Hypoculoside, a sphingoid base-like compound from Acremonium disrupts the membrane integrity of yeast cells

We have isolated Hypoculoside, a new glycosidic amino alcohol lipid from the fungus Acremonium sp. F2434 belonging to the order Hypocreales and determined its structure by 2D-NMR (Nuclear Magnetic Resonance) spectroscopy. Hypoculoside has antifungal, antibacterial and cytotoxic activities. Homozygous profiling (HOP) of hypoculoside in Saccharomyces cerevisiae (budding yeast) revealed that several mutants defective in vesicular trafficking and vacuolar protein transport are sensitive to hypoculoside. Staining of budding yeast cells with the styryl dye FM4-64 indicated that hypoculoside damaged the vacuolar structure. Furthermore, the propidium iodide (PI) uptake assay showed that hypoculoside disrupted the plasma membrane integrity of budding yeast cells. Interestingly, the glycosidic moiety of hypoculoside is required for its deleterious effect on growth, vacuoles and plasma membrane of budding yeast cells.


Results
Isolation and structure determination of hypoculoside and aglycone hypoculine. In a screen for natural compounds with antifungal activity, we obtained an active methanolic extract from the fungal strain Acremonium sp. F2434 ( Fig. 2A) that inhibited the growth of Candida albicans in the whole cell activity assay 18 . Bioassay-guided fractionation 18 led to the isolation of a hitherto unreported glycosidic amino hydroxy lipid, hypoculoside (compound 8: Fig. 2B), possessing a 24-membered linear chain. Hypoculoside (8) was assigned a molecular formula of C 30   which is consistent with one degree of unsaturation. Hypoculoside (8) did not show any absorption above λ 210 nm in its UV spectrum, indicating the absence of conjugation. Its 1 H, 13 C NMR and multiplicity-edited gradient Heteronuclear Single Quantum Coherence (HSQC) spectroscopy indicated the presence of two terminal methyl groups (δ H 1.12,1.20 and δ C 12.1, 12.2; Table 1), one oxygenated methylene, eight oxygenated methines and  (8) and its aglycone derivative hypoculine (9). (C) Logarithmically growing Candida albicans cells were exposed to hypoculoside and amphotericin B at various concentrations in triplicates in a 96-well microplate. Growth of the cells was quantified by recording the absorbance at 600 nm after 24 hours. Growth (normalized with respect to DMSOtreated cells) is plotted against log of concentration of the compound. A picture of the microplate after 24 hours of incubation at 30 °C is shown in Supplementary Fig. S2A. (D) Effect of hypoculoside on the growth of Saccharomyces cerevisiae cells grown in YPD medium was analyzed in the similar way as for Candida albicans described above in C.
Scientific RepoRts | (2019) 9:710 | https://doi.org/10.1038/s41598-018-35979-z one methine with amino group. The remaining carbon and hydrogen atoms were assigned to long alkyl chain based on the overlapping signals for methylene protons (δ H 1. 26-1.49) and the corresponding carbon signals (δ C 27.0-34.1). Analysis of 1 H NMR, 13 Fig. S1), which corresponded to the neutral loss of a glucosyl unit from hypoculoside. Treatment of hypoculoside with 4 M HCl followed with purification by C-18 column chromatography generated the aglycone unit, hypoculine (Compound 9: Fig. 2B) and sugar. The sugar was identified as D-glucose by HPLC (Shodex KS-G and KS-801 columns with mobile phase 100% water) in comparison with an authentic standard and has the α-configuration at the anomeric carbon as shown by the coupling constant for the anomeric proton at δ H 4.94 (d, J = 3.9 Hz). Site of linkage of the D-glucose group in hypoculoside (8) was determined on the basis of HMBC correlations from H-2 to C-1′, from H-1′ to C-2 and C-3′, thereby establishing the D-glucose moiety was linked to C-2. Hypoculoside has antifungal, antibacterial and cytotoxic activities. We evaluated the potency of hypoculoside by examining its effect on the growth of Candida albicans strain (SC5314) using the Clinical and Laboratory Standards Institute (CLSI) guidelines 19 . We used the well-known antifungal amphotericin B as a positive control. We exposed logarithmically growing Candida albicans cells to hypoculoside and amphotericin B at various concentrations and monitored the growth by recording the OD 600 nm after 24 hours. Consistent with published data 20 , amphotericin B completely inhibited the growth of C. albicans cells with an IC 50 of 32 nM ( Fig. 2C and Supplementary  Fig. S2A). Hypoculoside completely inhibited the growth of C. albicans cells with an IC 50 of 7.6 μM ( Fig. 2C and Supplementary Fig. S2A). We also tested the effect of hypoculoside on the growth of the Saccharomyces cerevisiae strain (BY4743). Hypoculoside prevented the growth of Saccharomyces cerevisiae cells with an IC 50 of 7.2 μM (Fig. 2D).
