Involvement of Irreversible Vacuolar Membrane Fragmentation in

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Involvement of Irreversible Vacuolar Membrane Fragmentation in the Lethality of Food Emulsifier Diglycerol Monolaurate against Budding Yeast Chikako Ikegawa,† Akira Ogita,†,§ Takeshi Doi,# Fumitaka Kumazawa,# Ken-ichi Fujita,† and Toshio Tanaka*,† †

Graduate School of Science, §Research Center for Urban Health and Sports, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan # Taiyo Kagaku Co., 1-3 Takaramachi, Yokkaichi, Mie 510−0844, Japan ABSTRACT: Diglycerol monolaurate (DGL) has been manufactured as a novel type of food emulsifier and is being considered for further application as a food preservative. DGL lethality was thus examined against Saccharomyces cerevisiae as a model of a yeast that causes food spoilage. In spite of its molecular structure as a nonionic surfactant, DGL could exhibit lethality at a concentration lower than that which caused disruptive damage to the yeast plasma membrane. DGL lethality was rather accompanied by a dynamic intracellular event such as a marked vacuolar membrane fragmentation. In DGL-treated cells, the tiny dots or particles of fragmented vacuolar membranes failed to fuse into the original large rounded architecture after its removal from medium, which were distinguished from those generated as a result of vacuolar fission normally accelerated under hyperosmotic conditions. Such an irreversible structural damage of the organelle membrane was considered a cause of DGL lethality. KEYWORDS: diglycerol monolaurate, surfactant, vacuole, Saccharomyces cerevisiae

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INTRODUCTION The control of fungal growth is one of the inevitable goals for reduction of food spoilage.1 In spite of the current developments in food preservation methods, microbial food spoilage cannot be fully prevented. This results in serious economic losses. Among microbial species, yeast species belonging to Zygosaccharomyces bailii and Z. rouxi are considered as the major cause of microbial spoilage of foods and beverages with high sugar content and acidic components.2,3 In addition to some other yeast species, even Saccharomyces cerevisiae is found as a food spoilage yeast, especially in the wine-making industry.4 The weak acids such as benzoic, sorbic, propionic, and acetic acids are often employed as food preservatives for protection against the growth of these yeast species. Although a number of theories have been proposed to inhibit yeast growth by this class of compounds, the exact mechanism is still unknown.5 Monoglycerol monolaurate (MGL) is a naturally occurring monoglyceride composed of lauric acid and one glycerol moiety. MGL, a hydrolysis product of human breast milk triglycerides, is considered a first line of defense in breast-fed infants against invading pathogenic bacteria.6,7 However, the industrial application of this oily compound in a liquid-type food may be limited because of MGL’s poor water solubility. Therefore, a food-grade microemulsion has been developed for stable and homogeneous solubilization of MGL in an aqueous solution, where Tween 80 serves as a surfactant together with other ingredients such as sorbic acid and ethanol.8 Such a microemulsion can indeed reduce the mycelial development and the conidiation of the filamentous fungi Aspergillus niger and Penicillium italicum and is lethal to yeasts S. cerevisiae and © XXXX American Chemical Society

Candida albicans by causing plasma-membrane-disruptive damage.9,10 Diglycerol monolaurate (DGL, Figure 1a) has been manufactured as a substitute for MGL, being characterized by a highly increased water solubility. The accompanying increase in its foaming ability is also effective in emulsification of nonpolar oils like squalane and even hexadecane at room temperature.11,12 The antifungal activity of DGL was examined against S. cerevisiae and C. utilis constituting human oral yeast flora and was detected at a quite lower dose than those of weak acids like benzoic acid.13 Indeed, the fungicidal concentration of DGL could be lower than needed for solubilization of fungal plasma membrane phospholipids by means of its possible surfactant-like action. In this study, we focused on the mechanism of DGL lethality against the budding yeast S. cerevisiae as a model of food spoilage-causing yeast, which may be distinguished from those of the conventionally used nonionic surfactants.

