A Drug-Sensitive Genetic Network Masks Fungi from the Immune System

Fungal pathogens can be recognized by the immune system via their β-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of “masking” this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying β-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing β-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its β-glucan, leading to increased β-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask β-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics.


Introduction
The innate immune system recognizes microbes based on their pathogen-associated molecular patterns (PAMPs), which provoke pathogen-specific responses tailored to meet the challenge [1].This ensures that parasites, viruses, bacteria, and fungi are each attacked appropriately.Fungi are opportunistic pathogens responsible for occasionally severe disease in individuals with systemic disease [2,3].Candida albicans, clinically the leading fungal pathogen, is recognized predominantly by two PAMPs (b-glucan and mannan), which account by weight for over 90% of its cell wall [4,5].The cell wall of these and other fungi is tiered, with an outer layer of mannoproteins covalently linked to an inner core of b-glucan (Figure 1A).
The inner b-glucan layer is an essential cell wall component targeted by fungicidal antibodies, immune receptors, and the echinocandin class of antifungal drugs.Recent work has shown that anti-b-glucan antibodies can directly kill fungi and assist in the clearance of fungal infection [6].The bglucan receptor Dectin-1 also recognizes fungi and mediates the innate immune system's proinflammatory response [7].The importance of this layer is also underscored by the effectiveness of the echinocandins, which bind and inhibit bglucan synthase to cause cell lysis [8].
The importance of this inner b-glucan layer for immune targeting belies an interesting paradox.Although it accounts for a major fraction of the cell wall, b-glucan is buried underneath a thin but dense mannan coat (Figure 1A) [9].This tiered arrangement of the cell surface raises several important issues: How do the cells of the immune system recognize b-glucan if it is coated with mannoproteins?Furthermore, what fungal genes function to ''mask'' the bglucan from the immune system?
In this study, we report the mechanisms and consequences of b-glucan masking and unmasking in the fungal-immune interaction.Growth of C. albicans in the presence of subinhibitory concentrations of the antifungal drug caspofungin exposes the normally masked b-glucan.We also define a conserved genetic network responsible for b-glucan masking in the genetically tractable, nonpathogenic model fungus Saccharomyces cerevisiae and confirm its requirement in the pathogen C. albicans.Exposure of b-glucan by drug treatment or mutation alters the way the fungi are recognized by immune cells, causing a b-glucan-dependent increase in the elicitation of tumor necrosis factor alpha (TNFa) from macrophages.For C. albicans, this unmasking transforms the fungi into cells that elicit a strong proinflammatory response.Thus, fungi camouflage the majority of their b-glucan from innate immune cells to lower the proinflammatory response to infection.

Results
The Cell Wall b-Glucan Is Masked in S. cerevisiae and C. albicans directed against pure b-glucan [10], purified tagged Dectin-1 carbohydrate recognition domain (Dectin-CRD), and Dectin-CRD preincubated with fluorescently labeled antiepitope antibody (CRD-Myc; see Materials and Methods).Each of these three reagents interacts only weakly with intact cells of both C. albicans and laboratory strains of S. cerevisiae.Consistent with the biological relevance of masking of this proinflammatory molecule, intact cells of C. albicans and S. cerevisiae elicit little immune response when presented to macrophages.
In agreement with previous reports [11], the primary areas of glucan-inhibitable binding of Dectin-1 and the anti-bglucan antibody are the sites of previous cell wall remodeling (i.e., bud scars, birth scars, and bud necks).Wild-type fungal cells have abundant b-glucan in their cell walls but present little b-glucan to the immune system, and this trace amount is restricted to the sites of cell division.As most of the cells in a culture are newly generated, they have completed only one division and would be less efficiently recognized by immune molecules.Thus, it seems that fungi have developed conserved pathways to conceal the potent proinflammatory molecule b-glucan from the immune system.

