Analysis of C. albicans cell wall β-1,6-glucans.

a, Percentages of cell wall polymers on total cell wall, distributed by fractions: SDS-β-ME, AI and AS. Cells were grown in SD medium at 37°C. Means and standard deviations were calculated from three independent experiments. b, Table of the mean percentages of each polymer in the cell wall from three independent experiments. c, Diagram of β-1,6-glucan structure. In blue are represented glucose residues linked in β-1,6 and in green glucose residues linked in β-1,3. According to NMR analysis and HPAEC after endo-β-1,6-glucanase digestion (Table S2), based on three independent experiments, an average of 6.4% (+/- 0.5%) of glucose units of the main chain are substituted by a single glucose residue (88-90%) or a laminaribiose (10-12%). d, Gel filtration analysis on a Superdex 200 column of β-1,6-glucan released by endo-β-1,3-glucanase digestion. The column was calibrated with dextrans (Tx: x kDa). e, HPAEC chromatographic analysis of the digestion products of the AI fraction treated with an endo-β-1,6-glucanase. Chromatographs in d and e are representative of three independent experiments. PED, pulsed electrochemical detector; nC, nanocoulombs; RI, refractive index; mV, millivolt; DP, degree of polymerization; Glc, glucose.

Comparative analyses of cell wall β-1,6-glucans produced in various environmental conditions.

a, Percentages of cell wall polymers (AI + AS fractions) on total cell wall. Cells were grown in liquid synthetic medium at 37°C under different conditions, as specified in Methods. b, β-1,6-glucan mean molecular weight (MW). Average molecular weight was estimated by gel filtration chromatography on a Superdex 200 column. c, Branching rate of β-1,6-glucans. Branching rate was determined by HPAEC after digestion of the AI fraction by an endo-β-1,6-glucanase (% expressed as number of glucose units of the main chain that are substituted by a side chain). For a, b, and c, means and standard deviations from three independent replicate experiments are shown. All data were compared to the control conditions and were analysed by one-way ANOVA with Dunnett’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001.

Comparative analysis of β-1,6-glucan content and structure produced by cell wall mutants.

a, d, g Percentages of cell wall β-1,6-glucans (AI and AS fractions) on total cell wall. b, e, h, β-1,6- glucans mean molecular weight (MW) and c, f, i Branching rate of β-1,6-glucans. Cells were grown in liquid SD medium at 30°C. Means and standard deviations from three independent replicate experiments are shown. All data were compared to the control conditions and were analysed by one-way ANOVA with Dunnett’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001; ns: non-significant; NA: non-applicable.

Phenotypic characterization of KRE6 family mutants: growth kinetics, drug susceptibility and filamentation.

a, Kinetic curve of all strains grown in liquid SD medium, 30°C. Optical density at 620 nm was measured every 10 min during 80 hours by TECAN SUNRISE. Means and standard deviations were calculated from three independent experiments.b, Doubling time of each strain was determined from three independent replicates. Statistical analyses were performed with one-way ANOVA with Tukey’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001; ns: non-significant. c, Spotting test of 10-fold serial dilution of yeast cells of all strains on SD medium, 30°C, 48h, with cell wall disturbing agents (CR, Congo red; CFW, CalcoFluor White) or drugs (nikkomycin, tunicamycin, caspofungin). These results are representative of three independent experiments. Pictures were taken with a Phenobooth (Singer Instruments).d, Filamentation assay of all strains. Top row, growth in liquid SD medium at 30°C; middle panels: growth in liquid YNB medium + 2% GlcNAc, buffered at pH 7.2 at 37°C during 6h. Picture were taken using an Olympus IX 83 microscope, 40x objective. Bottom row, cells were grown on agar YNB + 2% GlcNAc, buffered at pH 7.2, at 37°C for 6 days.

