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The Cell Wall of the Human Pathogen Candida glabrata: Differential Incorporation of Novel Adhesin-Like Wall Proteins

Piet W. J. de Groot, Eefje A. Kraneveld, Qing Yuan Yin, Henk L. Dekker, Uwe Groß, Wim Crielaard, Chris G. de Koster, Oliver Bader, Frans M. Klis, Michael Weig
Piet W. J. de Groot
1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
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Eefje A. Kraneveld
2Department of Molecular Biology and Preventive Dentistry, Academic Center for Dentistry Amsterdam, Amsterdam, The Netherlands
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Qing Yuan Yin
1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
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Henk L. Dekker
1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
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Uwe Groß
3Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Medical Center Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany
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Wim Crielaard
2Department of Molecular Biology and Preventive Dentistry, Academic Center for Dentistry Amsterdam, Amsterdam, The Netherlands
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Chris G. de Koster
1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
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Oliver Bader
3Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Medical Center Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany
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Frans M. Klis
1Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
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Michael Weig
3Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Medical Center Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany
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  • For correspondence: mweig@gwdg.de
DOI: 10.1128/EC.00284-08
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  • FIG. 1.
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    FIG. 1.

    The cell wall of C. glabrata has a bilayered structure. (A) The budding process and cytokinesis in C. glabrata visualized by transmission electron microscopy. I, formation of a new bud; II, isotropic bud growth and chitin deposition at the inner part of the wall in the mother/bud neck; III and IV, septum formation and cell separation; V, reinforcement of the bud scar; VI, formation of a new bud proximal to a previous budding event. (B) Close-up of a lateral wall fragment. Based on extrapolation using data from the well-studied yeast S. cerevisiae, the electron-dense outer layer of the cell wall is expected to predominantly consist of GPI-modified proteins (GPI-CWPs), which are covalently bound to 1,6-β-glucan, whereas the more transparent inner layer consists of a glycan network interspersed with ASL-CWPs or at least Pir proteins. This view is fully consistent with the data presented in this paper. CW, cell wall; EC, extracellular environment; PM, plasma membrane; C, cytosol.

  • FIG. 2.
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    FIG. 2.

    CWP-polysaccharide complexes in the cell wall of C. glabrata. (A and B) SDS-PAGE analysis of cell wall material extracted as indicated from isolated walls of C. glabrata grown in SC medium. Gels were analyzed by silver staining or by blotting and probing with the lectin ConA or anti-ScCwp1 antiserum (A) or with anti-1,6-β-glucan antiserum (B). (C) Identified cross-links between proteins and β-glucans in the cell wall of C. glabrata. The extraction methods used to solubilize CWPs are indicated. (D) Cell wall proteins identified in mild-alkali extracts by LC/MS/MS.

  • FIG. 3.
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    FIG. 3.

    Genomic organization for adhesin-like proteins encoded in the genome of C. glabrata ATCC 2001. GPI-modified adhesin-like proteins were primarily identified by a genome-wide in silico analysis, as described previously (29). Additional adhesin-like proteins were found by pattern searching using the conserved VSHITT motif, by BLAST analysis, and by analysis of telomeric regions, where most of the adhesin-like proteins are located. Chromosomes and ORFs are numbered following Génolevures' systematic ORF numbering. Adjacent ORF fragments belonging to a single gene, as also indicated by the NCBI genome browser, are connected. Unannotated ORF fragments identified by BLASTX and containing N- or C-terminal signal peptides were connected to CAGL0B00110g, CAGL0B05115g, and Epa11. ORF sizes are to scale, but distances between ORFs are not. Colors indicate subfamilies I to VII, sharing homology in the N-terminal putative ligand-binding parts, as presented in Fig. 4. CAGL0L09911g and CAGL0J05170g (white) are unrelated outgroups in Fig. 4. Numbers of proteins in each subgroup are indicated. For CAGL0H00110g (group VII, orange) and CAGL0E00187g (group IV, pink), only C-terminal parts of the proteins were identified; their classification is therefore based on BLASTP analysis of these regions. Numbers of nonadhesive ORFs separating adhesive-like proteins and telomeres and distances of terminal adhesive-like proteins to telomeres are indicated. Arrows indicate directions of transcription.

  • FIG. 4.
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    FIG. 4.

    Multiple subfamilies of adhesin-like proteins exist in C. glabrata. A neighbor-joining phylogenetic tree, with bootstrap values added (1,000 bootstraps performed), of adhesin-like wall proteins based on the putative functional domains (the 300 N-terminal amino acids or fewer in cases where the N-terminal ORF fragment is shorter) of the ORFs is shown. S/T-rich low-complexity regions within the first 300 amino acids of CAGL0C00209g and CAGL0G10175 were excluded from this analysis. CAGL0L09911g, which together with CAGL0J05170g is least related to other adhesin-like proteins, is plotted as an outgroup. The distantly related S. cerevisiae Flo1 and Dan1 and C. albicans Hyr1 are included for comparison. CAGL0H00110g (group VII) and CAGL0E00187g (group IV) were excluded from this analysis since their N-terminal parts are unidentified; see Fig. 3. The scale bar indicates phylogenetic distances, in number of amino acid substitutions per position.