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MATERIALS AND METHODS

Yeast Strain, Growth Conditions, and Assays of Cell Viability. Cells of S. cerevisiae W303−1A (MATa) were grown in the YPD medium (pH 7.0) consisting of 1.0% (w/v) of yeast extract (Difco Laboratories), 2.0% (w/v) of bacto-peptone (Difco Laboratories), and 2.0% (w/v) of D-glucose. Growth was performed overnight at 30 °C with vigorous shaking. Overnight-grown cells were collected by centrifugation, and were washed with and resuspended in Received: April 14, 2017 Revised: June 21, 2017 Accepted: June 23, 2017

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DOI: 10.1021/acs.jafc.7b01580 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

at 30 °C, with or without addition of each compound at various concentrations. The viable cell numbers were counted as colonyforming units (CFUs) at various incubation time points on YPD medium containing 1.8% (w/v) of agar.14 Leakage of K+ Ions and 260 nm-Absorbing Materials. Overnight-grown cells in YPD medium were harvested by centrifugation, and were washed with and resuspended in S-buffer at the cell density of 108 cells/mL. The cell suspensions, if supplemented, were supplemented with each compound and then incubated with vigorous shaking at 30 °C for 2 h. The supernatants obtained after removal of the cells by centrifugation were used for the quantification of K+ ions released from the cells. The quantification was performed with a K+ ion assay kit based on the tetraphenylborate method.15,16 The supernatants were also used for the measurement of 260 nmabsorbing materials as an index of plasma membrane disruption, representing the leakage of intracellular nucleosides, nucleotides, and other related compounds with absorption optima at 260 nm.17,18 Chemicals. DGL is a product of Taiyo Kagaku Co. (Yokkaichi, Mie, Japan). The purity of the commercially available preparation was found to be 91.1% (w/w).11 The nonionic surfactant polyoxyethylene sorbitan monolaurate (Tween 20, Figure 1c) is the product of Wako (Osaka, Japan), whereas polyethylene glycol tert-octylphenyl ether (Triton X-100, Figure 1d) is the product of Alfa Aesar (Lancashire, U.K.). The concentration of DGL was expressed as the weight concentration (w/v) in order to compare its dose-dependent lethality with the previously reported value,13 and those of Tween 20 and Triton X-100 were used as typical examples of nonionic surfactant. FM4-64 and K+ ion assay kit were purchased from Thermo Fisher Scientific (Kanagawa, Japan) and HACH Co. (Loveland, CO, U.S.A.), respectively. Amphotericin B (AmB, Figure 1b) was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). All other reagents were of analytical reagent grade. Vacuole Staining. Vacuoles were stained with the fluorescent probe FM4-64 (N-(3-triethylammoniumpropyl)-4-{6-[4-(diethylamino)-phenyl]hexatrienyl}pyridinium dibromide) according to published methods with some modifications.19−21 Briefly, overnight-grown cells (107 cells/mL) were incubated for another 1 h at 30 °C in YPD medium containing 5 μM FM4-64. The cells were then collected by centrifugation and resuspended in the malt extract medium at 107 cells/mL. The cell suspensions were then incubated with vigorous shaking at 30 °C for 2 h to examine the effects of various compounds on the vacuole morphology.

Figure 1. Structures of DGL (a), AmB (b), Tween 20 (c), and Triton X-100 (d). each of the following: distilled water (H2O), 50 mM sodium succinate buffer (S-buffer, pH 6.0), 2.5% (w/v) malt extract (Oriental Yeast Co., Ltd.) medium (pH 7.0), and YPD medium at the cell density of 107 cells/mL. The cell suspensions were incubated with vigorous shaking

Figure 2. Effects of DGL on the cell viability in the malt extract medium (a) and in H2O, S-buffer, or YPD medium (b). In (a), cells were incubated at 30 °C in malt extract medium alone (○), the medium containing AmB at 4.0 μM (Δ), the medium containing DGL at 31.3 μg/mL (●), the medium containing DGL at 62.5 μg/mL (□), and the medium containing DGL at 125 μg/mL (■). In (b), cells were incubated at 30 °C for 2 h in H2O, S-buffer, or YPD medium in the absence or presence of DGL at 62.5 μg/mL. The ratio of CFU/mL indicates the percentage of CFU/mL after 2 h incubation, in which the initial inoculum size corresponding to 107/mL was considered to be 100%. The data are means ± SD of triplicate experiments, in which the asterisk indicates significant differences. B


