RNA-dependent sterol aspartylation in fungi

Significance

Bacteria are known to add amino acids (aa) to membrane lipids to resist antimicrobials and escape immune responses. This surface lipid aminoacylation process requires diverting aminoacyl-tRNAs from protein synthesis. While widespread in bacteria, no analogous lipid remodeling system had thus far been evidenced in eukaryotes. We uncovered that most fungi tRNA-dependently add aspartate onto ergosterol (ergosteryl-3β- O- l-aspartate [Erg-Asp]), the major sterol found in fungal membranes. Asp addition is catalyzed by an ergosteryl-3β- O- l-aspartate synthase (ErdS) and its removal by a dedicated hydrolase (ErdH). This pathway is conserved across “higher” fungi, including pathogens. Given the central roles of sterols and derivatives in fungi, we propose that the Erg-Asp homeostasis system might impact membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.

Abstract

Diverting aminoacyl-transfer RNAs (tRNAs) from protein synthesis is a well-known process used by a wide range of bacteria to aminoacylate membrane constituents. By tRNA-dependently adding amino acids to glycerolipids, bacteria change their cell surface properties, which intensifies antimicrobial drug resistance, pathogenicity, and virulence. No equivalent aminoacylated lipids have been uncovered in any eukaryotic species thus far, suggesting that tRNA-dependent lipid remodeling is a process restricted to prokaryotes. We report here the discovery of ergosteryl-3β- O- l-aspartate (Erg-Asp), a conjugated sterol that is produced by the tRNA-dependent addition of aspartate to the 3β-OH group of ergosterol, the major sterol found in fungal membranes. In fact, Erg-Asp exists in the majority of “higher” fungi, including species of biotechnological interest, and, more importantly, in human pathogens like Aspergillus fumigatus. We show that a bifunctional enzyme, ergosteryl-3β- O- l-aspartate synthase (ErdS), is responsible for Erg-Asp synthesis. ErdS corresponds to a unique fusion of an aspartyl-tRNA synthetase—that produces aspartyl-tRNA ^Asp (Asp-tRNA ^Asp)—and of a Domain of Unknown Function 2156, which actually transfers aspartate from Asp-tRNA ^Asp onto ergosterol. We also uncovered that removal of the Asp modifier from Erg-Asp is catalyzed by a second enzyme, ErdH, that is a genuine Erg-Asp hydrolase participating in the turnover of the conjugated sterol in vivo. Phylogenomics highlights that the entire Erg-Asp synthesis/degradation pathway is conserved across “higher” fungi. Given the central roles of sterols and conjugated sterols in fungi, we propose that this tRNA-dependent ergosterol modification and homeostasis system might have broader implications in membrane remodeling, trafficking, antimicrobial resistance, or pathogenicity.