Identification of (1→6)-β-D-Glucan as the Major Carbohydrate Component of the Malassezia Sympodialis Cell Wall

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Members of the genus Malassezia are commensal fungi found on the skin of both human and domestic animals and are associated with skin diseases including dandruff/seborrheic dermatitis, pityriasis versicolor, and atopic eczema (AE) in humans. In this study we have characterized the cell-wall carbohydrates of Malassezia sympodialis, one of the species most frequently isolated from both AE patients and healthy individuals. Cells were grown in liquid Dixon media at 32 °C, harvested, and processed using a standard Fehling's precipitation methodology for the isolation of mannan and a standard base/acid extraction for (1→3)-β-d-glucans. Using these classic extraction methods we were unable to isolate precipitable mannan or insoluble (1→3)-β-d-glucan. However, acidification and addition of methanol to the remaining Fehling's-treated sample resulted in a very clean precipitate. This material was characterized by GPC-MALLS, 1D and 2D NMR, and GC-MS for monomer-type and linkage-type composition. We determined that trace amounts of both mannan and branched (1→3, 1→6)-β-d-glucan were present in the recovered precipitate, but not linear (1→3)-β-d-glucan. Surprisingly, NMR analysis indicated that (1→6)-β-d-glucan was the major carbohydrate component isolated from M. sympodialis cell wall. GC-MS linkage analysis confirmed the (1→6)-β-d-glucan structure. Based on these studies we have determined that the M. sympodialis cell wall contains (1→6)-β-d-glucan as the major carbohydrate component along with trace amounts of mannan and (1→3, 1→6)-β-d-glucan. In addition, these data indicate that modification of the classic mannan isolation methodology may be useful in the simultaneous isolation of both mannan and (1→6)-β-d-glucan from other fungi.