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. 2023 Aug 17;11(4):e0132723.
doi: 10.1128/spectrum.01327-23. Epub 2023 Jul 10.

Cellular Responses and Targets in Food Spoilage Yeasts Exposed to Antifungal Prenylated Isoflavonoids

Sylvia Kalli  1 , Cindy Vallieres  2 , Joseph Violet  2 , Jan-Willem Sanders  3 , John Chapman  3 , Jean-Paul Vincken  1 , Simon V Avery  2 , Carla Araya-Cloutier  1
Affiliations

Cellular Responses and Targets in Food Spoilage Yeasts Exposed to Antifungal Prenylated Isoflavonoids

Sylvia Kalli et al. Microbiol Spectr. .

Abstract

Prenylated isoflavonoids are phytochemicals with promising antifungal properties. Recently, it was shown that glabridin and wighteone disrupted the plasma membrane (PM) of the food spoilage yeast Zygosaccharomyces parabailii in distinct ways, which led us to investigate further their modes of action (MoA). Transcriptomic profiling with Z. parabailii showed that genes encoding transmembrane ATPase transporters, including Yor1, and genes homologous to the pleiotropic drug resistance (PDR) subfamily in Saccharomyces cerevisiae were upregulated in response to both compounds. Gene functions involved in fatty acid and lipid metabolism, proteostasis, and DNA replication processes were overrepresented among genes upregulated by glabridin and/or wighteone. Chemogenomic analysis using the genome-wide deletant collection for S. cerevisiae further suggested an important role for PM lipids and PM proteins. Deletants of gene functions involved in biosynthesis of very-long-chain fatty acids (constituents of PM sphingolipids) and ergosterol were hypersensitive to both compounds. Using lipid biosynthesis inhibitors, we corroborated roles for sphingolipids and ergosterol in prenylated isoflavonoid action. The PM ABC transporter Yor1 and Lem3-dependent flippases conferred sensitivity and resistance, respectively, to the compounds, suggesting an important role for PM phospholipid asymmetry in their MoAs. Impaired tryptophan availability, likely linked to perturbation of the PM tryptophan permease Tat2, was evident in response to glabridin. Finally, substantial evidence highlighted a role of the endoplasmic reticulum (ER) in cellular responses to wighteone, including gene functions associated with ER membrane stress or with phospholipid biosynthesis, the primary lipid of the ER membrane. IMPORTANCE Preservatives, such as sorbic acid and benzoic acid, inhibit the growth of undesirable yeast and molds in foods. Unfortunately, preservative tolerance and resistance in food spoilage yeast, such as Zygosaccharomyces parabailii, is a growing challenge in the food industry, which can compromise food safety and increase food waste. Prenylated isoflavonoids are the main defense phytochemicals in the Fabaceae family. Glabridin and wighteone belong to this group of compounds and have shown potent antifungal activity against food spoilage yeasts. The present study demonstrated the mode of action of these compounds against food spoilage yeasts by using advanced molecular tools. Overall, the cellular actions of these two prenylated isoflavonoids share similarities (at the level of the plasma membrane) but also differences. Tryptophan import was specifically affected by glabridin, whereas endoplasmic reticulum membrane stress was specifically induced by wighteone. Understanding the mode of action of these novel antifungal agents is essential for their application in food preservation.