  • FIG. 5.
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    FIG. 5.

    Strain- and growth phase-dependent cell surface hydrophobicity and in vitro adhesion. (A) In vitro adhesion of ATCC 2001 and ATCC 90876 was analyzed by growing cells in 25-μl spots on polystyrene plates for 3 days at 30°C. Nonadherent cells were washed away with water, and remaining adherent cells were stained with crystal violet. (B) Surface hydrophobicity of exponentially growing or stationary-phase cells of C. glabrata strains ATCC 2001 and ATCC 90876 was measured using a two-phase assay. Surface hydrophobicity can be inferred by subtraction of the fraction of cells measured in the aqueous phase. Significant differences (P values of <0.05, Student's t test) are indicated with asterisks.

  • FIG. 6.
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    FIG. 6.

    Awp2 and Awp4 have adhesin-specific intragenic tandem repeats. Multiple alignment of tandem repeats present in both Awp2 and Awp4 is shown. Awp2 is encoded by CAGL0K00110g. Awp4 is dispersed into five ORF fragments, CAGL0J11990g, CAGL0J12001g, CAGL0J12023g, CAGL0J12045g, and CAGL0J12056g. The two N-terminal parts (not shown) were identified by LC/MS/MS. The three C-terminal ORF fragments contain (parts of) repeat units. Numbers indicate the aligned regions of the ORFs (fragments).

Tables

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  • TABLE 1.

    Cell wall composition of C. glabrata in comparison to those of S. cerevisiae and C. albicans

    SpeciesCell wall contenta
    Protein (%)Chitin (%)Man (%)Glu (%)M/GAlkali-insoluble glucan (%)b
    1,6-β1,3-β
    C. glabrata 6.4 ± 0.11.2 ± 0.143.8 ± 0.554.0 ± 0.20.814.2 ± 0.116.7 ± 1.7
    S. cerevisiae 4.0 ± 0.11.4 ± 0.234.2 ± 1.660.3 ± 2.50.577.1 ± 0.226.8 ± 0.9
    C. albicans 3.5 ± 0.24.2 ± 0.126.6 ± 2.364.0 ± 4.90.4210.6 ± 0.626.2 ± 1.1
    • ↵ a Values are from exponentially growing cells (in YEPD), are the means and standard deviations for two independent samples measured in duplicate, and are expressed as percentages of freeze-dried cell walls. Man, mannose; Glu, glucose; M/G, ratio of mannose to glucose.

    • ↵ b Enzymatically released with 1,6-β-glucanase or 1,3-β-glucanase.

  • TABLE 2.

    Covalently bound C. glabrata CWPs identified by LC/MS/MSa

    Category and protein nameORF no.Properties and proposed functionbMS/MS resultConserved functional domain(s)dClosest S. cerevisiae homolog (SGD namee)Closest C. albicans homolog (CGD namee)Reference for C. glabrata protein name
    Sequence coverage (%)No. of peptides identifiedc
    Carbohydrate-active enzymes
        Crh1CAGL0G09449gSP, GPI, 452 aa. GH16 transglycosidase, involved in chitin incorporation2211GH16: 34-240Crh1fCrh11f 67
        Utr2CAGL0C02211gSP, GPI, 481 aa. GH16 transglycosidase, involved in chitin incorporation62CBM 18: 23-64; GH16: 93-303Utr2fUtr2f 67
        Gas1CAGL0G00286gSP, GPI, 559 aa. GH72 transglycosidase, elongation of 1,3-β-glucan81 (+ 2)GH72: 25-329; X8: 377-459Gas1fPhr2,f Phr1f 66
        Gas2CAGL0M13849gSP, GPI, 565 aa. GH72 transglycosidase, elongation of 1,3-β-glucan102 (+ 2)GH72: 26-330; X8: 378-460Gas1fPhr2,f Phr1f 66
        Gas4CAGL0F03883gSP, GPI, 480 aa. GH72 transglycosidase, elongation of 1,3-β-glucan123GH 2: 20-350Gas3fPhr3, Pga4f
        Gas5CAGL0F01287gSP, GPI, 523 aa. GH72 transglycosidase, elongation of 1,3-β-glucan2410GH72: 27-331Gas5fPga4f
        Scw4CAGL0G00308gSP, no GPI, 374 aa. GH17 transglycosidase, modification of 1,3-β-glucan288GH17: 118-372Scw4fMP65/Scw1f
    Other enzymatic activity
        Plb2CAGL0J11748gSP, GPI, 695 aa. phospholipase106PLAc: 33-550Plb2fPlb3
    Nonenzymatic CWPs
        Cwp1.1CAGL0F07601gSP, GPI, 218 aa. structural mannoprotein5313 (+ 26)NoneCwp1fNone 67
        Cwp1.2CAGL0F07579gSP, GPI, 212 aa. structural mannoprotein554 (+ 26)NoneCwp1fnone 67
        Ssr1CAGL0H06413gSP, GPI, 212 aa. contains CFEM domain276CFEM: 22-81Ccw14fSsr1f
        Tir1CAGL0F01463gSP, GPI, 221 aa. mannoprotein of the Srp1p/Tip1p family122NoneTir1fNone
        Pir1CAGL0I06204gSP, no GPI, 349 aa. conserved 4-cysteine domain211 (+ 9)4× Cys: 252-349Pir1-4fPir1f 67
        Pir2CAGL0I06182gSP, no GPI, 340 aa. conserved 4-cysteine domain262 (+ 10)4× Cys: 243-340Pir1-4fPir1f 67
        Pir3CAGL0M08492gSP, no GPI, 335 aa. conserved 4-cysteine domain2011 (+ 2)4× Cys: 238-335Pir1-4fPir1f 67
        Pir4CAGL0I06160gSP, no GPI, 233 aa. conserved 4-cysteine domain43114× Cys: 136-233Pir1-4fPir1f 67
    Unknown proteins
        Ecm33CAGL0M01826gSP, GPI, 421 aa. unknown role in cell wall biosynthesis187UnknownEcm33f, Pst1fEcm33f, Ecm331
        Pst1CAGL0E04620gSP, GPI, 429 aa. unknown role in cell wall biosynthesis63UnknownEcm33f, Pst1fEcm33f, Ecm331
    Adhesin-like wall proteinsh
        Awp1CAGL0J02508gSP, GPI, 870 aa. putative adhesin64UnknownAwa1, Hpf1, Hpf1′None
        Awp2CAGL0K00110gSP, GPI, 832 aa. putative adhesin85UnknownAwa1, Hpf1, Hpf1′Iff family, Hyr1
        Awp3CAGL0J11902g- CAGL0J11924gSP, GPI, unknown size putative adhesin6g2UnknownNoneNone
        Awp4CAGL0J11990-CAGL0J12056gSP, GPI, unknown size putative adhesin11 + 13g2 (+ 3)UnknownNoneIff family, Hyr1
        Epa6CAGL0C00110gSP, GPI, 715 aa. adhesin155 (+ 1)PA14: 139-247FlocculinsNone 4
    • ↵ a For mass spectrometric details, see Table S1 in the supplemental material.

