Sphingolipids Influence the Sensitivity of Lipid Bilayers to Fungicide, Syringomycin E

Abstract

Sphingolipids with long chain bases hydroxylated at the C4 position are a requisite for the yeast, Saccharomyces cerevisia, to be sensitive to the ion channel forming antifungal agent, syringomycin E (SRE). A mutant S. cerevisiae strain, Deltasyr2, having sphingolipids with a sphingoid base devoid of C4-hydroxylation, is resistant to SRE. To explore the mechanism of this resistance, we investigated the channel forming activity of SRE in lipid bilayers of varying composition. We found that the addition of sphingolipid-rich fraction from Deltasyr2 to the membrane-forming solution (DOPS/DOPE/ergosterol) resulted in lipid bilayers with lower sensitivity to SRE compared with those containing sphingolipid fraction from wild-type S. cerevisiae. Other conditions being equal, the rate of increase of bilayer conductance was about 40 times slower, and the number of SRE channels was about 40 times less, with membranes containing Deltasyr2 versus wild-type sphingolipids. Deltasyr2 sphingolipids altered neither SRE single channel conductance nor the gating charge but the ability of SRE channels to open synchronously was diminished. The results suggest that the resistance of the Deltasyr2 mutant to SRE may be partly due to the ability of sphingolipids without the C4 hydroxyl group to decrease the channel forming activity of SRE.

Fungicidal Activities and Mechanisms of Action of Pseudomonas syringae pv. syringae Lipodepsipeptide Syringopeptins 22A and 25A

“…In all cases, two types of single-channel conductance fluctuations were observed, small and large, differing in the levels of conductance five to sixfold as was described earlier for SRE (Malev et al, 2002; Kaulin et al, 2005). The dwell times of the large channels were longer than those of the small ones.…”

“…Mutant strain Δ fah1 defective in sphingolipid fatty acid α-hydroxylase was more susceptible to SP22A than to SRE or SP25A (Figure 1B) but nevertheless more resistant to SP22A concentrations below 2 μM that were inhibitory to isogenic wild type strain BY4741 (Table 1). The inhibitory patterns suggest influences of target membrane lipids on the fungicidal action of SP22A and SP25A similar to those for SRE (Grilley et al, 1998; Hama et al, 2000; Stock et al, 2000; Kaulin et al, 2005). Although needed at higher concentrations with the mutants, the same relative inhibitory capabilities of SRE > SP22A > SP25A were observed regardless of the lipid biosynthesis defect.…”

“…It was also observed that SP22A and SP25A induced voltage-dependent conductance in planar lipid bilayers and that their yeast inhibitory capabilities were influenced by sphingolipid and sterol biosynthesis – phenomena characteristic of SRE effects (Feigin et al, 1996; Grilley et al, 1998; Hama et al, 2000; Stock et al, 2000). Therefore, based on what is known of SRE’s mechanism of action (Malev et al, 2002; Takemoto et al, 2003; Kaulin et al, 2005), it is suggested that the syringopeptins form stable membrane pores comprised of and modulated by host membrane lipids and particularly sphingolipids and sterols.…”

“…Studies with yeast lipid mutants revealed that sphingolipids and sterols (lipids that predominate in the plasma membrane) modulate SRE’s fungicidal activity (Cliften et al, 1996; Grilley et al, 1998; Stock et al, 2000). Lipid-modulated clustering of single channels each composed of six SRE molecules has been proposed as the structural basis for SRE’s mechanism of action on membranes (Kaulin et al, 1998, 2005; Malev et al, 2002). …”

The yeast sphingolipid signaling landscape

“…Interestingly, the assortments of sphingolipid strains that confer resistance are specific to each peptide. The sur2 Δ (Kaulin et al, 2005), csg1 Δ, csg2 Δ, ipt1 Δ (Stock et al, 2000), and elo2 Δ (Soustre et al, 2000) strains are resistant to syringomycin, while scs7 Δ alone shows resistance to PM02734 (Herrero et al, 2008), and elo3 Δ (Pagé et al, 2003), tsc10 Δ (Pagé et al, 2003), lcb3 Δ (Pagé et al, 2003), and ipt1 Δ (Tomishige et al, 2003) strains show resistance K1 killer toxin. The csg1 Δ, skn1 Δ and ipt1 Δ strains are also resistant to the pore forming fungicide Dahlia merckii antimicrobial peptide 1 (Thevissen et al, 2000).…”