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Figure 3. Effects of DGL on K+ ion leakage from cells (a) and effects of Tween 20, Triton X-100, and DGL on the cell viability (b) and on leakage of 260 nm-absorbing molecules (c). In (a), cells were incubated at 30 °C for 2 h in S-buffer alone, and the buffer containing AmB or DGL at the indicated concentrations. In (b), cells were incubated at 30 °C for 2 h in malt extract medium in the absence or presence of each nonionic surfactant at the indicated concentrations. The ratio of CFU/mL indicates the percentage of CFU/mL after 2 h incubation, in which the initial inoculum size corresponding to 107/mL was considered to be 100%. In (c), cells were incubated at 30 °C for 2 h in S-buffer in the absence or presence of each nonionic surfactant at the indicated concentrations. The data are means ± SD of triplicate experiments, and n.s. indicates no significant differences, whereas the asterisk indicates significant differences. Microscopic Examination. After treatment with each compound, FM4-64-stained cells (107 cells/mL) were collected by centrifugation, washed, and resuspended in 100 μL of phosphate-buffered saline (137 mM NaCl, 8.1 mM Na2HPO4·12H2O, 2.68 mM KCl, 1.47 mM KH2PO4, pH 7.4) and imaged by bright-field and fluorescence microscopy. The cells labeled with FM4-64 were imaged with excitation at 520−550 nm and emission at 580 nm.21 Statistical Methods. The statistical evaluation involved Student’s t test, where data with p < 0.05 were considered statistically significant.

of DGL. As shown in Figure 2b, the cell viability was seriously reduced to a similar extent in H2O, S-buffer, and YPD medium, suggesting that DGL’s lethality is dependent on its disruptive effect on the yeast cellular architecture, which is maintained by plasma membrane and cell wall, rather than to an effect on a certain metabolic pathway essential for the yeast cell growth. Enhancing Effects of DGL on the Plasma Membrane Permeability. We next examined the relation between DGL lethality and the leakage of intracellular components like K+ ions as evidence of DGL-provoked plasma membrane disruption in comparison with the ion efflux from AmB-treated cells. This antifungal antibiotic can fully enhance the ion efflux from cells by creating the corresponding ion channel across the plasma membrane (Figure 3a), although K+ ion leakage is not considered a primary cause of AmB-induced cell death.14,21 As shown in Figure 3a, the extent of K+ ion efflux was significantly lower in cells treated with DGL at 62.5 μg/mL than those treated with AmB at 4.0 μM, although the cell viability was reduced upon each treatment to a similar extent (see Figure 2a). This result suggested that the plasma-membrane-disruptive damage is not the cause of cell death induced by DGL. We therefore compared the relation between the lethality and the extent of plasma membrane disruption among cell groups treated with DGL and the conventionally used nonionic surfactants Tween 20 and Triton X-100. In this experiment, the leakage of 260 nm-absorbing materials was employed as more

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RESULTS Effects of DGL on Cell Viability. On the basis of the molecular structure as nonionic surfactant, DGL was expected to cause a lethal effect on S. cerevisiae cells possibly by decomposing the ordered spatial arrangement of phospholipids essential for the maintenance of plasma membrane architecture. As shown in Figure 2a, DGL had a lethal effect on S. cerevisiae cells in the malt extract medium in a dose-dependent manner, and the apparent lethal dose of DGL (62.5 μg/mL, equivalent to 180 μM) was comparable to the value (50 μg/mL) reported in the previous study.13 However, the concentration needed for cell-death induction was much higher than that of AmB (4 μM), a typical polyene macrolide antibiotic, used as a positive control of fungicidal activity. We thus compared DGL lethality under various osmotic and nutritional conditions in order to estimate the molecular target C