Cell wall electron microscopy observations of KRE6 simple and multiple mutants.

a, Representative transmission electron microscopy images of the cell wall of each strains. After culture, cells were fixed and high-pressure frozen and freeze substituted with Spurr resin. Sections were cut and stained and then pictures were taken by a Tecnai Spirit 120Kv TEM microscope. Scale bar= 200 nm.

b, Measurement of the inner and outer cell wall layers of the mutants. Means and standard deviations are represented. 37-40 measurements were performed randomly on 7-13 cells.

Statistical analyses were performed with one-way ANOVA with Tukey’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001.

Stimulation of PBMCs and neutrophils in vitro by cell wall fractions and purified β- 1,6-glucans from C. albicans.

Cytokines, chemokines or acute phase proteins (IL-8, MCP-1, IL-6, MIP-1β, IL-1β, TNF-α, RANTES, C5a, IL-10) concentrations in culture supernatants of PBMCs (a) and neutrophils (b) stimulated by cell wall fractions of C. albicans (AI-Fraction, AI-Fraction OxP and β-1,6-glucan) at 25 µg/mL or LPS (positive control, 0.1 µg/mL). PBMC cells and neutrophils were isolated from healthy human donors (n=8). Three independent batches of each fractions were used. Means are represented and data were analyzed using nonparametric Friedman test with Dunn’s multiple comparisons: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001; ns: non-significant.

β-1,6-glucan in C. albicans is a major and dynamic cell wall polymer.

a, Scheme of the cell wall of C. albicans. The proportion of each cell wall polymer was representative the results obtained on C. albicans SC5314 grown in liquid SD medium at 37°C. b, Scheme representing the dynamic of β-1,6-glucan under different environmental factors. c, β-1,6-glucan is a compensatory pathway for mannan elongation defect. d, β-1,6-glucan is a PAMP. e, Scheme of the cell wall of KRE6 family deficient mutant.

Global cell wall composition produced by C. albicans in different environmental conditions.

Results are represented as the percentage of each polymer on total cell wall in AI fraction (a), AS fraction (b) and SDS-β-ME fraction (c). Means and standard deviations were obtained from three independent replicate experiments. All data were compared to the control conditions and were analysed using one-way ANOVA with Dunnett’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001.

Cell wall composition of C. albicans mutants.

Results are represented as the percentage of each polymer on total cell wall in AI fraction (a), AS fraction (b), and SDS-β-ME fraction (c). Means and standard deviations were obtained from three independent replicate experiments. All data were compared to the parental strain SC5314 and wereanalysed using one-way ANOVA with Dunnett’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001.

Branching rates of β-1,6-glucans and β-1,3-glucans produced by C. albicans under various environmental conditions.

a, Branching rate of β-1,6-glucans from AI fraction. The branching rate was estimated by HPAEC after digestion by an endo-β-1,6-glucanase.

b, Branching rate of β-1,3-glucans from AI fraction, The branching rate was estimated by HPAEC after digestion by an endo-β-1,3-glucanase.

Means and standard deviations from three independent replicate experiments are shown. All data were compared to the control conditions and were analysed using one-way ANOVA with Dunnett’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001.

Branching rates of β-1,6-glucans and β-1,3-glucans produced by different cell wall mutants of C. albicans.

a, Branching rate of β-1,6-glucans from AI fraction. The branching rate was estimated by HPAEC after digestion by an endo-β-1,6-glucanase.

b, Branching rate of β-1,3-glucans from AI fraction. The branching rate was estimated by HPAEC after digestion by an endo-β-1,3-glucanase.

Means and standard deviations from three independent replicate experiments are shown. All data were compared to the control conditions and were analysed using one-way ANOVA with Dunnett’s multiple comparisons test: *, P < 0,05; **, P < 0,01; ***, P < 0,001; ****, P < 0,0001; NA: non- applicable.

Stimulation of PBMCs and neutrophils in vitro by β-1,6-glucan with different size from C. albicans.

