Article Figures & Data
Figures
- Fig. 1.
Cell wall structure. (A) Transmission electron micrograph of a section of a C. albicans cell prepared by freeze-substitution, showing the cell wall as a thick, electron-dense, homogeneous structure. The presence of distinct layers was not evident in this preparation. Bar, 1 μm. Reprinted from reference3 with permission of the publisher. (B) Thin sections of cells treated with gold-conjugated concanavalin A, showing an intense labeling with gold particles of the external wall surface. The surface exhibits a fibrillar appearance (arrows), suggesting that concanavalin A-reactive cell wall components, i.e., mannoproteins, are particularly abundant at the most external wall layers. The remaining wall structure also appeared as a homogenous structure in this transmission electron micrograph. Bar, 0.5 μm. Reprinted from reference 549 with permission of the American Society for Microbiology. (C) Other procedures for transmission electron microscopy examination of thin sections of C. albicans cells revealed more clearly the presence of an outer floccular layer (arrow) and showed that the remaining cell wall structure is not homogeneous and that some layering exists. Bar, 200 nm. Reprinted from reference 240 with permission of the publisher. (D to F) Complexity of the wall ultrastructure and presence of distinct layers in the cell wall of C. albicansas revealed by different scanning electron microscopy-based procedures such as cryo-scanning electron microscopy (D) and freeze-fracture, freeze-etch analysis (E and F). The presence of well-ordered, regularly arranged, radiating fibrils in the outer layer is particularly evident in the micrographs shown in panels E (hydrophilic cells) and F (hydrophobic cells). Bar, 0.3 μm in both panels. Panel D reprinted from reference 246 with permission of the publisher. Panels E and F reprinted from reference 191 with permission of the publisher.
- Fig. 2.
Polypeptide composition of cell wall extracts and whole protoplast homogenates from C. albicans as revealed by different electrophoretic techniques (A and B). C. albicanscells from which samples were obtained were incubated in the presence of 14C-labelled protein hydrolysate and subsequently tagged with biotin. Double-labelled cell wall proteins and glycoproteins were extracted from intact blastoconidia (lanes 1 and 3) and germinated blastoconidia (lanes 2 and 4) by sequential treatment with βME (lanes 1 and 2) and digestion with Zymolyase 20T (lanes 3 and 4). Samples of protoplast homogenates from blastoconidia and germinated blastoconidia were run in lanes 5 and 6, respectively. Polypeptides were separated by SDS-PAGE and detected by fluorography (A) or transferred to nitrocellulose and detected with an avidin-peroxidase conjugate (B; lane S in this panel shows a mixture of prestained molecular weight standards run in parallel). Numbers and letters are used to identify and compare bands detected in the cell wall extracts by the different experimental procedures used. Although qualitative differences were observed (i.e., some polypeptides exhibited a strong radioactive label but were weakly biotinylated [band 6; star]), surface labelling of cells with biotin appeared to be a suitable technique to detect proteins in the wall of C. albicans. Thus, from the complex polypeptide pattern found in the protoplast homogenate samples as revealed by fluorography (A, lanes 5 and 6), only few species were labelled with biotin, indicating that most proteins released by βME and Zymolyase from intact cells are bona fide cell wall components. Brackets indicate a cluster of bands within a molecular weight range where many candidal moieties that represent receptors for host ligands have been identified (see the text). Reprinted from reference61 with permission of the American Society for Microbiology. (C) The complexity of the polypeptide pattern of the cell wall extracts was clearly evidenced when analysis was performed by two-dimensional PAGE and silver staining (the polypeptide pattern shown corresponds to the βME extract from blastoconidia). Reprinted from reference 486 with permission of the American Society for Microbiology.
- Fig. 3.