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Journal of Agricultural and Food Chemistry reliable evidence of plasma membrane disruption, instead of K+ ion leakage. As shown in Figure 3b, the lethality was found to be weak with Tween 20 and strong with Triton X-100 at a concentration of 1.0 mg/mL, whereas DGL lethality was more markedly observed even at the lower concentration of 62.5 μg/ mL. In contrast, the leakage of 260 nm-absorbing materials was found only in cells treated with Triton X-100, reflecting the leakage of intracellular molecules like nucleotides as evidence of disruption or solubilization of the plasma membrane phospholipids (Figure 3c).17 The leakage of 260 nm-absorbing materials was kept at the control level when the cells were treated with either Tween 20 or DGL, thus ruling out plasmamembrane-disruptive damage as a cause of their lethality. This finding also indicates the involvement of DGL in a toxic event controlling the yeast cell survival other than the simple change of plasma membrane permeability, in addition to the involvement of Tween 20 in a less-toxic event. DGL-Induced Vacuolar Membrane Structural Damage. It seems possible to clarify the difference in the mode of lethality between DGL and other nonionic surfactants by comparing the ability to penetrate the plasma membrane and the ability to interact with the intracellular membrane-enclosed architectures. The vacuole was thus chosen as one of these architectures because it has the largest size suitable for microscopic examination. As shown in Figure 4, the yeast

cells were visible with the normal rounded vacuoles when incubated in medium alone and were visible with tiny dots or particles of fragmented vacuolar membrane in the unstained image in medium with a lethal concentration of AmB.14 The fluorescent dye was scattered throughout the cytoplasm in AmB-treated cells. The organelles retained the original normal rounded architecture when treated with Tween 20 even at the lethal concentration of 1.0 mg/mL, in agreement with its failure to cause the plasma-membrane-disruptive damage (see Figure 3c). In contrast, Triton X-100 was partly effective in causing the vacuolar membrane structural damage, reflecting its mobilization to the organelle after passage through the plasma membrane, in which the spatial arrangement of phospholipids had to be disrupted enough as a result of the action of this surfactant (see Figure 3c). Unexpectedly, the vacuolar membrane structural damage was more markedly induced in DGL-treated cells even at the lethal concentration of 62.5 μg/ mL. These findings suggest that DGL, unlike Tween 20, can mostly pass across the plasma membrane without serious structural damage to the plasma membrane phospholipid architecture. In addition, the concentration of DGL effective for the vacuolar membrane structural damage seemed much lower than is effective for solubilization of the vacuolar membrane phospholipids by means of its molecular structure as a surfactant.11,12 Irreversible Vacuolar Membrane Fragmentation in DGL-Treated Cells. Yeast cells can reversibly alter the vacuole size in response to the environmental osmotic condition so that the vacuolar membrane fragmentation or fission is accelerated under hyperosmotic conditions.22,23 This suggested the possibility that the fragmented vacuolar membrane dots or particles in DGL-treated cells may be reversibly fused into the original swollen spherical architecture when DGL is fully removed from the medium. We therefore compared DGLtriggered vacuolar membrane morphological changes with those in cells incubated under hyperosmotic conditions with 1.2 M D-sorbitol. In the presence of D-sorbitol, cells were mostly observed with tiny dots or particles of fragmented vacuoles unstained or faintly stained, with the fluorescent probe together with some clearly stained small vacuoles (Figures 5a,b). After the removal of D-sorbitol and the following incubation in medium alone, instead of the tiny dots, the swollen spherical architecture of the vacuole was observed again, reflecting their reversible fusion into the original size. The presence of D-sorbitol may be slightly toxic to cells, as seen from the partly reduced CFU count during the initial 2 h incubation, but the surviving cells could grow during the following incubation in malt extract medium alone (Figure 5b,c). In most DGL-treated cells, however, the fragmented vacuolar membranes were prevented from assembly into the swollen spherical architecture, still being observed as fragmented dots or particles in the cytoplasm even after removal of DGL (Figure 5a,b). This should result in the failure in the osmotic stress response essential for initiating the growth of DGL-treated cells even after removal of this surfactant from medium (Figure 5c). These findings are in agreement with the idea of attributing DGL lethality to its vacuole-targeting action rather than the surfactant-like disruptive action on the plasma membrane.

