ABSTRACTβ-(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. UsingSaccharomyces cerevisiaeas a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. PermeabilizedS. cerevisiaeand 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, thebgl2Δ andgas1Δ 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 doubleS. cerevisiae gas1Δbgl2Δ mutant showed a drastically sick phenotype. AnScGas1p ortholog, Gel4p fromAspergillus fumigatus, also showed dual β-(1,3)-glucan elongating and branching activity. BothScGas1p andA. fumigatusGel4p 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.IMPORTANCEThe 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. UsingSaccharomyces cerevisiaeas 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 doubleScgas1Δbgl2Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition toScGas1p, GH72 familyScGas2p andAspergillus fumigatusGel4p, 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.
Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly
