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

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.

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.

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.

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.