Laboratoire de Génétique Moléculaire et Cellulaire, Institut National Agronomique Paris-Grignon, UMR-INRA216, URA-CNRS1925, BP01, 78850 Thiverval-Grignon, France1
Fungal Research Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands2
Aventis Pharma, 102 route de Noisy, 93235 Romainville cedex, France3
Laboratoire de Mycologie Fondamentale et Appliquée, INSERM EPI 9915, Université de Lille II, Faculté de Médecine H. Warembourg, Pôle Recherche, Place Verdun, 59037 Lille Cedex, France4
Author for correspondence: Mathias Richard. Tel: +33 1 30 81 54 53. Fax: +33 1 30 81 54 57. e-mail: richard{at}grignon.inra.fr
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ABSTRACT |
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Keywords: glycosylphosphatidylinositol, Pir proteins, Als proteins
Abbreviations: Als, agglutinin-like sequence; ConA, Concanavalin A; CWP, cell-wall protein; EtN, ethanolamine; EtN-P, phosphoethanolamine; GPI, glycosylphosphatidylinositol; Pir, protein with internal repeat
a Present address: Department of Microbiology, Columbia University, 701 West 168th Street, New York, NY 10032, USA.
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INTRODUCTION |
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S. cerevisiae is known to have a GPI core structure with a fourth mannose group; Smp3p, an essential protein, is described as being involved in the addition of this group (Grimme et al., 2001 ). Various studies have described three proteins involved in the transfer of EtN-Ps onto the three first mannose groups of the GPI anchor (Benachour et al., 1999
; Flury et al., 2000
; Taron et al., 2000
). Mcd4p adds an EtN-P on the first mannose, Gpi7p on the second and Gpi13p/Yll031c on the third one (Fig. 1
) (Flury et al., 2000
). Whereas both MCD4 and GPI13 are essential, GPI7 is not. The lethality of the GPI13 deletion is to be expected since its gene product is involved in the addition of the EtN-P, which links the anchor to the protein. The function of the other two EtN groups is unknown. Here we present evidence that the side chain added by Gpi7p participates in the proper anchoring of GPI-modified proteins targeted to the cell wall.
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METHODS |
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Quantazyme and chitinase sensitivity assays.
These assays were carried out to detect possible changes in the organization of the 1,3-ß-glucan and chitin layers of the cell wall. Quantazyme is a recombinant 1,3-ß-glucanase (Quantum Biotechnology). The Quantazyme sensitivity assay was conducted as described by Ovalle et al. (1998) . Briefly, cells in exponential phase (OD600=12) were resuspended in 40 mM ß-mercaptoethanol, 50 mM Tris/HCl, pH=7·5, to an OD600 of 1. After incubation for 1 h, Quantazyme dissolved in glycerol/water (1:1, v/v), was added to a final concentration of 400 U ml-1. Cell lysis was determined by following the decrease in turbidity at 600 nm over time. Quantazyme sensitivity was expressed as a percentage of the initial OD600. Streptomyces griseus chitinase (Sigma; C1525) was used in some experiments at 0·2 U ml-1 for 1 h before Quantazyme addition.
SDS hypersensitivity tests.
Exponential cultures were harvested and adjusted to an OD600 of 1; 5 µl of tenfold serial dilutions were spotted on YPD containing increasing amount of SDS: 0·01, 0·05 and 0·1%. Plates were read after 24 h culture at 30 °C for S. cerevisiae and 37 °C for C. albicans.
Isolation of cell-wall proteins (CWPs).
For these experiments, cultures were grown in SC medium at 30 °C, pH 5·5, for S. cerevisiae strains, and at 37 °C, pH 4·5, for C. albicans strains, since faint differences could only be detected at higher pH in the CWP content of the wild-type and the gpi7 mutants of C. albicans. After 16 h culture in SC, cells were harvested in exponential phase (OD600=2) and washed with 10 mM Tris/HCl, pH 7·6, as described previously (Kapteyn et al., 1995 ; Montijn et al., 1994
). Briefly, cell walls were isolated and washed extensively with 1 M NaCl. Isolated cell walls were boiled twice in the presence of SDS, EDTA and ß-mercaptoethanol to solubilize the non-covalently linked CWPs and to remove any contaminant derived from the cytosol and/or plasma membrane. SDS-extracted cell walls were freeze-dried after washing and 3 mg of the pellets were treated either with recombinant Trichoderma harzianum endo-1,6-ß-glucanase (0·8 U g-1) to release the GPI-CWPs (Kapteyn et al., 1996
) or with 30 mM ice-cold NaOH for 17 h to release the 1,6-ß-glucanase-resistant Pir-like-CWPs. Subsequently, 1,6-ß-glucanase-treated cell walls were treated with ice-cold 30 mM NaOH for 17 h to release the double linked CWPs. Each enzymic digestion was stopped by adding SDS at a final concentration of 0·4% and heating for 5 min at 100 °C. Prior to heating in SDS, NaOH-treated cell walls were first neutralized by adding acetic acid.