Figure 4. Effects of Tween 20, Triton X-100, and DGL on the vacuole morphology (a,b). Cells (107 cells/mL) were preincubated with FM464 followed by incubation at 30 °C for 2 h in malt extract medium in the absence or presence of each compound at the indicated concentrations, and were imaged by bright-field (top) and fluorescence microscopy (bottom). In (a), the most representative photographic images of bright-field (top) and fluorescence microscopy (bottom) are shown. Bars indicate 2 μm length. In (b), the ratios of cells with unfragmented vacuoles were determined by counting 10 cells at each of the 10 different stages. The data are expressed as means ± SD, in which the asterisk indicates significant differences.

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DISCUSSION MGL needs a nonaqueous delivery vehicle like ethanol to utilize it as a liquid preparation. The antibacterial and antiviral D


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illustrated in Figure 6, we observed various differences in the modes of lethality against S. cerevisiae cells among DGL and the conventional nonionic surfactants Tween 20 and Triton X-100. Tween 20 was weakly lethal at 1.0 mg/mL, though the leakage of 260 nm-absorbing materials was not observed at all in the cells treated with this surfactant (see Figure 3b,c). These results likely arise from the bulky hydrophilic head of Tween 20, which can slowly solubilize phospholipids only from the outer surface of the plasma membrane but not from the inner surface.28,29 Thus, the lethality of Tween 20 at this concentration is possibly due to its ability to extract some of plasma membraneembedded proteins essential for the yeast cell survival. At 1.0 mg/mL, Triton X-100 caused marked lethality with the accompanying leakage of 260 nm-absorbing materials (see Figures 3b,c). These findings may reflect rapid solubilization of phospholipids from both inner and outer surfaces of the plasma membrane, resulting in a highly disordered spatial arrangement of phospholipids owing to the intramembrane assembly of the surfactant. Such structural damage can be suitably elucidated by the hydrophilic−hydrophobic properties of polyoxyethylene chains, which together with the hydrophobic head constitutes the molecular structure of Triton X-100.28,29 In contrast to Triton X-100-treated cells, DGL-treated cells were not characterized by the leakage of 260 nm-absorbing materials, whereas DGL was much more lethal to the yeast cells than Triton X-100 was, clearly indicating that DGL’s lethal target is something other than the yeast plasma membrane. This study additionally points to the vacuole as the subcellular target of Triton X-100, possibly as a result of its mobilization into the yeast cytoplasm through the space generated within disrupted plasma membrane phospholipid bilayers (see Figure 4). However, the vacuolar membrane disruption was only partially achieved upon Triton X-100treatment in spite of its addition at the lethal concentration. Whereas, the vacuolar membrane structural damage could be more markedly observed at an increased ratio in response to the DGL lethality that was achieved at a significantly lower dose than Triton X-100. So far, the activities of fungal vacuolar membrane disruption have been detected with microbial metabolites such as niphimycin, AmB, and polymyxin B, and lately with polygodial as a first example of plant-derived metabolite.30−34 These compounds are more or less characterized by the enhancements’ effects on the plasma membrane permeability as reflected by the leakage of intracellular K+ ions. Among them, only AmB can selectively enhance the ion permeability by creating the corresponding ion-permeable channel across the plasma membrane, but not by nonspecific plasma membrane disruptive damage or by dysfunction such as that characterized by the concomitant leakage of 260 nmabsorbing materials. These plasma membrane-related toxic events were only slightly or not at all observed with DGLtreated cells, suggesting a highly selective interaction between this molecule and a molecular component constituting the vacuolar membrane. The yeast vacuoles are characterized by the lowest content of ergosterol among the subcellular organelles, and such low ergosterol content is likely to be related to the irreversible vacuolar membrane fragmentation in DGL-treated cells.35 Meanwhile, the coordination of reversible vacuole fission and fusion depends on the role of the vacuolar H+-ATPase (VATPase) in addition to the interactions between vacuolar SNARE proteins and the dynamin-like GTPase.36 Vacuole fusion requires the physical presence of V-ATPase, but not its