Cytokines, chemokines or acute phase proteins (IL-8, MCP-1, IL-6, MIP-1β, IL-1β, TNF-α, RANTES, C5a, IL-10) concentrations in culture supernatants of PBMCs (panel a) or neutrophils (panel b) stimulated by different β-1,6-glucans from C. albicans at 25 µg/mL or LPS (positive control, 0.1 µg/mL). β-1,6-glucans were isolated from cell wall AI fraction of C. albicans grown either at 37°C in SD medium (control, β-1,6-glucan size= 58 kDa), or in the presence of caspofungin at sublethal concentration 0.015 µg/mL (β-1,6-glucan size= 70 kDa) or in the presence of 2% lactate as sole carbon source (β-1,6-glucan size= 19 kDa). PBMCs and neutrophils were isolated from healthy human donors (n=8). Three independent batches of the different fractions were used. Means are represented and data were analyzed using nonparametric Friedman test with Dunn’s multiple comparisons: ns: non-significant.

Absence of β-1,6-glucans in the cell wall of the quadruple kre6/kre62/skn2/skn1Δ/Δ mutant.

HPAEC analysis of oligosaccharides released by the endo-β-(1,6)-glucanase digestions of AI fraction of WT (a), AI fraction of kre6/kre62/skn2/skn1Δ/Δ (b), AS fraction of WT (c), AS fraction of kre6/kre62/skn2/skn1Δ/Δ (d) and water (e). Experiments were performed in triplicates. PED, pulsed electrochemical detector; nC, nanocoulomb.

Cell disruption is essential to eliminate glycogen in AI and AS fractions.

HPAEC analysis of oligosaccharides released by α-amylase enzymatic digestion of AI and AS fractions.

(a) Control: glycogen, (b) AI fraction obtained after biomass cell disruption, (c) AI fraction from biomass with no cell disruption, (d) AS fraction obtained after biomass cell disruption, (e) AS fraction from biomass with no cell disruption. PED, pulsed electrochemical detector; nC, nanocoulomb.

Quantification methods of β-1,6-glucans in AI fractions.

a, The specific oxidation of β-1,6-glucans of the AI fraction by periodate was used for quantification. Briefly, IO4Na splits bonds between vivinal carbons bearing hydroxyl groups (only present in β-1,6- glucoside), which leads to the formation of aldehydes, which can react with 4-Hydroxybenzhydrazide (PAHBAH) to form a yellow compound measurable by absorbance at OD=405 nm.

b, Specificity of the periodate oxidation method for β-1,6-glucans (pustulan). The method is specific for β-1,6-glucans (pustulan and AI fraction) and inactive on β-1,3-glucans (curdlan).

c, Linearity of β-1,6-glucan assay after periodate oxidation. We showed that the response of the method described in (a) is proportional from 0 to 20 µg of pustulan.

Control PCR control of mutants obtained in this study.

a, Principle of PCR check done in b and c. P1 and P2 are PCR diagnostic primers. ORF = KRE6, KRE62, SKN2 or SKN1. Repair Templates contain either HygB or a SAT1-Flipper cassette for selection. FRT correspond to the scar after SAT1-Flipper cassette excision. b, Table of expected PCR bands according to mutant. c, PCR bands obtained.

Control PCR of the quadruple mutant complemented for KRE6 (kre6/kre62/skn2/skn1Δ/Δ+PACT1-KRE6)

KRE6 was reintegrated at the RPS1 locus under the control of ACT1 promoter, using SAT1 as a selection marker.

Exposure of β-1,6-glucans and β-1,3-glucans at the cell surface of C. albicans SC5314.

Cells were cultured in SD at 37°C. β-glucan exposure was detected by a polyclonal rabbit anti-β-1,6- glucan serum (top panel) and or monoclonal anti-β-1,3-glucan antibody (bottom panel).

Human proteome profiler done with culture supernatant from PBMCs stimulated with C. albicans cell wall fractions.