Fungal b-Glucan Is Exposed by Subinhibitory Doses of Caspofungin
We reasoned that antifungal drugs that specifically target cell-wall biosynthetic pathways would disrupt the intricate architecture of the cell wall and heighten b-glucan exposure, ''unmasking'' the cells through uncoating or cell wall disorganization.To examine this possibility, we grew wildtype C. albicans overnight in the presence of subinhibitory doses of caspofungin (CF), a potent echinocandin that is a recent addition to the antifungal arsenal [8].Remarkably, at CF concentrations that permit normal growth rates the drug caused exposure of b-glucan on the C. albicans cell surface (Figure 2A and 2B).At these drug concentrations, the cells had 103-303 greater reactivity with the anti-b-glucan antibody.It is important to note that the fungi grow at these drug concentrations without loss of viability (Figure 2C), suggesting that the increased b-glucan exposure is not due to generalized cell death.We also examined the effect of CF on cells grown in RPMI 1640, a culture medium that causes strong hyphal growth and b-glucan masking [11].Subinhibitory doses of CF caused a dramatic increase in the exposure of b-glucan on hyphae grown in this media (Figure 2D).These cells also did not show increased cell death, as measured by microscopic examination.
Caspofungin inhibits the synthesis of b-glucan, which leads to lower bulk levels of b-glucan in the cell [8]; however, the inhibition of b-glucan biosynthesis also upsets the equilibrium required to maintain the normal tiered cell wall architecture [12].This perturbation of the cell wall remodeling machinery can cause more exposure of b-glucan even in the presence of lower bulk levels of b-glucan.This fact also suggests that fungi have evolved a mechanism for masking bglucan that is compromised by the drug.

Caspofungin-Treated Cells Hyperelicit TNFa
Since b-glucan is the primary proinflammatory molecule on the fungal surface, we tested whether yeast unmasked by CF elicit larger proinflammatory responses from macrophages.This test requires inactivation of the fungi to prevent them from killing the macrophages before the end of the experiment.Previously used methods of inactivation (including heat, formaldehyde, and ethanol) artificially increase bglucan exposure and lead to high levels of TNFa elicitation (Figure S1).We discovered that ultraviolet light (UV)irradiated cells did not kill macrophages, retained an intact cell wall architecture, and elicited only low levels of TNFa (Figure S1).Therefore, we used UV-inactivated fungi for all subsequent experiments.
To test whether unmasked, CF-treated yeast elicit a greater proinflammatory response, we exposed CF-treated or -untreated (exposed or masked, respectively) C. albicans to primary bone marrow-derived macrophages (BMDMs) and assayed elicitation of the key proinflammatory cytokine TNFa.The CF-treated C. albicans elicited 3-to 4-fold higher levels of the key proinflammatory cytokine TNFa (Figure 2E) than did untreated C. albicans, which elicited undetectable levels of TNFa in this assay.Thus, subinhibitory doses of CF cause the pathogen to elicit a marked proinflammatory response.

An Interconnected Genetic Network Is Required for b-Glucan Masking
To identify the system used by fungi to mask their b-glucan from the immune system, we screened a genome-wide library of knockout mutants in S. cerevisiae for increased b-glucan exposure.Because subinhibitory levels of CF are able to cause increased b-glucan exposure and altered recognition of the fungi without killing the fungi, we reasoned that this library of nonessential gene knockouts should identify genes that specifically perturb the genetic network required for b-glucan masking.Further, due to similarity in cell wall structure between these two fungi, the genes we identified in Saccharomyces could guide us to functionally equivalent genes in Candida that serve the same masking function.
Using automated microscopy, we systematically screened the entire library of approximately 4,800 S. cerevisiae mutants for strains with increased b-glucan exposure.This platform

