Figure 1.
Fungal β-Glucan Is Buried in the Cell Wall and Largely Inaccessible
(A) Transmission electron micrograph demonstrating layer structure of fungal cell wall (courtesy of C. Rondeau). The plasma membrane is tightly connected to a thick layer of β-glucan network. Mannoproteins are linked to β-glucan and protrude outside of this layer to make up a dense coat. Schematic adapted from [5].
(B–D) There is little β-glucan on live C. albicans or S. cerevisiae that is exposed and accessible to the anti-β-glucan antibody (B), the Dectin-CRD (C), or the Dectin-CRD-anti-Myc probe (D). The staining with anti-β-glucan and Dectin-CRD-anti-Myc is nearly indistinguishable, and is more specific than that with the directly labeled Dectin-CRD. The Dectin-CRD-anti-Myc has the same size as an antibody and the same specificity as Dectin-1. The difference in staining between the β-glucan-binding reagents (B and D versus C) is likely due to the size of the reagents (IgG has dimensions of approximately 95 Å × 171 Å [33], while the CRD of CD69, which is similar to the Dectin-1 CRD, has dimensions of 44 Å × 32 Å × 30 Å [34]) relative to the estimated pore size of the S. cerevisiae cell wall (58 Å [23]), and thus the monomeric Dectin-CRD likely has more access to smaller areas of exposed β-glucan.
Figure 2.
Subinhibitory Concentrations of the Antifungal Drug CF Cause Exposure of β-Glucan
Wild-type C. albicans (CAF2) was grown overnight for ten generations in YPD medium, favoring yeast-form growth (A, B, C, and E) or RPMI medium, favoring hyphal growth (D). Cultures grown at one-quarter and one-eighth of the CF MIC50 were stained with anti-β-glucan antibody and Cy3-labeled secondary antibody (A and D) for visualization by microscopy or with PE-labeled secondary antibody (B) for FACS quantification. Mean fluorescence intensity (MFI) values were 9, 65, and 161, respectively, for no treatment, one-eighth the CF MIC50, and one-quarter the CF MIC50. Concurrently, cells were labeled briefly with propidium iodide to assess viability and visualized by epifluorescence microscopy or quantified by FACS (C). Cells grown overnight in YPD with or without CF were UV-inactivated and then exposed to BMDMs at a yeast:macrophage ratio of 10:1. Supernatants were taken at 6 h and assayed for TNFα (E).
Figure 3.
Automated Genome-Wide Screen for Increased β-Glucan Exposure
We screened the genome-wide set of S. cerevisiae knockout mutants by staining with anti-β-glucan antibody and quantifying β-glucan exposure using the Cellomics system.
(A) Images of yeast were taken at 20×, and object-finding software used the ConA fluorescence signal to identify cells (green outline); the software then applied a mask (blue outline) and quantified the average level of fluorescence from the anti-β-glucan channel for each cell. Wild-type yeast (left photomicrographs) show little or no fluorescence from the anti-β-glucan channel, whereas the unmasked vrp1Δ mutant shows high levels of fluorescence from this channel.
(B) Most of the mutants found with increased β-glucan exposure also showed greater binding to Dectin-CRD and increased TNFα elicitation from RAW 264.7 macrophages. Of the 76 mutants identified with increased anti-β-glucan binding (encircled with the black line), 48 (encircled with the dotted line) hyperelicited TNFα from macrophages and 65 (encircled with the red line) showed increased binding to the labeled Dectin-CRD. Numbers within the black oval show the intersections of each of these groups (e.g., 44 had increased anti-β-glucan binding and increased Dectin-CRD binding and increased TNFα elicitation from macrophages).
(C–E) To exemplify the methodology used, we chose the gas1Δ mutant, which has intermediate β-glucan exposure and TNFα elicitation. (C) Live wild-type (upper left image) or gas1Δ mutant (lower left image) S. cerevisiae were stained with anti-β-glucan antibody. To show specificity of the binding, gas1Δ cells were stained omitting primary antibody (upper right image) or antibody was preincubated in 100 μg/ml soluble glucan (laminarin) before and during the staining (lower right image). (D) Live wild-type or gas1Δ mutant cells were stained with Alexa Fluor-labeled Dectin-CRD. (E) Live wild-type or gas1Δ mutant cells were exposed to RAW264.7 macrophages at a ratio of 5:1 (yeast:macrophage), and supernatants were collected after 6 h and measured for TNFα levels.
Figure 4.
The Cell Wall Remodeling Network Required for β-Glucan Masking
A map of physical and genetic interactions among β-glucan masking genes was made using the Osprey v1.2.0 visualization program (available at: http://biodata.mshri.on.ca/osprey/servlet/Index). All genes of hypereliciting mutants identified in the screen are represented. Lines connecting genes are colored to represent the nature of known interaction(s) between a pair of genes (i.e., synthetic lethality, two-hybrid, or coimmunoprecipitation). Vertices are colored to represent the cellular processes directed by the gene product, as annotated by the Yeast Proteome Database (available at http://www.proteome.com). Vertices of genes required for caspofungin resistance [15,16] are circled in black.
Table 1.
β-Glucan Exposure Does Not Correlate with Bulk Level in S. cerevisiae Mutants
Figure 5.
C. albicans Mutants of Masking Genes Have More Exposed β-Glucan
Wild-type or mutant C. albicans strains were grown overnight in YPD, then stained with anti-β-glucan antibody and Cy3-labeled (A–C) or PE-labeled (D–F) secondary antibody.
(A–C) Upper photomicrographs show overlay of brightfield and anti-β-glucan staining of Cy3-labeled cells; lower photomicrographs show anti-β-glucan staining alone.
(D–F) Overlay histograms of FACS analysis of PE-labeled cells; data on 20,000 cells are shown. MFI values for wild-type and mutants are shown in insets. In parallel experiments, strains that were complemented with a wild-type copy of the gene showed full reversal of β-glucan exposure (for KRE5) or partial reduction in exposure (for PHR2) (Figure S5). This correlates with other phenotypes observed for these complemented strains [17,20,35].
Figure 6.
Unmasked C. albicans Mutants Hyperelicit TNFα from Macrophages through the β-Glucan Receptor
(A) C. albicans strains were exposed to RAW264.7 macrophages at a ratio of 2:1 (yeast:macrophage), and supernatants were collected after 6 h.
(B) Different numbers of C. albicans (wild-type or kre5Δ/Δ mutant) were exposed to BMDMs, and supernatants were collected after 6 h.
(C) BMDMs were pretreated for 20 min on ice with media or soluble β-glucan (laminarin); they were then exposed to different C. albicans strains at a ratio of 10:1 (yeast:macrophage). After unbound fungi were washed off, macrophages were incubated for 6 h at 37 °C, and supernatants were collected for TNFα quantitation.