Surface expression of cell wall proteins. Phase-contrast (A and C) and immunofluorescence (B, D, and E to I) of C. albicans blastoconidia (B) and mycelial filaments (M) reacted with different polyclonal and monoclonal antibody preparations raised to protein and glycoprotein cell wall constituents. Some antibodies recognized antigens that appeared to be specific for or preferentially expressed in germ tubes (A and D) or blastoconidia (E and F). Arrows in panels A to D point to the location of mother blastoconidia (A and C) that exhibited no fluorescence (B and D). Some antigens appeared to be homogenously distributed on the surface of mycelial filaments (B and D) or blastoconidia (G). However, patches of greater fluorescence intensity were observed with other antisera preparations (H and I), suggesting that antigens recognized by such antisera were heterogenously and randomly distributed within the cell wall structure. The pictures in panels B and D to G are from standard immunofluorescence microscopy observations. Panels H and I show single-focal-plane sections of different cells obtained by confocal fluorescence microscopy and associated software. Bar, 10 μm (except for panel G, which is 1.25 μm). Panels A to D reprinted from reference 59 with permission of the American Society for Microbiology. Panels E and F reprinted from reference24 with permission of the American Society for Microbiology. Panel G reprinted from reference 72with permission of the American Society for Microbiology. Panels H and I reprinted from reference 328 with permission of the American Society for Microbiology.
- Fig. 4.
Schematic diagram of the cell wall (CW) structure ofC. albicans, showing the presence of different layers enriched in particular components. The microfibrillar polymers of β-glucans (β-g, ) and chitin (c,
) appear to be more heavily concentrated in the inner cell wall domains; β-glucan–chitin complexes that appear to be formed by glycosidic linkages between both polymers will be located adjacent to the plasma membrane (PM) and the periplasmic space (ps). Proteins and glyco(manno)proteins (gp) appear to be dominant in the outermost cell wall layer, although they are also distributed through the entire wall structure. Once secreted through the plasma membrane, some protein and glycoproteins species will remain at the periplasmic space, possibly playing enzymatic roles (▵); some others will establish functional (i.e., β-glucanases [▾]) or structural covalent associations with β-glucans and possibly also with chitin (•—•) adjacent to the plasma membrane; and, finally, other moieties will constitute the most external layer, where the different molecular entities may be homogeneously (□) or heterogeneously (fimbriae, cluster of receptor-like molecules, etc. [○]) distributed or specifically released (i.e., extracellular enzymes) to the extracellular medium (EM) (•, ▪). Proteins and glycoprotein species in the outermost wall layer (□, ○) may establish different types of covalent (disulfide linkages) and noncovalent (hydrophobic and hydrogen ionic bonds) interactions. During their passage through the wall from the plasma membrane and periplasmic space to the outermost cell wall layers (□, ○) and possibly the extracellular environment (•, ▪), proteins and glycoproteins are most likely to be in equilibrium with other proteinaceous constituents, thus contributing, at least from a functional point of view, to the cell wall layering. In any case, protein and glycoprotein species other than those specifically secreted to the exocellular medium may also be released to such locations by dying (lysed) cells or as a consequence of unbalanced processes of synthesis and degradation of the cell wall structure, required for wall expansion during cell growth. To simplify the scheme, some aspects such as possible interactions of cell wall components with the plasma membrane and proteins retained in the cell wall, apparently by either covalent or noncovalent linkages, are not depicted.
Tables
- Table 1.
Hydrolytic enzymes and proteins with cell wall and extracellular targets
Enzyme Gene Location Comments Reference(s) Cell wall substrates Exo-β-(1,3)-glucanase EXG(XOG1) Cell wall, extracellular Cell wall morphogenesis 75, 307, 308, 361, 387, 428, 429 β-1,3-glucan transferase BGL2 Cell wall Cell wall metabolism 122, 178, 196, 466, 471 Chitinase CHT1-3 Periplasm, cell wall, extracellular Hydrolytic enzyme, cell wall morphogenesis 141, 170, 346, 347 β-N-acetylglucosaminidase HEX1 Periplasmic, extracellular Hydrolytic enzyme, virulence factor? 55,221, 365, 426, 519 Transglutaminase Cell wall Covalent cross-links? 456 Extracellular substrates Secreted aspartyl proteinase SAP1-9 Extracellular, cell surface Putative virulence factor, gene expression condition dependent 1, 3, 18, 31, 84, 99-101, 109, 147, 157, 205,206, 214, 215, 232, 233, 277, 348-350, 353, 359, 370, 372, 378, 379,381, 397, 433-435, 441, 445-448, 450, 451, 509-511, 514, 515, 552,570, 574 Phospholipase Phospholipase A Cell wall, surface, extracellular Hydrolytic enzyme 23, 174, 425, 427 Phospholipase B PLB1 Extracellular Hydrolytic enzyme, putative virulence factor 19, 217, 473, 533 Phospholipase C Extracellular Hydrolytic enzyme 425,427 Lysophospholipase Cell wall, surface, extracellular Hydrolytic enzyme 19, 23, 174, 355, 425,427, 533 Lysophospholipase-transacylase Extracellular Hydrolytic enzyme, putative virulence factor 19, 217, 533,534 Esterase Extracellular Hydrolytic enzyme 56, 419, 449, 551 Glucoamylase Extracellular Hydrolytic enzyme 82 Hemolytic factor Cell wall, extracellular Hydrolytic enzyme 317 Acid phosphatase Periplasmic, surface Hydrolytic enzyme 78, 105, 391, 547 Lipase LIP1 Extracellular Hydrolytic enzyme 148 Hyaluronidase Extracellular Hydrolytic enzyme, virulence factor? 499, 500 Chondroitan sulfatase Extracellular Hydrolytic enzyme, virulence factor? 499, 500 Metallopeptidase Cell wall, extracellular? Hydrolytic enzyme 120, 121 Trehalase Cell wall, extracellular Hydrolytic enzyme 362,454 - Table 2.