Figure 5. Irreversible fragmentation of vacuolar membrane (a,b) and the loss of cell viability (c) in DGL-treated cells. After preincubation with FM4-64, cells were incubated in malt extract medium containing none (N), the medium containing 1.2 M D-sorbitol (S), and the medium containing 62.5 μg/mL DGL (D) at 30 °C for 2 h. After centrifugation and washing the cells with fresh malt extract medium to remove D-sorbitol or DGL, cells were resuspended in the equal volume of fresh malt extract medium alone and were then incubated at 30 °C for the additional 2 h (N′, S′, D′). In (a), the most representative photographic images of bright-field (top) and fluorescence microscopy (bottom) are shown. Bars indicate 2 μm length. In (b), the ratios of cells with unfragmented vacuoles were determined by counting 10 cells at each of the 10 different stages. The data are expressed as means ± SD. In (c), the ratio of CFU/mL indicates the percentage of CFU/ mL after 2 h incubation (N, S, D) and that after the additional 2 h incubation (N′, S′, D′), in which the initial inoculum size corresponding to 107/mL was considered to be 100%. The data are expressed as means ± SD of triplicate experiments, and n.s. indicates no significant differences.

activities are suitably detected even after dilution of an MGLcontaining solvent in an aqueous medium or solution, indicating that its limited water solubility should be enough for exhibiting the biological activities.24−27 MGL can be alternatively dissolved in the form of a food-grade U-type microemulsion containing Tween 80 and some other ingredients, thus being uniformly and stably diluted with an aqueous medium to maintain the original antifungal activity.8−10 The major difference in the physicochemical properties between DGL and MGL is the much higher water solubility of DGL because of the addition of only one glycerol moiety to MGL, so that this newly developed surfactant is freely soluble in water at concentrations greater than 10 mg/mL, even at room temperature. It is therefore possible to examine the mechanism of DGL lethality in any of the given aqueous solutions or media without an interfering effect from the solvent or vehicle molecule such those needed for solubilization of MGL. Judging by the leakage of 260 nm-absorbing materials from S. cerevisiae cells treated with a food-grade microemulsion,10 the plasma membrane disruption and dysfunction seems to be the most probable fungicidal mechanism of MGL. As schematically E


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Figure 6. Schematic illustration of the relation between the lethality and vacuolar membrane-disruptive damage caused by Tween 20 (a), Triton X100 (b), and DGL (c). In (a), Tween 20 (●) interacts with phospholipids only from the outer surface of the plasma membrane, so that this surfactant extracts some of plasma membrane-embedded proteins essential for cell survival. In (b), Triton X-100 (■) interacts with phospholipids form both inner and outer surfaces, so that this surfactant can disturb their spatial arrangement to the extent sufficient for the leakage of 260 nmabsorbing materials (A260). The structural damage of the plasma membrane also enables the penetration of Triton X-100 itself partly into the cytoplasm, so that this surfactant can be mobilized to the vacuole in some cells. The goal was to cause the vacuolar-membrane-disruptive damage. In (c), DGL (▲) interacts with phospholipids to the extent permissive for the leakage of K+ ions to a limited extent, but not to the extent permissive for the leakage of 260 nm-absorbing materials (A260). In (a)−(c), the undotted circle indicates a normal vacuolar membrane, whereas the dotted circle indicates fragmented vacuolar membrane.

pump activity, while vacuole fission depends on proton translocation by V-ATPase. Vma1p, a subunit of the V-ATPase complex, similarly serves as the physical presence by being a molecular ligand, enhancing polymyxin B binding to the vacuole essential for its vacuolar membrane disruptive action.33 DGL is likely to cause irreversible fragmentation of the vacuolar membrane by inhibiting any of the molecular interactions between vacuolar SNARE proteins and dynamin-like GTPase, or by inhibiting unknown function of V-ATPase involved in the vacuole fusion. DGL may be utilized as a safe preservative against food spoilage-causing yeasts because of its possible degradation in mammalian digestive tract. This study also supports the safety of DGL as a food preservative because of its selective lethality against the food spoilage yeasts due to the irreversible vacuolar membrane structural damage.

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AUTHOR INFORMATION

Corresponding Author

*phone:+81 6 6605 3163; fax: +81 6 6605 3164; email:[email protected] ORCID

Akira Ogita: 0000-0002-1694-2356 Notes

The authors declare no competing financial interest.

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Involvement of Irreversible Vacuolar Membrane Fragmentation in

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