pH-DEPENDENT INFLUENCE OF A QUATERNARY AMMONIUM ...
CELLULAR & MOLECULAR BIOLOGY LETTERS
Volume 8, (2003) pp 105 – 110 http://www.cmbl.org.pl Received 12 December 2002 Accepted 24 January 2003 Short Communication pH-DEPENDENT INFLUENCE OF A QUATERNARY AMMONIUM SALT AND AN AMINOESTER ON THE YEAST Saccharomyces cerevisiae ULTRASTRUCTURE EWA OBŁĄK, RYSZARD ADAMSKI and TADEUSZ M. LACHOWICZ Institute of Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland Abstract: Quaternary ammonium salts inhibited the growth of yeast especially at pH higher (pH 8) than optimal. It was postulated that compounds integrate with the cell membrane and interfere with its functions. The yeast cell ultrastructure investigated under an electron microscope confirms this hypothesis. A relatively high percentage of cells treated at pH 6 with the quaternary ammonium salt of alanine derivative (DMALM-12) at the minimal inhibitory concentration showed an irregularity in the cell shape. No such irregularity was observed in the control. Besides, in the cells treated with the drug, practically no lipid droplets were seen at all. Inside the control cells, electron-dense round bodies were clearly seen and interpreted as vacuoles. These bodies were absent in the cells treated with DMALM-12. Although the yeast cells growing at pH 8 showed a more or less normal shape, they seemed to have difficulty in budding – no fully developed buds were found in the preparations. Only some convexities of the cell wall were seen that could be the beginning of budding which stopped early after the start. Some changes in the round bodies interpreted as vacuoles were visible: they were less dense and full of granules. Key Words: Quaternary Ammonium Salts, Lysosomotropic Aminoesters, Saccharomyces cerevisiae INTRODUCTION n-Alkyl N,N-dimethylalaninates (DMAL-n) as well as their corresponding quaternary ammonium salts (DMALM-n) were synthesized in a laboratory of the Department of Chemistry, Technical University of Wrocław, Poland. We investigated these compounds in order to find a possible relationship between Abbreviations used: DMAL-12 – the aminoester (n-alkyl N,N-dimethyl-alaninates) and DMALM-12 – its corresponding quaternary ammonium salt
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their chemical structure and biological activity on the yeast Saccharomyces cerevisiae. The growth-inhibiting activities of other quaternary ammonium salts and aminoesters were presented in previous publications [1-7]. The present paper is another contribution to the problem of the mechanism of inhibitory activity of quaternary ammonium salts and lysosomotropic amines on yeast cells. MATERIALS AND METHODS Compounds The general structure of aminoester DMAL-12 and its corresponding quaternary ammonium salt DMALM-12 is presented in Fig. 1. The compounds were synthesized according to the method described previously [8, 9]. The drugs were dissolved in water and added to a YPD medium buffered to pH 6 or pH 8 with Sörensen’s buffer (50 mM) in order to obtain a suitable final concentration. A) DMAL - 12 CH 3 CH 3 CH 3 N CH CO O C 12H25 · HCl−
B) DMALM - 12 CH 3 CH 3 + CH3 N CH CO O C 12H 25 Br− CH 3
Fig. 1. Chemical structure of aminoester DMAL-12 (n-alkyl N,N-dimethyl-alaninates) (A) and its corresponding quaternary ammonium salt DMALM-12 (B).