Morphology-associated and other cell wall proteins
Protein Gene Location Commentsa Reference(s) Morphology associated 4C12 antigen (180 kDa, 260 kDa) Hyphae Structural protein? 59, 62, 122, 128, 160, 327 3D9 antigen (110–170 kDa) Hyphae Structural protein? 324, 325 DC3:H10 antigen Hyphae Present in complex with other proteins 87, 300 Hwp1p (34 kDa) HWP1 Hyphae N-terminal signal sequence, tandem repeats, C terminus ST rich 513 Hyr1p (deduced 94 kDa) HYR1 Hyphae N-terminal signal sequence, C terminus ST rich, GPI anchor sequence 16 Cell wall protein (30 kDa) Hyphal cell wall, yeast? Probable posttranslation regulation of location 5 MAb determinants 106 Yeast cell (68, 82, 96, 104 kDa) Yeast cells and early germ tubes Proteins have one or two common epitopes Hyphae (104, 117 kDa) Germ tubes and hyphal tip Proteins have one or two common epitopes Other Hsp70 (70 kDa) SSA1 (HSP70),SSA2 Yeast cells, hyphae Chaperone?, translocation?; immunogenic, Ab protective? 278, 294, 303 Hsp90 47-kDa fragment HSP90 Yeast cells, hyphae? hsp90 degradation product, immunogenic, Ab protective 331, 334, 335, 337-340, 342, 532 Enolase (48 kDa) ENO1-2 Yeast cells, hyphae Binds to glucan, internal cell wall, lacks N-terminal signal sequence, N terminus blocked, immunogenic, antigen and Abs may be diagnostic, Ab protective? 8, 143, 177, 219, 221, 330, 356, 420, 520-522,556, 558, 564 Phosphoglycerate kinase (40 kDa) PGK1 Yeast cells, hyphae Lacks N-terminal signal sequence, enzymatic activity? immunogenic 6 GAPDH (33 kDa) TDH1 Yeast cells, hyphae Enzymatic activity, immunogenic 161 ADH ADH1? Cell wall? Fibronectin binding? immunogenic 27, 406, 489 Als1p ALS1 Cell wall? N-terminal signal sequence, variable tandem repeat, GPI-anchor sequence, member family 212, 473 ↵a Abbreviations: S, serine; T, threonine; Ab, antibody.
- Table 3.