To test whether hypoculoside has cytocidal or cytostatic activity, we examined the ability of Saccharomyces cerevisiae cells to recover following treatment with either hypoculoside (21 µM) or DMSO for 24 h. Unlike DMSO-treated cells, hypoculoside-treated cells were unable to grow after their transfer to hypoculoside-free YPD agar plates indicating that hypoculoside has cytocidal activity ( Supplementary Fig. S2B). We found that hypoculoside was inhibitory against the Gram-positive bacterium Staphylococcus aureus (IC 50 = 11.7 µM) but not against Gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella aerogenes ( Supplementary Fig. S2C). Hypoculoside also demonstrated cytotoxicity (IC 50 = 9-14 µM) against human lung and pancreatic carcinoma cell lines ( Supplementary Fig. S2D). These results indicate that hypoculoside has antifungal, antibacterial and cytotoxic activities.   Table 1. NMR spectral data a of hypoculoside (8) and hypoculine (9). a Assignments based on COSY, HSQCED and HMBC. b (8) and (9) were recorded at 100 MHz with CD 3 OD as internal standard at δ 49.0 Chemical shifts (δ) in ppm. c (8) and (9)    Hop analysis of hypoculoside. To gain insights into hypoculoside's Mode-Of-Action, we performed Homozygous Profiling (HOP) in Saccharomyces cerevisiae. This assay uses a collection of bar-coded yeast knockout strains created by the Saccharomyces cerevisiae Genome Deletion Project 21 , and can identify non-essential genes that confer either resistance or sensitivity to a compound in a single experiment [22][23][24] . Pooled mixture of bar-coded KO strains was grown in the absence or presence of hypoculoside for about five generations. Genomic DNA was isolated from the strains and the bar-codes were amplified by PCR and sequenced by Next Generation Sequencing methods. The sequences were then searched against the bar-code database and the Fitness co-efficient of 3702 deletion mutants were obtained (Supplementary Table S1). A negative logFC value indicates sensitivity to the compound and conversely a positive value indicates resistance. The logFC was plotted against P-value (Fig. 3A). Most of the 3702 mutants had logFC values close to 0 but there were some which had a significantly high positive or a negative value.

Validation of Hop data.
To test the validity of our HOP data, we first identified 336 'hypoculoside-sensitive' mutants (Supplementary Table S1) that had at least a 1.4-fold inhibitory effect on their growth rate (i.e. logFC ≤ −0.5) with a P-value ≤ 0.05. From the list of 336 mutants, we chose 24 mutants that represent different Gene Ontology categories (see below) and tested their sensitivity to hypoculoside. Eighteen of the total 24 mutants were sensitive to hypoculoside confirming the HOP data (Fig. 3B). We also tested mutants that were predicted to be resistant to hypoculoside (positive logFC ≥ 0.5 and P-value ≤ 0.05). But none of them were resistant to hypoculoside in comparison to wild type strain in the single-mutant sensitivity assays (data not shown). Hence, we focused on the hypoculoside-sensitive mutants for further analysis.