Synopsis
Opportunistic fungal pathogens such as Candida albicans often cause fatal infections in patients with a compromised immune system.Unfortunately, current drugs often fail to halt fungal disease, are ineffective against drug-resistant strains, and have severe side effects.Despite the clear clinical significance of fungal infections, it is still not understood how fungi are recognized by the immune system.Candida has high levels of the structural molecule b-glucan in its cell wall, but the majority of its b-glucan is masked by a mannoprotein coat and is therefore invisible to the immune system.Masking of b-glucan may be a fungal virulence factor, because exposed b-glucan provokes a proinflammatory response that is important for mounting an effective immune response against the fungus and clearing the infection.By surveying the genome of the model fungus Saccharomyces cerevisiae (bakers' yeast), the authors discovered a genetic network required for masking b-glucan from the immune system.Mutation of genes in this network in C. albicans caused unmasking of b-glucan and an increased immune response to the fungus.The authors also found that sublethal doses of the antifungal drug caspofungin cause unmasking and lead to a greater immune response.Drugs targeting this fungally conserved masking network may provide new tools to fight fungal infections.permits quantification of the fluorescence from high resolution pictures of the b-glucanstained yeast.This method of quantification is exemplified in the comparison of the vrp1D mutant to wild-type (Figure 3A).Strains with levels of antibody binding greater than two standard deviations from the mean of each plate were identified, and independently created mutants were rescreened using the same protocol (see Materials and Methods).
Emphasizing the connection between b-glucan exposure and immune recognition, most of the mutants with greater exposed b-glucan also showed increased binding to the Dectin-1 b-glucan receptor (Figure 3B).The greater exposure of b-glucan and better binding to Dectin-CRD of these mutants raised the possibility that they would have altered interaction with cells of the immune system.Each S. cerevisiae mutant was exposed to RAW264.7 murine macrophages and tested for TNFa elicitation.A large percentage (48 of 76) of the unmasked mutants triggered a significantly stronger proinflammatory response than did the wild-type lab strain of S. cerevisiae; some elicited up to ten times the amount of TNFa as did wild-type (Figure 3B and Table S1).Taking gas1D as a representative mutant with an intermediate phenotype, we found increased binding of the anti-b-glucan antibody to this mutant, and this binding is blocked by soluble b-glucan (Figure 3C).The gas1D mutant also binds Dectin-CRD better and elicits a higher level of TNFa from macrophages (Figure 3D and 3E).The strong correlation between b-glucan exposure and increased TNFa elicitation suggests that bglucan masking on the surface of Saccharomyces is a key factor in blocking the immune response to fungi.
Most of the genes identified in our screen for the masking of b-glucan from the immune system fit under an umbrella of interconnected gene networks that regulate polarized cell wall remodeling (Figure 4): polarization of the actin cytoske-leton, polarized secretion of proteins and polysaccharides, and polarized endocytosis of unwanted byproducts [13,14].The connections among these related pathways imply that coordination of polarized cell wall remodeling at sites of new cell wall addition is crucial for masking b-glucan.This interconnected network is sensitive to caspofungin.Three of the key hubs of the network (SLT2, SLA1, and MNN10) are required for caspofungin resistance [15,16].Furthermore, the Slt2p mitogen-activated protein kinase is a key mediator of the caspofungin-induced stress response [16].
In addition, several genes required for mannosylation of cell wall proteins (MNN10, MNN11, OCH1, OST3, and OST4) are also required for masking of b-glucan and immune recognition.The connection between mannoprotein processing and b-glucan masking buttresses the idea that a dense coat of mannosylated cell wall proteins masks b-glucan from recognition.Apparently the increase in exposure is due to uncoating or disorganization of the cell wall rather than bulk changes in b-glucan levels, because in those hypereliciting mutants for which the bulk level of b-glucan is known, the vast majority of mutants (14 of 18) has unchanged or lower levels of both types of b-glucan (Table 1).
As well as finding genes known to play a role in cell wall architecture (Figure S2), our screen identified many new genes that likely play roles in cell wall function (Figure 4).We identified four genes that encode global transcriptional regulatory proteins (ASF1, IES6, MOT2, and SSN8) and six genes with previously unassigned biological function (DIA2, VAC14, RDS2, YMR315W, YNL045W, and YPL158C) that were not expected a priori to be required for masking b-glucan or for recognition by the immune system.The stringency of our screen clearly implicates each of these genes in the cell wall remodeling process.
Several of the mutants with increased binding to anti-b- glucan antibody did not hyperelicit TNFa from macrophages.This could be due to different epitope specificity between antibody and the full-length receptor on the macrophage.To address this issue, we probed all of the ''unmasked'' mutants with a Dectin-CRD-anti-Myc probe (CRD-Myc) that colocalizes with Dectin-CRD and Dectin-1 (see Figure 1A).This CRD-Myc probe should have a similar size to anti-b-glucan and the same binding specificity as the Dectin-1 receptor.Only three of the 28 non-hyperelicitors showed even marginally increased binding to both Dectin-CRD and CRD-Myc (gup1D, kre11D, and ylr111wD), suggesting that differences in epitope recognition between the anti-b-glucan antibody and receptor account for a majority of the mutants that have greater anti-bglucan binding but no hyperelicitation (Table S2).
Alternatively, different levels of mannoprotein could independently alter immune response and confound these results.To examine the surface mannoprotein structure, we quantified the level of exposed mannose on the surface of live yeast by incubating with the mannose-specific lectin concanavalin A (ConA).Although two mutants with known roles in mannosylation (i.e., van1D, mnn2D) showed reduced binding to ConA, we did not find a preponderance of mutants with reduced binding among our set of exposed mutants and, importantly, did not see any correlation between ConA binding and TNFa elicitation (Table S2).Therefore, under these conditions, overall levels of mannan on the surface do not appear to regulate TNFa elicitation independently of bglucan recognition.