Strain, growth conditions and preparation of ultrathin slices Saccharomyces cerevisiae, the wild-type strain Σ1278b (α prototroph), was used in the investigation. The yeast cells were incubated for 48 hours in liquid YPD medium (1 % yeast extract Difco, 1 % Bacto peptone Difco, 2 % glucose) without and with the agent: at pH 6 in the final concentrations of DMALM-12 (10 µM), DMAL-12 (40 µM); at pH 8: DMALM-12 (2 µM), DMAL-12 (5 µM). The pH of the medium was adjusted with phosphate Sörensen’s buffer (50 mM). Yeast cells were collected by centrifugation, fixed in 2.5 % glutaraldehyde on 0.2 M phosphate buffer (PBS) of pH 7.4, rinsed several times in PBS, and fixed with 1 % OsO4. After dehydration in increasing concentrations of ethanol and acetone, the cells were embedded in Epone 812. Ultrathin slices were contrasted with uranyl acetate and then with lead citrate according to Reynolds [10]. The material prepared in this way was analyzed under transmission electron microscopes Tesla BS 540 and JEOL 100. RESULTS AND DISCUSSION The quaternary ammonium salt represented here by derivatives of alanine (DMALM-12) inhibits the growth of the yeast Saccharomyces cerevisiae at pH 6
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as well as at pH 8. However, the effects of their action, visible in the cell morphology, depend on the pH of the medium, as may be seen on the transmission electron micrographs of ultrathin sections. A considerable fraction of cells treated with this quaternary ammonium salt (DMALM-12) at pH 6, reveals irregular shapes. Their cell wall is wrinkled, the inside is filled with electron-dense grains. In such cells, it is hard to identify typical vacuoles and other cell organelles. In the cytoplasm, one can observe single lipid droplets (Fig. 2C). Cross-sections of control cells without the DMALM-12 treatment were of normal (oval) shape and, in their cytoplasm, we observed vacuoles with a uniform electron-dense structure, the cell nucleus, a large number of lipid droplets, and single mitochondria (Fig. 2A). The picture of cells treated with the quaternary ammonium salt (DMALM-12) agrees with the hypothesis concerning the mechanism of action of these compounds. According to this hypothesis, the first target of these compounds on the yeast Saccharomyces cerevisiae cells is the cell membrane system. Similarly, according to the structure, the aminoester (DMAL-12) under these conditions does not cause any deformation of yeast cell membranes. However, the compound accumulates in the vacuoles and changes their structure. Inside the vacuoles, we observed a large number of blisters which resemble lipid droplets; in some of them there appear electron-dense grains (Fig. 2B). We postulate that aminoesters, devoid of a polar “head”, penetrate freely the inside of the vacuoles. In these organelles, the molecules of the compound become protonated and, in this form, they acquire biological activity as lysosomotropic compounds. Saccharomyces cerevisiae cells incubated at pH 8, higher than the physiological optimum for these microorganisms, are characterized by an essentially smaller cell diameter. In larger cells, possibly older ones, there appear changes in the structure of the vacuole (Fig. 3A). If, under these conditions, the cells are treated with the quaternary ammonium salt (DMALM-12), a large number of cells at the stage of the budding initiation are observed. Their vacuoles are homogeneously electron-dense. However, lipid droplets show a tendency to accumulate around vacuoles. In the cytoplasm, single mitochondria are also visible (Fig. 3B). The appearance of cells can suggest that the quaternary ammonium salt (at pH 8) blocks the processes of budding. The analogous aminoester (DMAL-12), also examined at pH 8, does not cause any decrease in the yeast cell size. The process of their budding does not seem to be inhibited in the first stage. Lipid droplets are distributed evenly in the cytoplasm (Fig. 3C). Vacuoles in older cells undergo disintegration (Fig. 3D), while in younger ones they are homogeneously electron-dense (Fig. 3C). Transmission electron micrographs of the yeast cells suggest that the mechanism of action of quaternary ammonium salts as well as of aminoesters can be dependent on environmental pH.
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Fig. 2. Transmission electron micrographs of ultrathin sections of the yeast Saccharomyces cerevisiae which were incubated in liquid YPD medium at pH 6: A – without a drug (control) and in its presence in final concentrations: B – DMAL-12 (40 µM); C – DMALM-12 (10 µM). Legend: l – lipid; v – vacuole; m – mitochondria; N – cell nucleus. Bars = 10 µm.
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Fig. 3. Transmission electron micrographs of ultrathin sections of the yeast Saccharomyces cerevisiae which were incubated in liquid YPD medium at pH 8: A – without a drug (control) and in its presence in final concentrations: B – DMALM-12 (2 µM); C and D – DMAL-12 (5 µM). Legend: l – lipid; v – vacuole; m – mitochondria; N – cell nucleus. Bars = 10 µm.