Biological process, Cellular Component and Molecular Function enrichment analysis. To gain
insights into hypoculoside's mode of action, we performed Gene Ontology (GO) analyses of 336 genes that provide resistance to hypoculoside using the online tool DAVID 25,26 . We used REVIGO 27 to remove the redundant GO enrichment terms and for visualization ( Fig. 4). Biological Process enrichment analysis revealed that several hypoculoside-resistance gene products were involved in vesicle-mediated transport (GO:0016192, 44 genes), endosomal transport (GO:0016197,15 genes), vacuolar transport (GO:0007034, 22 genes) and protein localization (GO:0008104, 52 genes) ( Fig. 4A and Supplementary Table S1). Cellular Component enrichment analysis indicated that several gene products were localized to the endosomes (GO:0005768, 29 genes) and Golgi apparatus (GO:0005794, 25 genes) ( Fig. 4B and Supplementary Table S1). Molecular Function enrichment analysis showed that some of the hypoculoside-resistance gene products were protein transporters (GO:0008565, 12 genes) and phospholipid binding (GO:0005543, 11 genes) ( Fig. 4C and Supplementary Table S1). Taken together, our results suggest that hypoculoside exacerbates the growth defect of mutants impaired in vesicle-mediated transport.
structure-activity studies with aglycone and commercially available sphingosines. To understand the basis of hypoculoside's mode of action, we tested the effect of modifying the structure of hypoculoside on its activity and sensitivity of the various mutants. We firstly tested whether the sugar group is required for hypoculoside's inhibitory activity. Hypoculoside inhibited the growth of wild type cells at 11 μM and the vps35, pep8, vps20, vps27 and vps36 mutants at 5.5 μM. In contrast, wild type and mutants (except for pep8 and vps35) displayed significant growth even at 200 μM hypoculine (Fig. 5A). These results indicate that the sugar moiety is required for the antifungal activity of hypoculoside. We then compared the toxicity and mutant sensitivity patterns of hypoculoside with two sphingoid bases namely phytosphingosine (PHS) (compound 3: Fig. 1) and dihydrosphingosine (DHS) (compound 2: Fig. 1), which are intermediates in the yeast sphingolipid biosynthetic pathway. PHS and DHS completely inhibited the growth of wild type yeast cells at 40 μM ( Fig. 5B) which is about 4-fold higher in comparison to inhibitory concentration of hypoculoside (Fig. 5B). Consistent with earlier data, the vps35, pep8, vps20, vps27 and vps36 mutants were completely inhibited at 5.5 µM hypoculoside but the wild type strain was slightly (10%) inhibited. At 20 µM of PHS and DHS, the growth of the 5 hypoculoside-sensitive mutants was lower in comparison to wild type strain (Fig. 5B) suggesting that the toxicity mechanisms of hypoculoside and PHS/DHS are related.

Hypoculoside does not reverse the myriocin-induced inhibitory effect on sphingolipid biosynthesis.
Myriocin is an inhibitor of Serine palmitoyltransferase (SPT), which catalyzes the first step in the synthesis of sphingolipid biosynthesis. Addition of PHS or DHS can rescue inhibition caused by myriocin 28 . We tested whether hypoculoside can rescue the toxicity of myriocin-treated cells. Myriocin inhibited the growth of yeast cells at 3.11 μM (Fig. 6). Addition of either PHS or DHS at 10 μM completely rescued the myriocin-induced inhibition (Fig. 6). In contrast, hypoculoside did not rescue the myriocin-induced inhibition but had an additive effect (i.e. total effect of hypoculoside and myriocin = sum of their individual effects) on growth (Fig. 6). These results indicate that hypoculoside cannot compensate for other sphingosines in yeast cells and it inhibits a pathway distinct from the one targeted by myriocin.
