Syringomycin E Inhibition of Saccharomyces cerevisiae: Requirement for Biosynthesis of Sphingolipids with Very-Long-Chain Fatty Acids and Mannose- and Phosphoinositol-Containing Head Groups

Syringomycin E is a member of a family of small cyclic lipodepsinonapeptides (ca. 1,200 Da) produced by the plant bacterium Pseudomonas syringae pv. syringae (38). Other members include syringomycin A1 and G, the syringostatins, the syringotoxins, and the pseudomycins (2, 38). All possess a characteristic tetrapeptidyl sequence (dehydroaminobutanoic acid-hydroxyaspartic acid-chlorothreonine-serine) and a β-hydroxy fatty acid attached to the N-terminal serine. These metabolites are fungicidal to a broad range of fungi, including yeast and human pathogens (33), and they show relatively low levels of toxicity to plants (21) and cutaneous animal tissues (33). Syringomycin E was recently shown to be partly responsible for the biological control of fungal pathogens on postharvest citrus fruits by certain P. syringae pv. syringae strains (5). Syringomycin E interacts with the fungal plasma membrane, where it causes K+ efflux, Ca2+ influx, and changes in membrane potential by processes that are likely related to channel formation (14, 38). Molecular genetic studies with yeast were initiated to more precisely define the antifungal mechanism of action of syringomycin E. Syringomycin E-resistant mutants of Saccharomyces cerevisiae were generated to permit identification of the mutated genes by complementation (39). Two genes, representing two of eight syringomycin E-resistant complementation groups, have been characterized. SYR1/ERG3 encodes the sterol C-5,6 desaturase for the biosynthesis of ergosterol, the primary sterol in the yeast plasma membrane (39). SYR2 is required for sphingoid base C-4 hydroxylation, a nonessential step in sphingolipid biosynthesis, revealing that C-4 OH-phytoceramide-based sphingolipids are required for syringomycin E action (7, 17). Thus, sterols and sphingolipids appear to be important factors for the susceptibility of yeast to syringomycin E. Sphingolipids are involved in numerous cellular processes, such as protein anchoring, stress responses, and apoptosis (19, 20). In S. cerevisiae, the sphingolipids are predominantly located in the plasma membrane, and they constitute about 40% of the inositol-containing lipids in this membrane (10, 12). The three major species of S. cerevisiae sphingolipids differ by polar head group composition, and they are inositolphosphoryl-ceramide (IPC), mannosyl-inositolphosphoryl-ceramide (MIPC), and mannosyl-diinositolphosphoryl-ceramide [M(IP)2C] (20). Of the three, only IPC is essential for growth in standard laboratory growth media, and the specific functions of MIPC and M(IP)2C are not yet understood (10). In addition to SYR2, several other yeast sphingolipid biosynthetic genes that are nonessential for growth have been identified (10). ELO2 and ELO3 are responsible for the conversion of C16 and C18 fatty acids to the very long chain (C20 to C26) fatty acids that are N-acylated to the ceramide moieties of sphingolipid molecules (27). Both genes provide the ability to make C20 and C24 acyl chains, but only ELO3 gives the ability to convert fatty acids from C24 to C26. ELO2 is identical to GNS1 and FEN1, and mutations of these genes confer resistance to the echinocandins, the sterol isomerase inhibitor SR31747, and fenpropimorph (13, 31). ELO3 is identical to SUR4, SRE1, and APA1; when ELO3 is mutated it confers resistance to SR31747 and causes decreased activity of the plasma membrane ATPase (9, 16, 31). Mutants with mutations in ELO2 and ELO3 have reduced levels of sphingolipids (27). IPT1 encodes the enzyme that catalyzes the terminal yeast sphingolipid biosynthetic step, which involves the transfer of phosphorylinositol from phosphatidylinositol to MIPC to form M(IP)2C and diacylglycerol (11, 23). Two genes, CSG1/SUR1 and CSG2, are necessary for mannosylation of IPC to MIPC (4). Although nonessential for growth, both are needed for growth in the presence of 50 mM Ca2+. Here we describe findings that reveal the contributions of the biosynthetic genes described above to the susceptibility of yeast to syringomycin E. Two syringomycin E action genes (SYR3 and SYR4) are shown to be identical to ELO2 and IPT1, respectively, and mutants defective in these genes are shown to display resistance to syringomycin E. In addition, ELO3, CSG1/SUR1, and CSG2 are shown to promote susceptibility to syringomycin E. The findings reveal that production of sphingolipids with fully elongated very-long-chain fatty acids and with polar head groups that possess mannose and the terminal phosphorylinositol moieties promote the antifungal action of syringomycin E.