The Genetic Pathway for b-Glucan Masking Is Conserved in C. albicans
The screen in S. cerevisiae identified potential genes and pathways regulating b-glucan masking in pathogenic fungi, and suggested that their C. albicans homologs might have a similar masking function in this pathogen.Mutations in two of these homologs (PHR2 and KRE5) resulted in attenuated virulence in mice, and we reasoned that exposure of b-glucan may contribute to their reduced virulence.We found that mutations in each of three different b-glucan masking genes caused increased b-glucan exposure in C. albicans (Figure 5).
The PHR2 gene is the only C. albicans homolog of the S. cerevisiae GAS1 gene active under our growth conditions.It encodes a b-glucan transglycosylase required for b-glucan branching, cell wall integrity, and cell wall maintenance [17,18].When grown in our conditions, the phr2D/D mutant displays a strong increase in the exposure of b-glucan (Figure 5A and 5D).
The homozygous kre5D/D mutant of C. albicans also showed a dramatic increase in b-glucan exposure (Figure 5B and 5E).We examined its function in C. albicans because it is nonessential in this fungus yet has a similar function to KRE6, which was identified in the S. cerevisiae screen (the S. cerevisiae kre5D mutant is lethal and therefore is not in the deletion library).Both KRE5 and KRE6 play important roles in the synthesis of b1,6-glucan, which is a minor component of the cell wall by weight but is important for cell wall organization and for anchoring of mannoproteins in the wall [19].In C. albicans, homozygous kre5D/D mutants are avirulent in the mouse model of infection and have defects in filamentous growth and adherence to epithelial cells [20].
C. albicans has a single homolog of the S. cerevisiae gene, which encodes a global transcriptional regulator.The homozygous ssn8D/D mutant displays a mild increase in bglucan exposure that is reproducibly found constrained to the tips of cells and filaments (Figure 5C and 5F).This polarized exposure of b-glucan is identical to that found in the phenotype of the S. cerevisiae ssn8D mutant, which is also altered in filamentation.The increased exposure of b-glucan at the tips and junctions, which are sites of cell wall remodeling, suggests that in wild-type growth there must be genes such as SSN8 that direct the reconstitution of the wall.The homozygous C. albicans ssn8D/D mutant is mildly filamentous even in rich (yeast peptone dextrose [YPD]) media, which normally promotes yeast-form growth, showing that filamentation and b-glucan masking appear to be separable phenomena.

Unmasked C. albicans Mutants Elicit More Proinflammatory Cytokines through the b-Glucan Receptor
To test whether increased exposure of Candida b-glucan in these mutants also leads to altered immune recognition, we exposed wild-type or mutant Candida to RAW 264.7 macrophages and examined the elicitation of TNFa.As shown in Figure 6A, the Candida mutants with increased b-glucan exposure elicited higher levels of TNFa from macrophages.The relationship between b-glucan exposure and increased proinflammatory response is biologically relevant because unmasked mutants of S. cerevisiae (Figure S3) and C. albicans (Figure 6B) also elicited higher levels of TNFa and interleukin 6 (IL-6) (Figure S4) from BMDMs.This difference in elicitation is not simply a dosage effect, because this phenomenon occurs at different ratios of fungi to macrophages (Figure 6B).The altered signaling is also dependent on the b-glucan receptor, because the great majority of the increased elicitation of proinflammatory cytokines could be blocked by preincubation of the macrophages with the soluble b-glucan laminarin (Figure 6C).These data suggest that the majority (if not all) of the increased elicitation of proinflammatory cytokines occurs through the b-glucan receptor.