Hypoculoside disrupts the vacuolar structure of yeast cells. As several vacuolar transport mutants were sensitive to hypoculoside (Fig. 3), we examined the effect of hypoculoside on the vacuolar structure of yeast cells. We incubated wild type yeast cells with FM 4-64, a dye that stains the vacuolar membranes 29 , for 1 hour. We then washed off the excess FM 4-64 from the medium and transferred the cells into fresh medium with either hypoculoside or hypoculine or DMSO. We classified the cells into three categories namely those that had a single vacuole or those with multiple vacuoles or those with defective vacuoles (Fig. 7A). About 80% of DMSO-treated cells had multiple vacuoles and the remaining 20% cells had a single vacuole (Fig. 7B)    and vacuolar structure. In DMSO pre-treated cells, FM 4-64FX first stained the plasma membrane (Fig. 8A) at t = 0 and stained the cytoplasmic structures after 30′ indicating its uptake by endocytosis (Fig. 8A). However, in hypoculoside pre-treated cells, FM 4-64FX stained the cytoplasmic contents even at t = 0 (Fig. 8A). These results indicate that hypoculoside compromised the membrane integrity of yeast cells.  Hypoculoside has membrane disrupting activity. We then tested whether hypoculoside causes membrane leakage using the membrane impermeant dye propidium iodide (PI). Yeast cells were treated with either hypoculoside or its non-toxic aglycone derivative hypoculine at different concentrations (1.8, 3.5 and 5.3 µM) or with DMSO, for 30′ and 2 hours. We then incubated the cells with PI and visualized its uptake by fluorescence microscopy. 98% of DMSO-treated cells failed to take up PI as expected (Fig. 8B,C). However, about 60% of cells treated with hypoculoside (5.3 µM) had internalized PI after 2 hours (Fig. 8B,C). In contrast, less than 10% of aglycone-treated cells had PI staining (Fig. 8B,C). These results indicate that hypoculoside disrupts the membrane integrity of yeast cells.

Discussion
We report the discovery of a new deoxysphingoid base-related compound from Acremonium with antifungal, antimicrobial and cytotoxic activities. To the best of our knowledge, such a deoxysphingoid base has never been reported from any organism. Chemogenomic profiling analysis of hypoculoside in budding yeast indicated that it is two-fold more toxic to several vesicular trafficking mutants in comparison to the wild type strain. Interestingly, the sugar residue in hypoculoside is essential for its toxicity. This is in stark contrast to oceanipiside, another deoxysphingoid derivative whose aglycone derivative is more toxic 16 . Multiple lines of evidence suggest that hypoculoside disrupts the membranes of yeast cells. Firstly, hypoculoside has a cytocidal activity on yeast cells. Secondly, hypoculoside renders yeast cells permeable to propidium iodide which normally cannot cross the yeast plasma membrane. Thirdly, pre-treatment of yeast cells with hypoculoside resulted in immediate staining of intracellular membranes by the lipophilic dye FM 4-64. This is in contrast to control cells in which intracellular staining is only detected after 30 minutes as internalization of the dye requires endocytosis. Fourthly, hypoculoside disrupts the vacuolar structure of yeast cells. Vacuoles in yeast disintegrate in response to osmotic stress, which is thought to help in their adaptation 30 . Our observations are consistent with the idea that hypoculoside causes osmotic stress by causing membrane damage. Moreover, a number of vacuolar transport mutants were sensitive to the inhibitory action of hypoculoside.
Although we have focused on the antifungal activity of hypoculoside, it also displays antibacterial and cytotoxic activities. Hypoculoside inhibited the growth of the Gram-positive bacterium Staphyloccus aureus but not the Gram-negative bacteria Pseudomonas aeruginosa and Klebsiella aerogenes. It would be interesting to test whether hypoculoside also inhibits bacteria and mammalian cells by perturbing their plasma membranes.   Interestingly, sphingosine, dihydrosphingosine and phytosphingosine have been shown to inhibit the growth of Staphylococcus aureus and disrupt cell membrane 31 . It would be interesting to compare how hypoculoside and hypoculine interact with the plasma membrane and assess their relative effects on membrane fluidity and structure. The sugar group in hypoculoside might make it more amphipathic in comparison to hypoculine and facilitate its insertion into the lipid bilayer. It is equally intriguing to determine the biological function(s) of hypoculoside in Acremonium sp. F2434. It could either modulate the membrane structure or act as a signaling molecule or confer protection from other microbes.