Keywords: Saccharomyces cerevisiae; Zygosaccharomyces parabailii; antifungal; chemogenomics; glabridin; prenylated isoflavonoids; transcriptomics; wighteone.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Molecular structure, antifungal activity against strains of Z. parabailii at pH 4.0 (MIC values from reference 11) and relevant physicochemical properties, namely, hydrophobicity (logP) and hydrogen bond (HB) acceptor (A) and donor (D) capacity, of the prenylated isoflavonoids wighteone and glabridin. The IUPAC numbering is the same for the two prenylated isoflavonoids.
FIG 2
FIG 2
Up- and downregulated GO terms (biological processes) in Z. parabailii (ATCC 60483) cells upon exposure to wighteone (A and B) and glabridin (C and D) after 30 min (A and C) and 120 min (B and D), respectively. GO terms encompass genes with both a significant (P < 0.05) and log2 fold change of >|1.0|. Biological process categories broader than those shown on the figure’s x axes are differentiated by color, as defined by the legend below the panels. Only nonredundant GO terms are shown (a semantic similarity threshold of <0.7 was used according to reference 80). Diamonds represent the number of significantly up- or downregulated genes per overrepresented biological function. Genes represented by the different GO terms, together with their description and significance (P value), are shown in Table S2.
FIG 3
FIG 3
Overview of chemogenomic screening using S. cerevisiae deletants. (A) Distribution of sensitive and resistant deletants to both prenylated isoflavonoids (solid green and red, respectively, with darker colors denoting a higher level of sensitivity or resistance) together with the fractions of affected deletants that were unique for each prenylated isoflavonoid (patterned colors). Unaffected deletants are depicted in gray. (B and C) GO enrichment analysis for biological processes in the annotations of significant genes (P < 0.05) where deletion produced sensitive (GR > 2.0) or resistant (GR ≤ 0.5) phenotypes to wighteone and glabridin. Broader biological process categories than those shown on the figures’ x axes are differentiated by color, as defined by the legend at the bottom right of the figure. Only nonredundant GO terms are shown (a semantic similarity threshold of <0.7 was used according to reference 80). Diamonds represent the number of significant genes per overrepresented biological function. Genes represented by the different GO terms together with their description and significance (P value) are shown in Table S3.
FIG 4
FIG 4
Intracellular biosynthesis pathways for ergosterol, sphingolipids, and phospholipids in S. cerevisiae based on references . In the case of blocked sphingolipid or phospholipid biosynthesis, long-chain (unsaturated) fatty acids [LC(U)FA] are imported to the cell (dashed arrows). Proteins involved in the biosynthesis of the three major lipid classes are colored based on the observed phenotype of their gene deletants upon exposure to the two prenylated isoflavonoids according to the chemogenomic screening: green indicates sensitive deletants, whereas red shows resistant deletants. “(W)” or “(G)” indicates that the specific deletant phenotype was only observed in response to either wighteone or to glabridin, respectively. The corresponding GR and P values of the deletants can be found in Table S4. Faa1 and Faa4, long-chain fatty acyl-CoA synthetases; Erg2, Δ8-sterol isomerase; Erg5, Δ22-sterol desaturase; Erg6, Δ24-sterol C-methyltransferase; Erg11, lanosterol 14α-demethylase; Spt1, serine C-palmitoyltransferase; Ydc1, alkaline dihydroceramidase; Ipt1, inositol phosphotransferase; Elo2, FA elongase of sphingolipid biosynthesis, which acts on FAs of up to C24 FAs from C18-CoA; Elo3, FA elongase of sphingolipid biosynthesis, which acts on FAs of up to C20 to C26 FAs from C18-CoA; Ale1, lysophospholipid acyltransferase; Pah1, phosphatidic acid (PA) phosphatase; Dpp1, diacylglycerol pyrophosphate; Lpp1, lipid phosphate phosphatase; Tgl3, bifunctional triacylglycerol lipase and lysophosphatidylethanolamine acyltransferase; Ept1, sn-1,2-diacylglycerol ethanolamine and choline phosphotransferase; Cho2, phosphatidylethanolamine methyltransferase; Opi3, methylene-fatty acyl-phospholipid synthase; Pct1, choline phosphate cytidylyltransferase; Cpt1, choline phosphotransferase; VLCFA, very-long-chain fatty acids; IPC, inositol phosphoryl ceramide; MIPC, mannosyl inositol phosphoryl ceramide; M(IP)2C, mannosyl-di-(inositolphosphoryl)ceramide; PI, phosphoinositol; (CDP)-DAG, (CDP) diacylglycerol; PS, phosphatidylserine; P(M)E, (N-methyl)phosphatidylethanolamine; PDE, phosphatidyldimethylethanolamine; PC, phosphatidylcholine.
FIG 5
FIG 5
Resistance of S. cerevisiae deletants lacking the genes encoding the fatty acyl-CoA synthetases FAA1 (A and B) and FAA4 (C) and sensitivity of deletants lacking the gene encoding the FA elongase, ELO2 (D), to the prenylated isoflavonoids wighteone (W) and glabridin (G). Treated S. cerevisiae faa1Δ and faa4Δ strains were cultured in the presence of 5.0 μg/mL wighteone or 10.0 μg/mL glabridin, whereas the treated elo2Δ strain was cultured in the presence of 3.8 μg/mL wighteone or 7.5 μg/mL glabridin. Filled bars represent the WT strain, and patterned bars represent the deletant strains. Values are the means ± standard deviation (SD) from three biological replicates, each performed in triplicate. Asterisks denote significant differences (P < 0.05). For the full growth curves, refer to Fig. S3A to D.
FIG 6
FIG 6
Effect of wighteone (W) and glabridin (G) on the growth of Z. parabailii (ATCC 60483) cells preadapted to myriocin (A and B) and fluconazole (C and D). Different symbols indicate different prenylated compound concentrations. The depicted curves are an example of the three biological replicates (data points are the means ± SD from technical duplicates). All biological replicates can be found in Fig. S4 and S5. Control cells and cells preadapted to myriocin or fluconazole without the addition of wighteone or glabridin are shown in Fig. S1C and D, respectively.
FIG 7
FIG 7
Phenotypes of ATPase transmembrane transporter yor1Δ, pdr53Δ, and lem3Δ deletion strains, to wighteone (W) or glabridin (G). Treated S. cerevisiae yor1Δ (A) and pdr5Δ (B) strains were cultured in the presence of 3.8 μg/mL wighteone or 7.5 μg/mL glabridin and the lem3Δ strain (C) in the presence of 5.0 μg/mL wighteone or 10.0 μg/mL glabridin. Filled bars represent the WT strain, and patterned bars represent the deletant strains. Values are the means ± SD from three biological replicates, each performed in triplicate. Asterisks denote significant differences (P < 0.05). For the full growth curves, refer to Fig. S6.
FIG 8
FIG 8
Sensitivity of trp1Δ cells to glabridin (G [7.5 μg/mL]), but not to wighteone (W [3.8 μg/mL]), (A) and rescue by TAT2 overexpression or tryptophan (T) supplementation (B). Treated S. cerevisiae WT, trp1Δ and trp1Δ′ (trp1Δ plus empty vector) strains were cultured in the presence of 1 mM tryptophan and/or 7.5 μg/mL glabridin. The values shown are the means ± SD from three biological replicates, each performed in triplicate. For the full growth curves, refer to Fig. S7A and B.
FIG 9
FIG 9
Overview of the most dominant and/or distinguishing cellular processes in food spoilage yeasts along with main associated genes that were either differentially expressed (transcriptomics) or whose deletion resulted in altered phenotype (chemogenomics) in the presence of prenylated isoflavonoids wighteone (highlighted in orange) and glabridin (highlighted in green). PM, plasma membrane; ERM, endoplasmic reticulum membrane.
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