107108.
Gozalbo, D., Elorza, M. V., Sanjuan, R., Marcilla, A., Valentin, E. & Sentandreu, R. (1994). Critical steps in fungal cell wall synthesis: strategies for their inhibition. Pharmacol Ther 60, 337345.[CrossRef]
Guillum, A. M., Tsay, E. Y. H. & Kirsch, D. R. (1984). Isolation of the Candida albicans gene from oritidine-5'-phosphate decarboxylase by complementation of Saccharomyces cerevisiae ura3 and Escherichia coli pyrF mutations. Mol Gen Genet 198, 21002112.
Hamada, K., Terashima, H., Arisawa, M. & Kitada, K. (1998). Amino acid sequence requirement for efficient incorporation of glycosylphosphatidylinositol-associated proteins into the cell wall of Saccharomyces cerevisiae. J Biol Chem 273, 2694626953.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557580.[Medline]
Kapteyn, J. C., Van Den Ende, H. & Klis, F. M. (1999). The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1426, 373383.[Medline]
Klis, F. M. (1994). Review: cell wall assembly in yeast. Yeast 10, 851869.[Medline]
Kopecka, M. & Gabriel, M. (1992). The influence of congo red on the cell wall and (13)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae. Arch Microbiol 158, 115126.[Medline]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105132.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lipke, P. N., Wojciechowicz, D. & Kurjan, J. (1989). AG1 is the structural gene for the Saccharomyces cerevisiae
-agglutinin, a cell surface glycoprotein involved in cellcell interactions during mating. Mol Cell Biol 9, 31553165.[Medline]
Lu, C. F., Kurjan, J. & Lipke, P. N. (1994). A pathway for cell wall anchorage of Saccharomyces cerevisiae -agglutinin. Mol Cell Biol 14, 48254833.[Abstract]
Marcilla, A., Elorza, M. V., Mormeneo, S., Rico, H. & Sentandreu, R. (1991). Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and -mercaptoethanol. Arch Microbiol 155, 312319.[CrossRef][Medline]
Montero, M., Marcilla, A., Sentandreu, R. & Valentin, E. (1998). A Candida albicans 37 kDa polypeptide with homology to the laminin receptor is a component of the translational machinery. Microbiology 144, 839847.[Abstract]
Montijn, R. C., van Rinsum, J., van Schager, F. A. & Klis, F. M. (1994). Glucomannoproteins in the cell wall of Saccharomyces cerevisiae contain a novel type of carbohydrate side chain. J Biol Chem 269, 1933819342.
Moukadiri, I., Armero, J., Abad, A., Sentandreu, R. & Zueco, J. (1997). Identification of a mannoprotein present in the inner layer of the cell wall of Saccharomyces cerevisiae. J Bacteriol 179, 21542162.[Abstract]
Mrsa, V., Seidl, T., Gentzsch, M. & Tanner, W. (1997). Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast 13, 11451154.[CrossRef][Medline]
Nakai, K. & Horton, P. (1999). PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24, 3436.[CrossRef][Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 16.[Abstract]
Nuoffer, C., Horvath, A. & Rietzman, H. (1993). Analysis of the sequence requirements for glycosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein. J Biol Chem 268, 1055810563.
Odds, F. C. (1988). Candida and Candidosis, a Review and Bibliography, 2nd edn. London: Baillière Tindall.
Odds, F. C. (1994). Candida species and virulence. ASM News 60, 313318.
Orlean, P., Ammer, H., Watzele, M. & Tanner, W. (1986). Synthesis of an O-glycosylated cell surface protein induced by alpha factor. Proc Natl Acad Sci U S A 83, 62636266.[Abstract]
Pastor, F. I. J., Valentin, E., Herrero, E. & Sentandreu, R. (1984). Structure of the Saccharomyces cerevisiae cell wall: mannoproteins released by zymolyase and their contribution to wall architecture. Biochim Biophys Acta 802, 292300.
Ram, A. F. J., Wolters, A., Ten Hoopen, R. & Klis, F. M. (1994). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10, 10191130.[Medline]
Ramón, A. M., Gil, R., Burgal, M., Sentandreu, R. & Valentin, E. (1996). A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica. Yeast 12, 15351548.[CrossRef][Medline]
Richard, M., Ibata-Ombetta, S., Dromer, F., Bordon-Pallier, F., Jouault, T. & Gaillardin, C. (2002). Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages. Mol Microbiol 44, 841853.[CrossRef][Medline]
Roy, A., Lu, C. F., Marykwas, D. L., Lipke, P. N. & Kurjan, K. (1991). The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin. Mol Cell Biol 11, 41964206.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sentandreu, R., Elorza, M. V., Mormeneo, S., Sanjuan, R. & Iranzo, M. (1993). Possible roles of mannoproteins in the construction of Candida albicans cell wall. In Dimorphic Fungi in Biology and Medicine, pp. 169175. Edited by H. van den Bossche, F. C. Odds & D. Kerridge. New York: Plenum.
Sentandreu, R., Mormeneo, S. & Ruiz-Herrera, J. (1994). Biogenesis of fungal cell wall. In The Mycota I. Growth, Differentiation and Sexuality, pp. 111124. Edited by J. G. H. Wessels & F. Meinhardt. Berlin: Springer.
Sherman, F. (1991). Getting started with yeast. In Guide to Yeast Genetics and Molecular Biology, pp. 321. Edited by C. Guthrie & G. R. Fink. San Diego, CA: Academic Press.
Sigrist, C. J., Cerutti, L., Hulo, N., Gattiker, A., Falquet, L., Pagni, M., Bairoch, A. & Buchner, P. (2002). PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform 3, 265274.[Medline]
Staab, J. F., Bradway, S. D., Fidel, P. L. & Sundstrom, P. (1999). Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 283, 15351538.
Teunissen, A. W. R. H., Holub, E., van der Hucht, J., van der Berg, J. A. & Steensma, H. Y. (1993). Sequence of the open reading frame of the FLO1 gene from Saccharomyces cerevisiae. Yeast 9, 423427.[Medline]
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 43504354.[Abstract]
Valentin, E., Herrero, E., Pastor, F. I. J. & Sentandreu, R. (1984). Solubilization and analysis of mannoprotein molecules from the cell wall of Saccharomyces cerevisiae. J Gen Microbiol 130, 14191428.
Valentin, E., Mormeneo, S. & Sentandreu, R. (2000). The cell surface of Candida albicans during morphogenesis. In Contributions in Microbiology, vol. 5, Dimorphism in Human Pathogenic and Apathogenic Yeasts, pp. 138150. Edited by J. F. Ernst & A. Schmidt. Basel: Karger.
van der Vaart, J. M., Caro, L. H., Chapman, J. W., Klis, F. M. & Verrips, C. T. (1995). Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol 177, 31043110.[Abstract]
Van Rinsum, J., Klis, F. M. & van den Ende, H. (1991). Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9. Yeast 7, 717726.[Medline]
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14, 46834690.[Abstract]
Wojciechowicz, D., Lu, C. F., Kurjan, J. & Lipke, P. N. (1993). Cell surface anchoring and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein -agglutinin, a member of the immunoglobulin superfamily. Mol Cell Biol 13, 25542563.[Abstract]
Received 13 February 2003;
revised 17 April 2003;
accepted 8 May 2003.
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