Determining the biosynthetic pathway of hypoculoside in Acremonium sp. F2434 would be informative. Based on the biosynthetic pathways of related molecules like sphingosine and fumonisin B1, we hypothesize that hypoculoside biosynthesis involves addition of alanine to a hydrocarbon chain. This would explain the presence of -NH 2 and -CH 3 groups attached to the C23 in hypoculoside (compound 8: Fig. 2B). Two enzymes Fum8 and Serine palmitoyltransferase (SPT) that belong to the family of 2-oxoamine synthases have been shown to catalyze the addition of alanine to a hydrocarbon chain. Fum8, an aminotransferase involved in fumonisin B1 biosynthesis in the fungal maize pathogen Fusarium verticillioides, adds alanine onto the polyketide chain 32,33 . SPT catalyzes the first step in sphingosine biosynthesis namely the condensation of serine with palmitoyl CoA 34 . Although serine is the preferred substrate of SPT, alanine can be utilized at a low frequency and mutations in SPT have been shown to promote the use of alanine instead of serine in the condensation reaction 35 . It is therefore tempting to speculate that a homologue of Fum8/SPT in Acremonium might be involved in hypoculoside biosynthesis. The hydrocarbon chain itself could be synthesized either by a fatty acid synthase or a Polyketide synthase (PKS). Uncovering genes involved in hypoculoside biosynthesis in Acremonium and testing the phenotypic consequences of their inactivation are exciting but formidable challenges for the future.

Methods
Fermentation, extraction and isolation of hypoculoside. The fungal strain F2434 was isolated from a soil sample collected in Singapore. F2434 was sub-cultured on Malt Extract Agar (Oxoid, CM0059) for 7 days at 24°C. Three agar plugs of 5 mm diameter from the culture plate was then used to inoculate each of 40 × 250 mL Erlenmeyer flasks, each containing 50 mL of fermentation medium [sucrose 30 g/L (Merck), soluble starch 10 g/L (Sigma), malt extract 5 g/L (Sigma), yeast extract 5 g/L (Becton Dickinson), calcium carbonate 5 g/L (Sigma), vegetable juice 200 mL/L (Campbell V8), pH 6.5]. These cultures were allowed to grow for 14 days at 24 °C with shaking at 200 rpm. At the end of the incubation period, cultures from all 40 flasks were harvested and freeze dried. The freeze-dried cultures were then extracted overnight with an equal volume of 1:1 dichloromethane: methanol. The entire extraction mixture was then passed through cellulose filter paper (Whatman Grade 4) to remove the solid materials, and the filtrate was subsequently dried using a rotary evaporator.
The dried crude extract (9 g) was then re-dissolved in 8 mL of methanol and separated by reverse-phase preparative HPLC column with the gradient elution program (15% B isocratic for 5 minutes; 15% to 20% B over 5 minutes, followed by 20% to 40% B over 50 minutes, and an increase from 40% to 60% B over 20 minutes, and to 100% B in 10 minutes) generating forty fractions. Bioactivity-guided analysis of various fractions was performed by testing the effect of individual fraction on the growth of Candida albicans (ATCC 900280) as previously described 18 . Antifungal activity against Candida albicans was found to be concentrated in fraction eluting at 46-46.5 min. This fraction was concentrated and dried under reduced pressure to give hypoculoside (16 mg) as a white amorphous solid.
Purification and chemical characterization of hypoculoside. Preparative HPLC analysis was performed on the Agilent 1260 Infinity Preparative-Scale LC/MS Purification System, completed with Agilent 6130B single quadrupole mass spectrometer for LC and LC/MS Systems. The samples were separated on an Agilent Prep C18 column (100×30 mm) at flowrate of 30 mL/min by gradient elution with a mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The HRESIMS and MS/MS spectra were acquired on Agilent UHPLC 1290 Infinity coupled to Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer equipped with a splitter and an ESI source. The analysis was performed with a C18 4.6 × 75 mm, 2.7 µm column at a flowrate of 2 mL/min, under standard gradient condition of 5% to 100% acetonitrile with 0.1% formic acid over 14 minutes. The typical QTOF operating parameters were as follows: positive ionization mode; sheath gas nitrogen, 12 L/min at 295 °C; drying gas nitrogen flow, 8 L/min at 275 °C; nebulizer pressure, 30 psig; nozzle voltage, 1.5 kV; capillary voltage, 4 kV. Lock masses in positive ion mode: purine ion at m/z 121.0509 and HP-921 ion at m/z 922.0098. MS/MS data of precursor ions were acquired using collision energies between 10-40 eV and acquisition rate at 2 spectra/s. MS/MS analysis was performed with a Zorbax Eclipse Plus 2.1 × 50 mm 1.8 µm column at flowrate of 0.5 mL/min, under standard gradient condition of 5% to 100% acetonitrile with 0.1% formic acid over 10 minutes. NMR spectra were collected on a Bruker DRX-400 NMR spectrometer with Cryoprobe, using 5-mm BBI ( 1 H, G-COSY, multiplicity-edited G-HSQC, and G-HMBC spectra) or BBO ( 13 C spectra) probe heads equipped with z-gradients. Spectra were calibrated to residual protonated solvent signals (CD 3 OD δ H 3.30 and CD 3 OD δ C 49.0). Optical rotations were recorded on a JASCO P-2000 digital polarimeter. UV spectra were obtained on a GE Healthcare Ultrospec 9000 spectrophotometer.