Analysis of CWPs.
CWPs were separated by electrophoresis using linear 2·620% polyacrylamide gels and transferred onto Immobilon-P polyvinylidene difluoride (PVDF, 0·45 µm) membrane (Millipore) using a wet blotting apparatus. CWPs were visualized by probing the membrane with peroxidase-labelled Concanavalin A (ConA; 0·3 µg ml-1) in PBS containing 3% bovine serum albumin, 2·5 mM CaCl2 and 2·5 mM MnCl2 (Kapteyn et al., 2000 ). Western immunoblot analyses were performed with polyclonal antisera directed against 1,6-ß-glucan, agglutinin-like sequence (Als) proteins, Cwp1p, Pir2p, Ssr1p and Gas1p. For the antisera directed against Als proteins, Cwp1p, Pir2p, Ssr1p and Gas1p, the membranes were treated with 50 mM periodic acid and 100 mM sodium acetate for 30 min before the blocking step with 5% milk powder in PBS to enhance the specificity of the antisera. Binding of rabbit antibodies was assessed with goat anti-rabbit IgG peroxidase (Pierce) at a dilution of 1:10000 in PBS/5% milk powder. The blots were visualized with ECL Western blotting detection reagents (Amersham Pharmacia Biotech) and Fuji Medical X-Ray film.
Determination of glucose, mannose and glucosamine content in cell walls.
Isolated cell walls (about 11·5 mg) were hydrolysed as described previously with minor modifications (Dallies et al., 1998 ). Briefly, cell walls were hydrolysed with 50 µl 72% sulfuric acid for 3 h at 25 °C. Then 650 µl distilled water was added to obtain a 1 M H2SO4 solution, followed by incubation at 100 °C for 3 h. The solution was diluted by adding 3 ml distilled water and precipitation of sulfate was carried out using 3·3 ml 40 g barium hydroxide l-1. After centrifugation, the sugars were quantified using a Dionex Bio-LC system with a CarboPac PA 1 anion-exchange column. In this experiment 5 mM galactose was used as internal standard.
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RESULTS |
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Composition of the mutant cell wall is modified
To better characterize the cell-wall modification in the mutants, cell-wall preparations were hydrolysed with sulfuric acid and the released sugars were separated and quantified by high performance anion-exchange chromatography. The following results represent the mean of two independent experiments and are expressed as a percentage of the respective wild-type levels (standard error on these measurements is less than 5%). The sugar composition of the C. albicans Cagpi7 null mutant cell wall was 70, 102 and 412% for mannose, glucose and glucosamine, respectively, and 86, 116 and 261%, respectively, for the S. cerevisiae gpi7 null mutant. The Cagpi7 null mutants showed a fourfold increase in the chitin level, suggesting compensation of a cell-wall defect by chitin deposition, as often observed in cell-wall-defective mutants of S. cerevisiae (Dallies et al., 1998 ) and C. albicans (Kapteyn et al., 2000
). Moreover, the mannose content of the cell wall decreased by 30%, indicating a defect in protein mannosylation or a decrease of mannoproteins in the mutant cell wall compared to the wild-type. Similar results were observed on cell walls of a gpi7 mutant of S. cerevisiae, with a greater than twofold increase in chitin content, although the mannose level was marginally affected. Overall, disruption of GPI7 seems to modify the cell wall of both organisms in a qualitatively similar way, resulting in a decrease of mannoproteins, which in turn is compensated by increased chitin deposition.
We reasoned that the excess of chitin in mutant cell walls might protect cells against lytic enzymes. Indeed, we found a higher resistance of the mutant cells of C. albicans and S. cerevisiae to Quantazyme, a recombinant 1,3-ß-glucanase (data not shown). Furthermore, we checked whether a chitinase pre-treatment of the mutant may restore Quantazyme susceptibility. Although a 1 h treatment with chitinase alone had no effect on cell lysis under our experimental conditions, it induced a significant increase in mutant cell lysis, comparable to wild-type, during a further Quantazyme treatment in both C. albicans and S. cerevisiae (data not shown).