Adhesins and binding proteins
Adhesin-ligand interaction Gene Location Comments Reference(s) Protein-protein CR2 (C3d binding proteins) 194, 395,563 60, 50 kDa Hyphal surface (60 kDa), yeast cell membrane? (50 kDa) Isolated whole cell hyphal extracts; binding species differ in glycosylation 52, 60, 235, 289,563 55–60 kDa (minor 67–68, 20 kDa) Hyphae Hyphal extract, Western blot with antiserum to CR2 235 66, 40, 20 kDa Yeast cells, hyphae Western blot of cell wall extract with antiserum to CR2; 40 kDa ubiquitinated 300, 486 94, 68, 60, 50, 31 kDa Hyphae Western blot of cell wall extract with antiserum to CR2 145 iC3b binding proteins Yeast cells, hyphae Epithelial, endothelial cell adhesin 25, 26, 115, 146, 162, 176, 194 130 kDa (also 100, 50 kDa) Hyphae Immunoprecipitation of surface proteins by MAb OKM-1 117 165 kDa INT1? Yeast cells Western blot of yeast cell cytosol, membrane, but not cell wall extracts with MAb OKM-1 153, 210 66, 55, 42 kDa Hyphae C3 affinity chromatography of soluble hyphal cell extract; 42 kDa binds C3, C3b, iC3b; 55 kDa binds C3 >C3b, iC3b; 66 kDa no binding 4 Fibrinogen binding proteins 34, 358, 548 mp58 (58 kDa) FBP1 Some yeast cells, hyphae Fibrinogen binding; ubiquitinated, collagen-like domains 5, 60, 484,486 68, 60–62 kDa Hyphae Multifunctional binding; fibrinogen, plastic, laminin, C3d 33-35,550 Laminin binding proteins p37 (37 kDa), 67 kDa Yeast cell, hyphal cell wall 67 kDa only yeast cell wall; p37 of yeast but not hyphal cells binds laminin; reacts with human high-affinity receptor Ab; p37 ubiquitinated, collagen-like domains 296, 484, 486 68, 62, 60 kDa Hyphae > yeast cells Multifunctional binding: fibrinogen, plastic, laminin, C3d 33-35 Multiple species Ligand affinity blot of yeast cell wall extract 164 Fibronectin binding protein Differences reported for fibronectin binding site 222, 248, 253, 257, 380, 407, 466, 507 62, 72 kDa (60, 105 kDa unreduced) Yeast cells, hyphae Affinity chromatography gelatin or fibronectin; 60-kDa species reacts with MAb OKM-1; 60-kDa species highly glycosylated; more species identified Ab to human fibronectin receptor 256,259 Multiple species Yeast cells Yeast cell wall extract; anti-human fibronectin receptor antibody three proteins 40–50 kDa, polydisperse >143; ligand affinity blot additional proteins 164 Collagen binding proteins 249, 255, 259 62, 72 kDa (60, 105 kDa unreduced) Yeast cells, hyphae Affinity chromatography gelatin or fibronectin; 60-kDa species reacts with MAb OKM-1 259 Entactin binding proteins 65, 44, 25 kDa Yeast cells, hyphae Ligand affinity blot; RGD dependent and independent 293 Vitronectin binding protein 30 kDa Yeast cells Ligand affinity blot with cell wall extract 222, 286, 505 Ala1p ALA1 ? Confers collagen IV, laminin and fibronectin binding on S. cerevisiae, member agglutinin-like gene family 154a Protein-sugar Fucose binding protein Yeast cells, hyphae? Buccal, vaginal epithelial cell adhesin; strain variation 37, 53, 92, 93, 345, 544 >15.7 kDa 53,544 GlcNAc binding protein Yeast cells, hyphae? Buccal, vaginal epithelial cell adhesin; strain variation 37, 53, 92, 93, 345, 544 190 kDa Yeast cells Cell wall extracts of galactose- but not glucose-grown cells; not known if is the same protein in reference345 129 Protein-glycosphingolipid Fimbrial protein 66 kDa? Yeast cells, hyphae? Epithelial cell adhesin; adhesintope shared with P. aeruginosapili 279, 583-586 Protein-presumed protein Secreted aspartyl proteinase,
42–45 kDaSAP1-9 Yeast cells, hyphae Binding endothelial cells, corneocytes 147, 433; Table 1 Protein-plastic Plastic binding proteins Binding to many plastics, environmental influences 181, 243, 344, 458 60, 68, 200, >200 kDa Hyphae 60, 68 kDa may be multifunctional binding fibrinogen, laminin, C3d 33, 34, 549 30 kDa AAF1 Yeast cells, hyphae Bind polystyrene and epithelial cells 20, 21 Carbohydrate-protein Acid labile mannan β-1,2 mannotetraose Yeast cells, hyphae Mannose receptor, splenic, lymph node macrophage 73, 285 Carbohydrate-unknown Acid-stable mannan Factor 6 Yeast cells, hyphae BEC adhesion 357 Unknown Yeast cells, hyphae Splenic, lymph node macrophage 234