Hydrolysis of hypoculoside. Acid-hydrolysis was performed as previously described with minor modifications 36 . Hypoculoside (8, 10 mg) was hydrolyzed in 4 M HCl (2 mL) and refluxed for 5 hours. Then the reaction flask was cooled on ice water for 3 min and 2 mL of water was added to the flask. The reaction mixture was then neutralized with ammonia. The sugar was separated from the aglycone by C18 Reversed-phase column chromatography (Phenomenex, Sepra C18-E, 50 µm, 65 A). The sugar was first eluted by using 20 mL water. The aglycone was subsequently eluted by addition of 40 mL methanol. The identity of the sugar was determined by comparing its retention time (peak detected at 20 min) with that of the authentic standard sugars by HPLC on Shodex KS-G and KS-801 columns with 100% water as mobile phase. A perfect match with the retention time of D-glucose established that hypoculoside contains D-glucose. The aglycone obtained from the 40 mL methanol eluted fraction was dried under reduced pressure to give a white amorphous solid (7 mg) which we named as hypoculine (9).
Chemical structural data. The 39 using Mega X 40 and bootstrap values based on 500 replications 41 and the results showed that F2434 was a novel species belonging to the genus Acremonium ( Supplementary Fig. S14).
Inhibition of growth assays in C. albicans and S. cerevisiae. Susceptibility of Candida albicans strain SC5314 to hypoculoside and amphotericin B was determined by a broth microdilution assay using Clinical Laboratory Standards Institute (CLSI) guidelines 42 . Cells grown to exponential phase in YPD medium (1% Yeast extract, 2% Bacto peptone and 2% glucose) were harvested, washed and resuspended in RPMI 1640 medium at a density of 2×10 3 cells/mL. RPMI 1640 medium with L-glutamine without sodium bicarbonate (Sigma) was buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) to a pH of 7.0. Two-fold dilutions of hypoculoside and amphotericin B were prepared such that final concentration of DMSO was 1% in all cultures. Cell suspension was distributed in microtiter plate (200 µL/well) and incubated at 35 °C with 220 rpm for 24 h. The Saccharomyces cerevisiae diploid wild-type strain (BY4743) was used for determining the Inhibitory Concentration (IC) of hypoculoside. Frozen yeast cells were allowed to recover on YPD agar plates and grown in YPD medium for nine generations (OD 600 nm ≤ 2). Cells were diluted to OD 600 nm of 0.0625 in YPD medium. 200 μL of the diluted yeast culture was transferred into the 96-well microtiter plate having 2-fold serially diluted concentrations of hypoculoside. Cells were incubated in a microplate reader for 16-24 hours at 30 °C with shaking. IC of hypoculoside was computed by comparing the growth of treated versus control cells.
Antibacterial and mammalian cell cytotoxicity assays. Effect of hypoculoside on the growth of three bacterial strains Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 9027) and Klebsiella aerogenes (ATCC 13048) was assessed as previously described 43 . Cytotoxicity of hypoculoside on the A549 human lung cell line and the pancreatic carcinoma cell lines MIA PaCa-2 and PANC-1 was determined as described before 43 . A549 and MIA PaCa-2 were seeded at 1,500 cells per well, and PANC-1 cells were seeded at 2,500 cells per well in a 384-well microplate. Cells were treated with hypoculoside at various concentrations and incubated for 72 hours at 37 °C in the presence of 5% CO 2 . Cytotoxic effect of hypoculoside was measured using the PrestoBlue ™ cell viability reagent (Life Technologies). Following incubation of the microplates with the dye for 2 hours, the fluorescence reading (Excitation/Emission: 560 nm/590 nm) was recorded using the Tecan Infinite M1000 Pro reader.
