Dataset Information

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

ABSTRACT: ?-(1,3)-Glucan, the major fungal cell wall component, ramifies through ?-(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 ?-(1,6)-branching on ?-(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 bgl2? and gas1? mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced ?-(1,6)-branching on the ?-(1,3)-oligomers following its ?-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear ?-(1,3)-oligomers as well as Bgl2p-catalyzed products [short ?-(1,3)-oligomers linked by a linear ?-(1,6)-linkage]. The double S. cerevisiae gas1? bgl2? mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual ?-(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 ?-(1,3)-glucan elongating and branching activity. Our report unravels the ?-(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 ?-(1,3)-glucan ramification, is poorly understood; the ramified ?-(1,3)-glucan interconnects other cell wall components. Once linear ?-(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 ?-(1,3)-glucan branching. The sick phenotype of the double Scgas1? bgl2? mutant suggested that ?-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual ?-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall ?-(1,3)-glucan branching mechanism. The essentiality of ?-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.

SUBMITTER: Aimanianda V

PROVIDER: S-EPMC5478894 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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

Aimanianda Vishukumar V Simenel Catherine C Garnaud Cecile C Clavaud Cecile C Tada Rui R Barbin Lise L Mouyna Isabelle I Heddergott Christoph C Popolo Laura L Ohya Yoshikazu Y Delepierre Muriel M Latge Jean-Paul JP

mBio 20170620 3

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 delet ...[more]

PMID: 28634239

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Project description:Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

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Dataset Information

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

Publications

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

Similar Datasets

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