Discussion
We found that ''unmasking'' the inner b-glucan layer of fungi causes their altered recognition by innate immune cells.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 MIC 50 were stained with anti-b-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 MIC 50 , and one-quarter the CF MIC 50 .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 TNFa (E).DOI: 10.1371/journal.ppat.0020035.g002 Using the genome knockout library of S. cerevisiae as a guide, we identified the set of genes that establish and maintain the cell wall architecture in such a way that the majority of bglucan is not recognized.The mutants identified in this screen are recognized better than the wild-type by the Dectin-1 b-glucan receptor, and they hyperelicit the proinflammatory cytokine TNFa from macrophages.Furthermore, mutation of cognate genes in the pathogen C. albicans leads to unmasking of b-glucan and hyperelicitation of proinflammatory cytokines in primary macrophages in a b-glucandependent manner.We also found that caspofungin unmasks fungal b-glucan at subinhibitory concentrations of the drug and alters recognition of C. albicans.This unanticipated activity may assist in fungal clearance during treatment of an infection.
The fungal mutants with exposed b-glucan and increased binding to Dectin-1 stimulate increased levels of proinflammatory cytokines independent of their bulk level of b-glucan.By creating barriers on their surfaces, fungal pathogens may mask certain PAMPs from the immune system and subvert the immune response.The immune system may also be able to counteract this fungal defense by unmasking the signature components of the fungus during the course of infection.For example, soluble or phagosomal proteases could remove the mannoprotein coat, which is known to expose b-glucan [21].Differential exposure and unmasking could in part explain the diverse potential of fungi for pathogenesis.We screened the genome-wide set of S. cerevisiae knockout mutants by staining with anti-b-glucan antibody and quantifying b-glucan exposure using the Cellomics system.(A) Images of yeast were taken at 203, 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-b-glucan channel for each cell.Wild-type yeast (left photomicrographs) show little or no fluorescence from the anti-b-glucan channel, whereas the unmasked vrp1D mutant shows high levels of fluorescence from this channel.(B) Most of the mutants found with increased b-glucan exposure also showed greater binding to Dectin-CRD and increased TNFa elicitation from RAW 264.7 macrophages.Of the 76 mutants identified with increased anti-b-glucan binding (encircled with the black line), 48 (encircled with the dotted line) hyperelicited TNFa 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-b-glucan binding and increased Dectin-CRD binding and increased TNFa elicitation from macrophages).(C-E) To exemplify the methodology used, we chose the gas1D mutant, which has intermediate b-glucan exposure and TNFa elicitation.(C) Live wildtype (upper left image) or gas1D mutant (lower left image) S. cerevisiae were stained with anti-b-glucan antibody.To show specificity of the binding, gas1D cells were stained omitting primary antibody (upper right image) or antibody was preincubated in 100 lg/ml soluble glucan (laminarin) before and during the staining (lower right image).(D) Live wild-type or gas1D mutant cells were stained with Alexa Fluor-labeled Dectin-CRD.(E) Live wildtype or gas1D 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 TNFa levels.DOI: 10.1371/journal.ppat.0020035.g003Figure 4.The Cell Wall Remodeling Network Required for b-Glucan Masking A map of physical and genetic interactions among b-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.DOI: 10.1371/journal.ppat.0020035.g004In parallel experiments, strains that were complemented with a wild-type copy of the gene showed full reversal of b-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].DOI: 10.1371/journal.ppat.0020035.g005 The architecture of the fungal cell wall must be taken into account when assessing how fungi are detected by immune cells.The relative accessibility of b-glucan versus mannan on the surface could favor quite different responses from immune cells [22].In this light, the finding that heat inactivation alters the cell wall to expose b-glucan suggests that experiments using heat-killed fungi or zymosan may benefit from complementary studies using live and/or UVinactivated fungi.
The exposure of b-glucan on the cell surface might be altered by environmental conditions that affect the regulation of the genes we have shown to affect cell wall architecture.This effect could explain the reported greater binding of Dectin-1 to the yeast form of C. albicans as compared with its hyphal form [11].In those experiments the fungus was grown on one medium to foster growth of the yeast form, and a different medium to induce the hyphal form.The different media could affect b-glucan masking independently of fungal morphotype [23].Consistent with this view, preliminary results with C. albicans mutants locked in the yeast form suggest that simply modifying the media is sufficient to change the level of b-glucan masking (unpublished data).As all of our comparisons were done with strains grown on the same medium, the unmasking we observed cannot be explained by alterations in environmental conditions.Although yeast and hyphal forms showed no fundamental differences with respect to b-glucan accessibility and CF-induced unmasking, the morphotypes have other cell wall differences that result in differential immune recognition [22,24].
The weak proinflammatory response elicited by wild-type cells of C. albicans may prevent recruitment of effector cells and clearance of the fungus, and explain its persistence commensally in the gut in a large percentage of the population [25].Both TNFa and IL-6 have been shown to be protective in the mouse model of disseminated candidiasis, suggesting that unmasking b-glucan could provoke a proinflammatory response and attenuate virulence in disseminated disease [26].It is noteworthy that two of the unmasked C. albicans mutants (phr2D/D and kre5D/D) are avirulent [17,20], and it is possible that b-glucan unmasking contributes to their attenuated virulence.However, these mutations result in additional phenotypes [17,20] that cannot be excluded as a cause of their virulence defects.Evaluating the relevance of b-glucan masking to the immune response will require further experiments in an informative wholeanimal model.
The finding that b-glucan unmasking by CF promotes the proinflammatory response suggests that antimycotics such as CF may strike at fungi in a dual way, killing them at high concentrations and unmasking their b-glucan at lower concentrations.In addition to enhancing nonopsonic, bglucan-mediated uptake and signaling, drug-induced exposure of b-glucan may also target fungi for recognition by natural anti-b-glucan antibodies, which have been characterized in mouse and human sera [27].Many other fungal pathogens are sensitive to CF, have homologs of b-glucanmasking genes, and can be sensed by Dectin-1 and/or targeted via their b-glucan [6,8,28,29].The proteins encoded by these b-glucan-masking genes may provide useful drug targets for broad-spectrum treatment of fungal infection.

Materials and Methods
Fungal strains and growth.S. cerevisiae strains in the BY4741 or BY4742 background were used to make the complete deletion library [30].S. cerevisiae knockout libraries were purchased from Open Biosystems (Huntsville, Alabama, United States).C. albicans strains were derived from clinical isolate SC5314 [31].Strain details are shown in Table S3.Fungi were grown overnight in YPD rich medium for yeast-form growth and RPMI 1640 for hyphal growth.C. albicans was grown at 37 8C; S. cerevisiae was grown at 30 8C.For CF treatment, overnight cultures of C. albicans CAF2 were diluted 1:1,000 into fresh YPD or RPMI 1640 containing dilutions of CF (caspofungin acetate, Cancidas formulation, Merck, Whitehouse Station, New Jersey, United States) and grown overnight.MIC 50 was 2.5 ng/ml.For viability, cells were stained with propidium iodide (Sigma, St. Louis, Missouri, United States).
Inactivation of fungi for macrophage interaction experiments.For UV inactivation, the equivalent of 2.5 3 10 7 cells from a culture were washed and resuspended in 1 ml of PBS in a six-well plate.The fungi were exposed to four doses of 100,000 ljoules/cm 2 in a CL-1000 UVcrosslinker (UVP, Upland, California, United States), with agitation between each dose to treat cells evenly.For heat inactivation, 2.5 3 10 7 cells in 1 ml of PBS were boiled for 10 min.After UV-or heat inactivation, cells were washed and renormalized by OD 600 .In S. cerevisiae, we found greater b-glucan exposure on cells that were heattreated at 65 8C for 15 min, fixed for 30 min on ice in 3.7% formaldehyde, or fixed for 30 min on ice in 70% ethanol (unpublished data).
Screen for b-glucan exposure.Overnight cultures of strains were grown in YPD and stained with anti-b-glucan primary antibody (Biosupplies Inc., Parkville, Australia) and Cy3-labeled goat-antimouse secondary.This antibody is specific for b1,3-glucan [10], and costaining with this antibody and the purified Dectin-CRD shows colocalization (unpublished data).Stained cells were adhered to clear-bottom plates with concanavalin A (Sigma) and scanned with Cellomics VTI fluorescence microscopic imager (Cellomics, Pittsburgh, Pennsylvania, United States) using Target Acquisition software (Zeiss, Oberkochen, Germany).Mean average intensity and standard deviation of average intensity measurements were used to identify strains with over two standard deviations greater b-glucan exposure.Initially matA mutant strains were screened, then the independently constructed mata counterparts were screened.Dectin-CRD was expressed from pTrcHis2 vector (Invitrogen, Carlsbad, California, United States), purified from E. coli as described [11], and labeled directly with Alexa Fluor 555-succinimidyl ester (Molecular Probes, Eugene, Oregon, United States).Fungi were labeled with pure protein and showed glucan-inhibitable binding and similar staining pattern to that described [11].
Macrophage-yeast interaction.Fungi were grown overnight, washed extensively to remove any shed proteins or polysaccharides, and normalized by OD 600 or hemacytometer (these two methods produced equivalent results).For S. cerevisiae, live fungi were added to RAW264.7 macrophages at equivalent effector:target (E:T) ratio of 1:5 in 24-well plates and incubated for 6 h, when supernatants were collected for ELISA.For exposure of C. albicans, UV-inactivated fungi were exposed either to RAW264.7 macrophages at an equivalent E:T of 1:2 or to BMDMs at an E:T of 1:10.For RAW264.7 exposures, supernatants were collected after 2 h; for BMDM exposures, supernatants were collected after 6 h.For blocking, BMDMs were preincubated with 100 lg/ml laminarin for 20 min on ice, exposed to fungi for 30 min at 37 8C, washed extensively, then incubated for 6 h more at 37 8C.ELISAs were performed according to the manufacturer's instructions (R&D Systems).Note that soluble b-glucan (laminarin) alone does not stimulate the macrophages (Figure 6C).
Staining, microscopy, and FACS.Fungi were grown overnight, washed, blocked in PBS þ 2% BSA, and stained with either the anti-bglucan antibody (followed by Cy3-or PE-labeled secondary antibody), Alexa Fluor-labeled Dectin-CRD, or Dectin-CRD-anti-Myc.Staining with antibody and Dectin-CRD is described above for the screen.For staining with Dectin-CRD-anti-Myc, Dectin-CRD was preincubated with FITC-labeled anti-Myc (Invitrogen) at a ratio of 3.4 lg of Dectin-CRD þ 10 ll of anti-Myc, and then added to fungi at 1:100 on ice.Preincubation of Dectin-CRD with anti-Myc creates a probe with the same approximate size as the anti-b-glucan antibody but with the specificity of Dectin-1.Images of stained cells were taken using a Nikon TE2000-S microscope (Nikon, Tokyo, Japan) equipped with Spot RT camera (Diagnostic Instruments, Sterling Heights, Michigan, United States) and processed in Photoshop (Adobe Systems, Palo Alto, California, United States).Fluorescence was quantified on a FACScalibur cytometer (Becton-Dickinson, Palo Alto, California, United States); cells were gated by forward and side scatter based on wild-type cell size and shape, and mean fluorescence intensity of 20,000 labeled cells was calculated using Cellquest software (Becton-Dickinson).We examined the intersection (A) between the genes required for bglucan masking (shown in green) and those identified by any one of four other genome-wide cell wall-directed screens (shown in dark red).These included (B) a screen to find mutants with synthetic lethality with genes required for b1,3-glucan biosynthesis (shown in dark blue [1]), (C) a screen for mutants with altered sensitivity to caspofungin (shown in light blue [1]), (D) two screens for mutants with altered sensitivity to the chitin-binding drug calcofluor white (shown in orange [2]), and (E) a screen for mutants with altered sensitivity to the cell wall-directed K1 killer toxin (shown in purple [3]).Found at DOI: 10.1371/journal.ppat.0020035.sg002(489 KB PDF).