    • ↵ b Predicted signal peptides for secretion (SP) (http://www.cbs.dtu.dk/services/SignalP/ ) and C-terminal signatures for GPI anchoring (http://mendel.imp.ac.at/gpi/fungi_server.html ) are indicated; see also reference 67. Conserved functional domains were identified using CDD v2.03 (http://www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml ), CAZy (http://www.cazy.org/ ), and reference 15. GH, glycoside hydrolase; aa, amino acids.

    • ↵ c Numbers in parentheses indicate nonunique peptides.

    • ↵ d Identified as outlined in footnote b.

    • ↵ e SGD, Saccharomyces Genome Database (http://www.yeastgenome.org/ ); CGD, Candida Genome Database (http://www.candidagenome.org/ ).

    • ↵ f Identified as covalently bound cell wall protein using LC/MS/MS (13, 58, 70, 72).

    • ↵ g Sequence coverage of identified ORF fragments.

    • ↵ h Not identified in ATCC 90876 cells that were grown in YEPD to mid-log phase.

  • TABLE 3.

    The genome of C. glabrata strain ATCC 2001 harbors 67 genes putatively encoding adhesin-like proteins

    SubgroupaNo. of proteinsNo. of proteins with subtelomeric localizationPresence of PA14 domainNo. of proteins with Awp2 repeatsNo. of proteins present in NCBI protein databaseb
    I, Epa family (blue)1714Yes411
    II, Pwp family (green)71Yes14
    III (yellow)1311No115
    IV (pink)33No03
    V, includes Awp2 and -4 (red)1312No84
    VI, includes Awp1 and -3 (purple)71No15
    VII (orange)52No44
    Others (white)20No21
    Total67443137
    • ↵ a Colors corresponding to Fig. 3 are given in parentheses.

    • ↵ b As of 21 August 2008.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - LC/MS/MS identification of covalently linked cell wall proteins in C. glabrata.
      PDF document, 65.7K.
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The Cell Wall of the Human Pathogen Candida glabrata: Differential Incorporation of Novel Adhesin-Like Wall Proteins
Piet W. J. de Groot, Eefje A. Kraneveld, Qing Yuan Yin, Henk L. Dekker, Uwe Groß, Wim Crielaard, Chris G. de Koster, Oliver Bader, Frans M. Klis, Michael Weig
Eukaryotic Cell Nov 2008, 7 (11) 1951-1964; DOI: 10.1128/EC.00284-08

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The Cell Wall of the Human Pathogen Candida glabrata: Differential Incorporation of Novel Adhesin-Like Wall Proteins
Piet W. J. de Groot, Eefje A. Kraneveld, Qing Yuan Yin, Henk L. Dekker, Uwe Groß, Wim Crielaard, Chris G. de Koster, Oliver Bader, Frans M. Klis, Michael Weig
Eukaryotic Cell Nov 2008, 7 (11) 1951-1964; DOI: 10.1128/EC.00284-08
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KEYWORDS

Candida glabrata
Candidiasis
Cell Adhesion Molecules
Cell Wall
Fungal Proteins

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