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REFERENCES 1. Lachowicz T.M., Obłąk E. and Piątkowski J. Auxotrophy – stimulated sensitivity to quaternary ammonium salts and its relation to active transport in yeast. Bul. Pol. Acad. Sci. 40 ( 1992) 173-182. 2. Obłąk E., Ułaszewski S., Morawiecki A., Witek S., Witkowska R., Majcher K. and Lachowicz T.M. Quaternary ammonium salt resistant mutants in yeast Saccharomyces cerevisiae. Yeast 5 (Special Issue) (1989) 273-278. 3. Obłąk E., Ułaszewski S. and Lachowicz T.M. Mutants of Saccharomyces cerevisiae resistant to a quaternary ammonium salt. Acta Microbiol. Polon. 37 (1988) 261-269. 4. Lachowicz T.M., Witkowska R. and Obłąk E. Amino acid auxotrophy increases sensitivity of Saccharomyces cerevisiae to a quaternary ammonium salt IM. Acta Microbiol. Polon. 39 (1990) 157-162. 5. Obłąk E., Bącal J. and Lachowicz T.M. A quaternary ammonium salt as an inhibitor of plasma membrane H+-ATPase in yeast Saccharomyces cerevisiae. Cell. Mol. Biol. Lett. 5 (2000) 315-324. 6. Obłąk E., Lachowicz T.M., Łuczyński J. and Witek S. Comparative studies of biological activities of the lysosomotropic aminoesters and quaternary ammonium salts on yeast Saccharomyces cerevisiae. Cell. Mol. Biol. Lett. 6 (2001) 871-880. 7. Obłąk E., Lachowicz T.M. and Witek S. DL - leucine transport in a Saccharomyces cerevisiae mutant resistant to quaternary ammonium salts. Folia Microbiol. 41 (1996) 116-119. 8. Thompson R. Amphiphilic glycine-based esters as soft antimicrobial agents, PhD Thesis, Goeteborg 1992. 9. Obłąk E., Lachowicz T.M., Łuczyński J. and Witek S. Lysosomotropic N,N- dimethyl α-aminoacid n-alkylesters and their quaternary ammonium salts as plasma membrane and mitochondrial ATPases inhibitors. Cell. Mol. Biol. Lett. 7 (2002) 1121-1129. 10. Reynolds E.W. The use of lead citrate at high pH as an electron – opaque stain electron microscopy. J. Cell. Biol. 17 (1963) 208-212.
Volume 8, (2003) pp 105 – 110 http://www.cmbl.org.pl Received 12 December 2002 Accepted 24 January 2003 Short Communication pH-DEPENDENT INFLUENCE OF A QUATERNARY AMMONIUM SALT AND AN AMINOESTER ON THE YEAST Saccharomyces cerevisiae ULTRASTRUCTURE EWA OBŁĄK, RYSZARD ADAMSKI and TADEUSZ M. LACHOWICZ Institute of Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland Abstract: Quaternary ammonium salts inhibited the growth of yeast especially at pH higher (pH 8) than optimal. It was postulated that compounds integrate with the cell membrane and interfere with its functions. The yeast cell ultrastructure investigated under an electron microscope confirms this hypothesis. A relatively high percentage of cells treated at pH 6 with the quaternary ammonium salt of alanine derivative (DMALM-12) at the minimal inhibitory concentration showed an irregularity in the cell shape. No such irregularity was observed in the control. Besides, in the cells treated with the drug, practically no lipid droplets were seen at all. Inside the control cells, electron-dense round bodies were clearly seen and interpreted as vacuoles. These bodies were absent in the cells treated with DMALM-12. Although the yeast cells growing at pH 8 showed a more or less normal shape, they seemed to have difficulty in budding – no fully developed buds were found in the preparations. Only some convexities of the cell wall were seen that could be the beginning of budding which stopped early after the start. Some changes in the round bodies interpreted as vacuoles were visible: they were less dense and full of granules. Key Words: Quaternary Ammonium Salts, Lysosomotropic Aminoesters, Saccharomyces cerevisiae INTRODUCTION n-Alkyl N,N-dimethylalaninates (DMAL-n) as well as their corresponding quaternary ammonium salts (DMALM-n) were synthesized in a laboratory of the Department of Chemistry, Technical University of Wrocław, Poland. We investigated these compounds in order to find a possible relationship between Abbreviations used: DMAL-12 – the aminoester (n-alkyl N,N-dimethyl-alaninates) and DMALM-12 – its corresponding quaternary ammonium salt
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their chemical structure and biological activity on the yeast Saccharomyces cerevisiae. The growth-inhibiting activities of other quaternary ammonium salts and aminoesters were presented in previous publications [1-7]. The present paper is another contribution to the problem of the mechanism of inhibitory activity of quaternary ammonium salts and lysosomotropic amines on yeast cells. MATERIALS AND METHODS Compounds The general structure of aminoester DMAL-12 and its corresponding quaternary ammonium salt DMALM-12 is presented in Fig. 1. The compounds were synthesized according to the method described previously [8, 9]. The drugs were dissolved in water and added to a YPD medium buffered to pH 6 or pH 8 with Sörensen’s buffer (50 mM) in order to obtain a suitable final concentration. A) DMAL - 12 CH 3 CH 3 CH 3 N CH CO O C 12H25 · HCl−
B) DMALM - 12 CH 3 CH 3 + CH3 N CH CO O C 12H 25 Br− CH 3
Fig. 1. Chemical structure of aminoester DMAL-12 (n-alkyl N,N-dimethyl-alaninates) (A) and its corresponding quaternary ammonium salt DMALM-12 (B).
