Sphingofungins are sphinganine analog mycotoxins acting as inhibitors of serine palmitoyl transferases, enzymes responsible for the first step in the sphingolipid biosynthesis. Eukaryotic cells are highly organized with various structures and organelles to facilitate cellular processes and chemical reactions, including the ones occurring as part of the secondary metabolism. We studied how sphingofungin biosynthesis is compartmentalized in the human-pathogenic fungus Aspergillus fumigatus, and we observed that it takes place in the endoplasmic reticulum (ER), ER-derived vesicles, and the cytosol. This implies that sphingofungin and sphingolipid biosynthesis colocalize to some extent. Automated analysis of confocal microscopy images confirmed the colocalization of the fluorescent proteins. Moreover, we demonstrated that the cluster-associated aminotransferase (SphA) and 3-ketoreductase (SphF) play a bifunctional role, supporting sphingolipid biosynthesis, and thereby antagonizing the toxic effects caused by sphingofungin production.

IMPORTANCE: A balanced sphingolipid homeostasis is critical for the proper functioning of eukaryotic cells. To this end, sphingolipid inhibitors have therapeutic potential against diseases related to the deregulation of sphingolipid balance. In addition, some of them have significant antifungal activity, suggesting that sphingolipid inhibitors-producing fungi have evolved mechanisms to escape self-poisoning. Here, we propose a novel self-defense mechanism, with cluster-associated genes coding for enzymes that play a dual role, being involved in both sphingofungin and sphingolipid production.