Figure 1 .
Figure 1.Fungal b-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 b-glucan network.Mannoproteins are linked to b-glucan and protrude outside of this layer to make up a dense coat.Schematic adapted from [5].(B-D) There is little b-glucan on live C. albicans or S. cerevisiae that is exposed and accessible to the anti-b-glucan antibody (B), the Dectin-CRD (C), or the Dectin-CRD-anti-Myc probe (D).The staining with anti-b-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 b-glucan-binding reagents (B and D versus C) is likely due to the size of the reagents (IgG has dimensions of approximately 95 A ˚3 171 A ˚[33], while the CRD of CD69, which is similar to the Dectin-1 CRD, has dimensions of 44 A ˚3 32 A ˚3 30 A ˚[34]) relative to the estimated pore size of the S. cerevisiae cell wall (58 A ˚[23]), and thus the monomeric Dectin-CRD likely has more access to smaller areas of exposed b-glucan.DOI: 10.1371/journal.ppat.0020035.g001

Figure 2 .
Figure 2. Subinhibitory Concentrations of the Antifungal Drug CF Cause Exposure of b-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 MIC 50 were stained with anti-b-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 MIC 50 , and one-quarter the CF MIC 50 .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 TNFa (E).DOI: 10.1371/journal.ppat.0020035.g002

