Characterization of cerebrosides from the thermally dimorphic mycopathogenHistoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-Delta(3)-unsaturation correlates with the yeast-mycelium phase transition

Cerebroside (monohexosylceramide) components were identified in neutral lip ids extracted from both the yeast and mycelial forms of the thermally dimor phic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectr ometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta -g lucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadie nine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl c omponents. However, while the fatty acid of the yeast form GlcCer was virtu ally all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characteri zed by almost exclusive expression of N-2'-hydroxy-(E)-Delta (3)-octadeceno ate. These results suggest that the yeast-mycelium transition is accompanie d by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-h ydroxy fatty N-acyl (E)-Delta (3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide b iosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), t he other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-h ydroxy fatty acids, In addition to identification of the major glucocerebro side components, minor components (<5%) detectable by molecular weight diff erences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID -MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-<Delta>(8)-unsaturation, and are hypothesized to be either biosynthe tic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these fin dings with respect to chemotaxonomy, compartmentalization of fungal glycosp hingolipid biosynthetic pathways, and regulation of morphological transitio ns in H.capsulatum and other dimorphic fungi are discussed.