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. 2024 Dec 5;13:RP100569. doi: 10.7554/eLife.100569

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

(a) Percentages of cell wall polymers on total cell wall, distributed by fractions: sodium-dodecyl-sulfate-β-mercaptoethanol (SDS-β-ME), alkali-insoluble (AI), and alkali-soluble (AS). Cells were grown in synthetic dextrose (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 nuclear magnetic resonance (NMR) analysis and high-performance anion exchange chromatography (HPAEC) after endo-β-1,6-glucanase digestion (Figure 1—figure supplement 1), 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: × kDa). (e) HPAEC 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.

Figure 1—source data 1. Raw data for panels a and b.

Figure 1.

Figure 1—figure supplement 1. 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 alkali-insoluble (AI) fraction.

Figure 1—figure supplement 1.

Chemical shifts are expressed in ppm and coupling constants in Hz.
Figure 1—figure supplement 2. Cell disruption is essential to eliminate glycogen in alkali-insoluble (AI) and alkali-soluble (AS) fractions.

Figure 1—figure supplement 2.

High-performance anion exchange chromatography (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, and (e) AS fraction from biomass with no cell disruption. PED, pulsed electrochemical detector; nC, nanocoulomb.
Figure 1—figure supplement 3. Quantification methods of β-1,6-glucans in alkali-insoluble (AI) fractions.

Figure 1—figure supplement 3.

(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.