Figure 3 .
Figure3.Automated Genome-Wide Screen for Increased b-Glucan Exposure We screened the genome-wide set of S. cerevisiae knockout mutants by staining with anti-b-glucan antibody and quantifying b-glucan exposure using the Cellomics system.(A) Images of yeast were taken at 203, 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-b-glucan channel for each cell.Wild-type yeast (left photomicrographs) show little or no fluorescence from the anti-b-glucan channel, whereas the unmasked vrp1D mutant shows high levels of fluorescence from this channel.(B) Most of the mutants found with increased b-glucan exposure also showed greater binding to Dectin-CRD and increased TNFa elicitation from RAW 264.7 macrophages.Of the 76 mutants identified with increased anti-b-glucan binding (encircled with the black line), 48 (encircled with the dotted line) hyperelicited TNFa 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-b-glucan binding and increased Dectin-CRD binding and increased TNFa elicitation from macrophages).(C-E) To exemplify the methodology used, we chose the gas1D mutant, which has intermediate b-glucan exposure and TNFa elicitation.(C) Live wildtype (upper left image) or gas1D mutant (lower left image) S. cerevisiae were stained with anti-b-glucan antibody.To show specificity of the binding, gas1D cells were stained omitting primary antibody (upper right image) or antibody was preincubated in 100 lg/ml soluble glucan (laminarin) before and during the staining (lower right image).(D) Live wild-type or gas1D mutant cells were stained with Alexa Fluor-labeled Dectin-CRD.(E) Live wildtype or gas1D 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 TNFa levels.DOI: 10.1371/journal.ppat.0020035.g003

Figure 5 .
Figure 5. C. albicans Mutants of Masking Genes Have More Exposed b-Glucan Wild-type or mutant C. albicans strains were grown overnight in YPD, then stained with anti-b-glucan antibody and Cy3-labeled (A-C) or PE-labeled (D-F) secondary antibody.(A-C) Upper photomicrographs show overlay of brightfield and anti-b-glucan staining of Cy3-labeled cells; lower photomicrographs show anti-b-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 b-glucan exposure (for KRE5) or partial reduction in exposure (for PHR2) (FigureS5).This correlates with other phenotypes observed for these complemented strains[17,20,35].DOI: 10.1371/journal.ppat.0020035.g005

Figure 6 .
Figure 6.Unmasked C. albicans Mutants Hyperelicit TNFa from Macrophages through the b-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 kre5D/D 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 bglucan (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 8C, and supernatants were collected for TNFa quantitation.DOI: 10.1371/journal.ppat.0020035.g006

Figure S1 .
Figure S1.Heat Inactivation of C. albicans Causes Greater b-Glucan Exposure and Greater Elicitation of TNFa Wild-type (CAF2) fungi were grown overnight in YPD medium at 37 8C.Cells were killed by UV irradiation or by heat inactivation (10 min at 100 8C).(A)Live or killed cells were probed with anti-b-glucan antibody and PE-labeled secondary antibody and with Alexa Fluor 488-labeled Dectin-CRD, then subjected to FACS analysis.(B) UV-or heat-killed cells were then exposed to BMDMs at a ratio of 10:1 (yeast:macrophage), and supernatants were taken after 6 h for measurement of TNFa levels.Found at DOI: 10.1371/journal.ppat.0020035.sg001(271 KB PDF).

Figure S2 .
Figure S2.Overlap of b-Glucan Cell Wall Architecture Screen with Other Cell Wall-Related ScreensWe examined the intersection (A) between the genes required for bglucan masking (shown in green) and those identified by any one of four other genome-wide cell wall-directed screens (shown in dark red).These included (B) a screen to find mutants with synthetic lethality with genes required for b1,3-glucan biosynthesis (shown in dark blue[1]), (C) a screen for mutants with altered sensitivity to caspofungin (shown in light blue[1]), (D) two screens for mutants with altered sensitivity to the chitin-binding drug calcofluor white (shown in orange[2]), and (E) a screen for mutants with altered sensitivity to the cell wall-directed K1 killer toxin (shown in purple[3]).Found at DOI: 10.1371/journal.ppat.0020035.sg002(489 KB PDF).

Figure S4 .
Figure S4.Unmasked C. albicans Mutants Hyperelicit IL-6 from Macrophages through the b-Glucan Receptor BMDMs were pretreated for 20 min on ice with medium or soluble bglucan (laminarin), and 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 8C, and supernatants were collected for IL-6 quantitation.Found at DOI: 10.1371/journal.ppat.0020035.sg004(527 KB PDF).