Strain, growth conditions and preparation of ultrathin slices Saccharomyces cerevisiae, the wild-type strain Σ1278b (α prototroph), was used in the investigation. The yeast cells were incubated for 48 hours in liquid YPD medium (1 % yeast extract Difco, 1 % Bacto peptone Difco, 2 % glucose) without and with the agent: at pH 6 in the final concentrations of DMALM-12 (10 µM), DMAL-12 (40 µM); at pH 8: DMALM-12 (2 µM), DMAL-12 (5 µM). The pH of the medium was adjusted with phosphate Sörensen’s buffer (50 mM). Yeast cells were collected by centrifugation, fixed in 2.5 % glutaraldehyde on 0.2 M phosphate buffer (PBS) of pH 7.4, rinsed several times in PBS, and fixed with 1 % OsO4. After dehydration in increasing concentrations of ethanol and acetone, the cells were embedded in Epone 812. Ultrathin slices were contrasted with uranyl acetate and then with lead citrate according to Reynolds [10]. The material prepared in this way was analyzed under transmission electron microscopes Tesla BS 540 and JEOL 100. RESULTS AND DISCUSSION The quaternary ammonium salt represented here by derivatives of alanine (DMALM-12) inhibits the growth of the yeast Saccharomyces cerevisiae at pH 6
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as well as at pH 8. However, the effects of their action, visible in the cell morphology, depend on the pH of the medium, as may be seen on the transmission electron micrographs of ultrathin sections. A considerable fraction of cells treated with this quaternary ammonium salt (DMALM-12) at pH 6, reveals irregular shapes. Their cell wall is wrinkled, the inside is filled with electron-dense grains. In such cells, it is hard to identify typical vacuoles and other cell organelles. In the cytoplasm, one can observe single lipid droplets (Fig. 2C). Cross-sections of control cells without the DMALM-12 treatment were of normal (oval) shape and, in their cytoplasm, we observed vacuoles with a uniform electron-dense structure, the cell nucleus, a large number of lipid droplets, and single mitochondria (Fig. 2A). The picture of cells treated with the quaternary ammonium salt (DMALM-12) agrees with the hypothesis concerning the mechanism of action of these compounds. According to this hypothesis, the first target of these compounds on the yeast Saccharomyces cerevisiae cells is the cell membrane system. Similarly, according to the structure, the aminoester (DMAL-12) under these conditions does not cause any deformation of yeast cell membranes. However, the compound accumulates in the vacuoles and changes their structure. Inside the vacuoles, we observed a large number of blisters which resemble lipid droplets; in some of them there appear electron-dense grains (Fig. 2B). We postulate that aminoesters, devoid of a polar “head”, penetrate freely the inside of the vacuoles. In these organelles, the molecules of the compound become protonated and, in this form, they acquire biological activity as lysosomotropic compounds. Saccharomyces cerevisiae cells incubated at pH 8, higher than the physiological optimum for these microorganisms, are characterized by an essentially smaller cell diameter. In larger cells, possibly older ones, there appear changes in the structure of the vacuole (Fig. 3A). If, under these conditions, the cells are treated with the quaternary ammonium salt (DMALM-12), a large number of cells at the stage of the budding initiation are observed. Their vacuoles are homogeneously electron-dense. However, lipid droplets show a tendency to accumulate around vacuoles. In the cytoplasm, single mitochondria are also visible (Fig. 3B). The appearance of cells can suggest that the quaternary ammonium salt (at pH 8) blocks the processes of budding. The analogous aminoester (DMAL-12), also examined at pH 8, does not cause any decrease in the yeast cell size. The process of their budding does not seem to be inhibited in the first stage. Lipid droplets are distributed evenly in the cytoplasm (Fig. 3C). Vacuoles in older cells undergo disintegration (Fig. 3D), while in younger ones they are homogeneously electron-dense (Fig. 3C). Transmission electron micrographs of the yeast cells suggest that the mechanism of action of quaternary ammonium salts as well as of aminoesters can be dependent on environmental pH.