On the left, membrane blots obtained after incubation with supernatants from PBMCs cultures stimulated by different C. albicans cell wall fractions: AI, AI-OxP, β-1,6-glucans. Incubation with the culture medium was used as a control (top). On the right, coordinate of each protein (cytokines, chemokines, acute phase proteins) detected on the membranes. The experiment was performed once using a pool of 24 supernatants from the stimulation of PBMCs isolated from 8 healthy donors, each stimulated with 3 independent batches of fractions.

Human proteome profiler done with culture supernatant from neutrophils stimulated with C. albicans cell wall fractions.

On the left, membrane blots obtained after incubation with supernatants from neutrophils cultures stimulated by different C. albicans cell wall fractions: AI, AI-OxP or β-1,6-glucans; incubation with the culture medium was used as a control (top). On the right, coordinate of each protein (cytokines, chemokines, acute phase proteins) detected on the membranes. The experiment was performed once using a pool of 24 supernatants from the stimulation of neutrophils isolated from 8 healthy donors, each stimulated with 3 independent batches of fractions.

β-1,6-glucan from C. albicans activates complement system.

a, Normal human serum (NHS) enhances the immunostimulatory capacity of β-1,6-glucan from C. albicans. PBMCs isolated from healthy human donors (n=2) were stimulated with three independent batches of β-1,6-glucan at 25 µg/mL with (w/) or without (w/o) NHS (10%). Immune response was analyzed by measuring IL-8 released into the culture medium. Means are represented and data were analyzed with an unpaired parametric t-test: ****, P < 0,0001.

b, Complement factor C3b binds to β-1,6-glucan purified from C. albicans cell wall. Three cell wall fractions (AI, AI-OxP and β-1,6-glucan) from C. albicans were coated on microtiter plates at 50 µg, 25 µg or 12.5 µg per well. Human normal serum, diluted in Gelatin-Veronal Buffer (GVB), was added to activate complement pathways. The amount of deposited C3b on each fraction (=level on complement activation) was determined by using anti-human C3b and peroxidase-conjugated anti- mouse IgG antibodies. 3,3’,5,5’-Tetramethylbenzidine (TMB) was used as the peroxidase substrate and the reaction was stopped with 4% H2SO4 and optical density (OD) was measured at 450 nm.

The experiment was done with three independent batches of each cell wall fractions. Blank value was subtracted from the values presented. Statistical analyses were performed with one-way ANOVA with Tukey’s multiple comparisons test: ****, P < 0,0001.

A model for β-1,6-glucan biosynthetic pathway and putative role of Kre6 family members in this process in yeast.

The cellular location of β-1,6-glucan synthesis in yeast is still unknown. We assume that synthesis begins intracellularly with the polymerisation of linear β-1,6-glucan chain (step 1), which requires a β-glucosyltransferase and UDP-glucose as a donor33,92 and a putative acceptor (sugar, protein, lipid). Our data (Fig. 3g, S6) suggest that Kre6 and its homologues (Kre62, Skn1, Skn2) act at this stage, but their function remains unknown. Two proposed hypotheses are: 1) Kre6 family members are β- glucosyltransferases and 2) they have glycosylhydrolase and transglycosidase activity essential for polymerisation. Step 2 is the branching of nascent β-1,6-glucan where glucosides and laminaribiosides are added to form side chains. The enzymes (β-glucosyltransferase or transglycosidase) involved in this branching remain unknown. According to our data, members of the Kre6 family are not involved in branching (Fig. 3i). Next, the polysaccharide is secreted (step 3) and then cross-linked to β-1,3-glucans in the cell wall space by an unknown transglycosidase (step 4). The transfer of GPI-anchored proteins onto β-1,6-glucan (step 5), leading to the formation of the outer layer of the cell wall, appears to be driven by Dfg5/Dcw1130. The chronology between these two cross-links (Steps 4 and 5) has not been established.

C. albicans strains used in this study.

1H and 13C NMR resonance assignments, 3JH1/H2 and 1JH1/C1 coupling constants of the monosaccharide residues of cell wall β-1,6-glucan purified from the AI fraction.

Chemical shifts are expressed in ppm and coupling constants in Hz.

Primers used in this study.