Homozygous profiling (HOP) assay. HOP assay was done as described previously 44 with a commercially available collection of pooled yeast homozygous Knock out collection (Invitrogen). For the assay, hypoculoside was used at 2.7 µM which caused a 60% reduction in growth in YPD medium after 10 hours at 30 °C. The uptag barcode was amplified from genomic DNA prepared from DMSO-treated and hypoculoside-treated yeast cells using a common reverse oligo R c and a variable forward oligo F v containing a sample-specific TruSeq index. Sequence of R c is 5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT GTCCACGAGGTCTCT 3′. Sequence of F v is 5′ CAAGCAGAAGACGGCATACGAGATNNNNNN GTGACTGGA GTTCAGACGTGTGCTCTTCCGATCTGTCGACCTGCAGCGTACG 3′. Nucleotides indicated in green in R c and F v are complementary to sequences flanking the bar-code in the Yeast knockout strains. Nucleotides indicated in red in R c is the sequence of the Universal Illumina Adaptor. Nucleotides in blue in F v is the sequence of the Illumina PCR Primer 2.0. Hexanucleotide NNNNNN in F v is the sample-specific TruSeq index. Nucleotides indicated in orange in F v is sequence of the Multiplexing Read 2 sequencing primer. PCR products were pooled and sequenced on MiSeq System (Illumina) with 1 × 51 single-end reads. Sequencing data was analyzed as Scientific RepoRts | (2019) 9:710 | https://doi.org/10.1038/s41598-018-35979-z described previously 44 . NGS data was deposited at the NCBI's Sequence Read Archive (SRA) database (accession: PRJNA498909).
Bioinformatic methods. Gene Ontology analysis was performed using the online tools DAVID 25,26 and REVIGO 27 .

FM 4-64FX Fluorescence microscopy.
Labelling of yeast cells with FM4-64FX (Invitrogen) was done as previously reported 45 . Overnight culture of yeast cells was diluted into fresh nutrient medium at OD 600 nm = 0.2 and grown for few hours until OD 600 nm = 0.8. Then, the cells were treated with either DMSO or 5.4 µM of hypoculoside for 30′, at 30 °C with shaking. Following that, the cells were spun down at 3000 × g, 5′ at 4 °C and washed once with 1 mL of cold YPD media. 20 μM of FM 4-64FX was then added to the cells and incubated on ice for 10′ 46 . After that, the cells were washed with 1 mL of cold YPD medium and then incubated at 30 °C for 0′, 30′ and 120′ in a shaker, before fixation. The cells were fixed with 3.7% formaldehyde for 10 min on ice and washed with Phosphate Buffer Saline (PBS). The images were acquired using an inverted fluorescence microscope ZEISS LSM 5 LIVE (Carl Zeiss, Oberkochen, Germany).

FM 4-64 Fluorescence microscopy.
Labeling of yeast cells with FM4-64 (Invitrogen) was done as previously reported 47 . Overnight culture of yeast cells was diluted into fresh nutrient medium at OD 600 nm = 0.2 and grown for few hours until OD 600 nm = 0.8. Then, the cells were labeled with 15 µM FM4-64 in YPD for 1 hour, at 30 °C with shaking. After that, the cells were washed and treated with either DMSO or various concentrations of either hypoculoside or hypoculine for 30′, at 30 °C with shaking. Following that, the cells were spun down at 3000 × g, 5′ and washed once with 1 mL of Synthetic Defined (SD) media. After that, the cells were resuspended in 30 µL of SD media. The images were acquired using an inverted fluorescence microscope ZEISS LSM 5 LIVE (Carl Zeiss, Oberkochen, Germany).
Propidium Iodide (PI)-staining assay. BY4743 cells (OD 600 nm ≈ 1) were exposed to various concentrations of hypoculoside or hypoculine or DMSO in YPD for 30′ and 2 hours. Cells were washed twice and resuspended in PBS and treated with 5 μg/mL PI for 20′ in the dark with shaking at 25 °C. PI uptake by cells was assayed by bright-field and fluorescence microscopy [Excitation/Emission (nm): 535/617] and the percentage of PI-staining cells (N ≥ 100) was calculated.