Fewer proteins are linked to the cell wall in the mutant cells
To investigate whether fewer mannoproteins were indeed present in the cell wall, as suggested by previous data on cell-wall composition, we analysed different fractions from hot SDS cell-wall extracts of C. albicans and S. cerevisiae mutant and wild-type cells grown in SC medium in the yeast form. Indeed, in both S. cerevisiae and C. albicans two types of CWPs have been identified that are covalently linked to ß-glucan, the GPI-dependent CWPs (GPI-CWPs) and the Pir-CWPs (Fig. 3). In S. cerevisiae, Pir-CWPs are cross-linked to cell-wall 1,3-ß-glucan by a covalent link sensitive to mild alkali treatments (see Methods). GPI-CWPs are cross-linked by a 1,6-ß-glucan to 1,3-ß-glucan (and to a lesser extent to chitin), or by an alkali-sensitive bond to 1,3-ß-glucan (Pir-CWPs), or by both types of bond (Kapteyn et al., 1999
). A recent publication demonstrated that the general organization of the C. albicans cell wall is very similar to that of S. cerevisiae (Kapteyn et al., 2000
). To characterize the effect of CaGPI7 deletion on CWPs, we compared the fractions obtained after 1,6-ß-glucanase digestion, mild alkali treatment, or mild alkali treatment after 1,6-ß-glucanase digestion. Lectin-blotting of wild-type C. albicans 1,6-ß-glucanase digests, using peroxidase-linked ConA, revealed a large smear and three mannosylated protein bands with apparent molecular masses of 220, 180 and 100 kDa, respectively (Fig. 4
). As expected, anti-1,6-ß-glucan antiserum did not recognize any of this material, indicating that the 1,6-ß-glucanase did not leave any detectable 1,6-ß-glucan epitope attached to the proteins (data not shown). Cagpi7 mutants exhibited a marked reduction of ConA stained material in this test, suggesting that fewer proteins were linked to the cell wall in the mutant (Fig. 4
).
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CWPs are released into the medium of gpi7 disrupted strains
The above results show that mutant cell walls generally contain less GPI-CWP than the wild-type, suggesting that these proteins are either repressed, degraded or mislocalized. Preliminary results (not shown) indicate that transcription of eight ALS genes tested was not modified in the mutant. We thus checked whether GPI-CWPs were released into the medium. Culture supernatants of the C. albicans strains were concentrated by TCA precipitation and analysed by Western blotting. Anti-Als protein and anti-Pir protein antisera revealed increased levels of putative CWPs in the culture medium of the mutant (Fig. 5a). These results indicate that these proteins are not normally linked to the cell wall in the Cagpi7 null mutant.
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Taken together, these data demonstrate that disruption of GPI7 in C. albicans or in S. cerevisiae results in a defective cell-wall linkage of GPI-CWPs and Pir-CWPs.
GPI membrane proteins are not affected by the GPI7 deletion
We then wanted to test whether the GPI7 deletion specifically disrupted localization of GPI-CWPs, or whether it also affected proteins anchored by GPI to the cell membrane (GPI-MP). We followed the behaviour of one GPI-MP in each organism.
In S. cerevisiae we chose Gas1p, a GPI-modified 1,3-ß-glucanosyltransferase, predominantly anchored to the plasma membrane with only traces in the cell wall (Popolo et al., 1993 ). SDS washes of cell-wall preparations (see Methods), known to be heavily contaminated by cell membranes (Schreuder et al., 1993
), were used as a non-purified plasma membrane fraction. This crude fraction, as well as culture supernatants from S. cerevisiae mutant and wild-type cells, were probed with an anti-Gas1p antiserum (Fig. 6a
). No signal was detected in the culture supernatants from wild-type and mutant strains. In contrast, a protein band was present in the membrane fraction of both strains at the expected size of 125 kDa and in nearly identical amounts. These data suggest normal anchorage of at least one GPI-MP to the cell surface in the S. cerevisiae gpi7 mutant.
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DISCUSSION |
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This defect in cell-wall structure correlates with a lower protein content of the cell wall in C. albicans and an increased release of CWPs in the growth medium of the mutant. Moreover, mutations in GPI7 do not affect localization of the cell membrane-attached GPI protein Gas1p.
The lower levels of GPI-dependent CWPs can be explained in different ways. First, the lack of EtN-P on the second mannose of the GPI core structure might affect the transport of GPI-dependent CWPs through the secretory pathway. This does not seem to be the case since the growth rate and the quantity of Gas1p in the plasma membrane were not affected. Second, the lack of EtN-P might block the release of the plasma-membrane-bound intermediate form of GPI proteins destined for the cell wall. This seems unlikely because in the mutant more soluble forms were found in the culture medium. Third, this side-chain might be essential for efficient linkage of GPI-CWPs to other cell-wall components. Thus, the lack of this side-chain may trigger mislocalization of GPI-CWPs, including some proteins involved in cell-wall synthesis and in the linkage of Pir proteins. Consequently, a defective cell wall is formed, the cell aggregates, morphogenesis is affected and virulence and the resistance to macrophages are also affected (Richard et al., 2002 ).
Finally, although gpi7 mutants of S. cerevisiae and C. albicans share common phenotypes, like secretion of GPI-CWPs, resistance to Quantazyme and increased chitin deposition, they also differ. In particular, no SDS hypersensitivity was detected in S. cerevisiae mutants and the amount of proteins in the cell wall was negligibly affected. Possibly, these phenotypic differences are due to the different growth temperatures used for S. cerevisiae (30 °C) and C. albicans (37 °C).
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ACKNOWLEDGEMENTS |
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Received 4 December 2001;
revised 6 February 2002;
accepted 4 March 2002.