The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall

Abstract

β-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(¹⁴C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual β-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core β-(1,3)-glucan ramification, is poorly understood; the ramified β-(1,3)-glucan interconnects other cell wall components. Once linear β-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the β-(1,3)-glucan branching. The sick phenotype of the double Scgas1Δ bgl2Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual β-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall β-(1,3)-glucan branching mechanism. The essentiality of β-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.

Keywords: Aspergillus fumigatus; Saccharomyces cerevisiae; beta-glucan; cell wall; remodeling.

FIG 1

S. cerevisiae cell wall β-(1,3)-glucan is β-(1,6)-branched. (A) Dionex profile of the endo-β-(1,3)-glucanase (LamA) solubilized alkali-insoluble (AI) fraction from the wild-type S. cerevisiae strain (pulse electrochemical detector [PED], gradient run I). (B) TLC of the LamA-solubilized AI fraction on a silica plate (solvent, ethyl acetate/acetic acid/water [2:1:1]). Lane 1, glucose (G) and laminarioligo standards (L₂ to L₅) containing 2 to 5 β-(1,3)-linked glucose units; lane 2, LamA-digested AI fraction, revealed by orcinol-H₂SO₄ treatment. (C) ¹H,¹³C HSQC spectrum of the gel permeation chromatography (GPC)-purified branching oligomers [linear β-(1,3)-oligomers were coisolated due to negligible differences in the M_w; corresponding signals are seen on the HSQC map]. (D) Dionex profile of the LamA-solubilized AI fraction from permeabilized S. cerevisiae incubated with UDP-(¹⁴C)glucose. (E) TLC profile of the GPC-purified ¹⁴C-labeled branched oligomers (solvent, ethyl acetate/acetic acid/water [2:1:1]). Lane 1, glucose/laminarioligo standards L₂ to L₅; lanes 2 and 3, purified radiolabeled branched trimer and tetramer, respectively. All samples were run on the same TLC plate. Lane 1 was separated and revealed by orcinol-H₂SO₄ treatment, whereas lanes 2 and 3 were subjected to autoradiography. After the samples were revealed, lane 1 was aligned with lanes 2 and 3 based on the sample application points on the TLC plate before migration. (F) Branching activity is localized in the cell wall. The cytosolic fraction did not incorporate radioactivity upon incubation with UDP-(¹⁴C)glucose followed by LamA treatment. (G) Membrane fractionation released glucose and laminaribiose, and the cell wall fraction profile showed peaks corresponding to branched oligomers (gradient run I, radiometric detection).

FIG 2

β-(1,3)-Glucan branching was decreased in the Scgas1Δ and Scbgl2Δ mutants, while the Scgas1Δ bgl2Δ mutant was devoid of branching on β-(1,3)-glucan. (A and B) Dionex profiles of the LamA-digested cell wall AI fractions from the wild-type strain, single gas1Δ mutant, single bgl2Δ mutant, and double gas1Δ bgl2Δ mutant (A) and AI fractions from the corresponding permeabilized cells incubated with UDP-(¹⁴C)glucose (B) (gradient run II for panel A and gradient run I for panel B).

FIG 3

The gas1Δ bgl2Δ deletion mutant showed a sick phenotype. (A) Growth curves. (B) Cell wall composition. Mannose, glucose, and glucosamine (GlcN) data represent mannan, β-(1,3)-glucan/β-(1,6)-glucan, and chitin content in the cell wall. (C) Calcofluor white (CFW) staining results showing dispersed bud scars and increased labeling intensity with the single gas1Δ mutant and the double gas1Δ bgl2Δ mutant. (D) Sensitivity to cell wall-perturbing agents, CFW, and Congo red (CR). Images were taken after 48 h of growth at 30°C.

FIG 4

Recombinant Gas1p and Bgl2p showed β-(1,3)-glucan branching activity. β-(1,3)-Oligomer (DP11) was incubated with Gas1p, with Bgl2p, or with both (37°C), followed by LamA digestion and Dionex profiling. (A) Bgl2p+DP11 (24 h) plus LamA addition. (B) Gas1p+DP11 (24 h) plus LamA addition. (C) Gas1p+DP24 (24 h) plus LamA addition. (D) Gas1p+Bgl2p+DP11 (20 h) plus LamA addition (PA1 column, gradient run II; the oligomeric substrates used were reduced [“r”], which released reduced glucose rG and reduced laminaribiose rL₂ [rG/rL₂] upon LamA digestion).

FIG 5

Recombinant Gas1p could utilize transferred Bgl2p products as the substrate. The figure presents Dionex profiles of Bgl2p incubated with β-(1,3)-oligomer of DP8 overnight (A) followed by either addition of LamA and analysis of the sample (B) or by heat inactivation of Bgl2p and addition of Gas1p, a further incubation performed overnight, and direct analysis of the products (C) or analysis performed after LamA treatment (D) (gradient run II and gradient run III for the PA1 [profiles C and D] and PA200 [profiles A and B] columns, respectively; rG/rL₂ [see Fig. 4]).

FIG 6

A. fumigatus Gel4p also showed β-(1,3)-glucan branching activity. (A) Similar to Gas1p, a recombinant AfGel4p could introduce β-(1,6)-linkages on the β-(1,3)-oligomers of DP11. (B) There was an increase in the amount of β-(1,6)-linkages introduced when DP24 was used as the substrate (gradient run II; rG/rL₂; see Fig. 4).

FIG 7

Dual elongating-branching activity was associated with a putative CBM, CBM43. Similar to ScGas1p and AfGel4p, ScGas2p, but not ScGas5p, AfGel1p, or AfGel2p, showed elongating-branching activity. ScGas1p, ScGas2p, and AfGel4p, but not ScGas5p, AfGel1p, and AfGel2p, are characterized by the presence of a CBM (CBM43), suggesting that the dual elongating-branching activity is associated with glycosyltransferases with a putative CBM, CBM43 (gradient run II; rG/rL₂; see Fig. 4).

FIG 8

Mechanism of S. cerevisiae cell wall β-(1,3)-glucan branching—a model. Short linear β-(1,3)-glucans are synthesized by a plasma membrane-bound glucan synthase complex using UDP-glucose as the substrate. The short linear glucans entering cell wall space undergo further elongation by Gas1p or are linked to another short β-(1,3)-glucan by Bgl2p through a linear β-(1,6)-linkage. Gas1p utilizes self-elongated glucan for branching, or it can elongate a Bgl2p-catalyzed product which contains a free carbon(C)-3 hydroxyl (−OH) group(s) on the β-(1,6)-linked glucose unit. In the following step, Gas1p further elongates and branches β-(1,3)-glucan, resulting in the formation of a ramified β-(1,3)-glucan.