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Fig. 2. Transmission electron micrographs of ultrathin sections of the yeast Saccharomyces cerevisiae which were incubated in liquid YPD medium at pH 6: A – without a drug (control) and in its presence in final concentrations: B – DMAL-12 (40 µM); C – DMALM-12 (10 µM). Legend: l – lipid; v – vacuole; m – mitochondria; N – cell nucleus. Bars = 10 µm.
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Fig. 3. Transmission electron micrographs of ultrathin sections of the yeast Saccharomyces cerevisiae which were incubated in liquid YPD medium at pH 8: A – without a drug (control) and in its presence in final concentrations: B – DMALM-12 (2 µM); C and D – DMAL-12 (5 µM). Legend: l – lipid; v – vacuole; m – mitochondria; N – cell nucleus. Bars = 10 µm.
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REFERENCES 1. Lachowicz T.M., Obłąk E. and Piątkowski J. Auxotrophy – stimulated sensitivity to quaternary ammonium salts and its relation to active transport in yeast. Bul. Pol. Acad. Sci. 40 ( 1992) 173-182. 2. Obłąk E., Ułaszewski S., Morawiecki A., Witek S., Witkowska R., Majcher K. and Lachowicz T.M. Quaternary ammonium salt resistant mutants in yeast Saccharomyces cerevisiae. Yeast 5 (Special Issue) (1989) 273-278. 3. Obłąk E., Ułaszewski S. and Lachowicz T.M. Mutants of Saccharomyces cerevisiae resistant to a quaternary ammonium salt. Acta Microbiol. Polon. 37 (1988) 261-269. 4. Lachowicz T.M., Witkowska R. and Obłąk E. Amino acid auxotrophy increases sensitivity of Saccharomyces cerevisiae to a quaternary ammonium salt IM. Acta Microbiol. Polon. 39 (1990) 157-162. 5. Obłąk E., Bącal J. and Lachowicz T.M. A quaternary ammonium salt as an inhibitor of plasma membrane H+-ATPase in yeast Saccharomyces cerevisiae. Cell. Mol. Biol. Lett. 5 (2000) 315-324. 6. Obłąk E., Lachowicz T.M., Łuczyński J. and Witek S. Comparative studies of biological activities of the lysosomotropic aminoesters and quaternary ammonium salts on yeast Saccharomyces cerevisiae. Cell. Mol. Biol. Lett. 6 (2001) 871-880. 7. Obłąk E., Lachowicz T.M. and Witek S. DL - leucine transport in a Saccharomyces cerevisiae mutant resistant to quaternary ammonium salts. Folia Microbiol. 41 (1996) 116-119. 8. Thompson R. Amphiphilic glycine-based esters as soft antimicrobial agents, PhD Thesis, Goeteborg 1992. 9. Obłąk E., Lachowicz T.M., Łuczyński J. and Witek S. Lysosomotropic N,N- dimethyl α-aminoacid n-alkylesters and their quaternary ammonium salts as plasma membrane and mitochondrial ATPases inhibitors. Cell. Mol. Biol. Lett. 7 (2002) 1121-1129. 10. Reynolds E.W. The use of lead citrate at high pH as an electron – opaque stain electron microscopy. J. Cell. Biol. 17 (1963) 208-212.
