Molecular and Biochemical Parasitology, 44 ( 1991 ) 97-108
97
Elsevier MOLBIO 01436
Phospholipid metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus David H. Beach l, George G. Holz, Jr. l, Bibhuti N. Singh I and Donald G. Lindmark 2 1Department of Microbiology and Immunology, SUNY Health Science Center, Syracuse, NY, U.S.A. and 2Department of Biology, Cleveland State University, Cleveland, OH, U.S.A. (Received 30 March 1990; accepted 27 July 1990)
Trichomonas vaginalis and Tritrichomonasfoetus grown in a fetal calf serum-based culture medium were exposed to radiolabeled phospholipids and lipid precursors to determine the extent to which these organisms can incorporate complex lipids and/or de novo synthesize their major membrane phosphoglycerides. Phosphatidylethanolamine and phosphatidylcholine were the dominant phospholipids (40-50% of extractable phospholipids), with acidic lipids, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and O-acylphosphatidylglycerol accounting for the remaining phosphoglycerides. T. vaginalis was rich in sphingomyelin while T. foetus lacks significant amounts of this lipid. Incubation with [32p]orthophosphate resulted in only modest incorporation into extractable phospholipids; the most striking observation being the failure to label choline-containing lipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin). Phosphatidylethanolamine was heavily labeled with modest labeling observed in the acidic phosphoglycerides. [U-14C]Glucose failed to label choline-containing lipids in T. foetus but did so in T. vaginalis, with phosphatidylethanolamine again being heavily labeled. Choline, phosphorylcholine, ethanolamine, serine, inositol, glycerol and methionine were incorporated poorly or failed to label the expected phosphoglycerides in either of the trichomonads, demonstrating an impairment in synthesis. Intact phosphoglycerides, labeled in the fatty acyl groups, labeled most phospholipids indicating that turnover of membrane lipids can occur with respect to the acyl component of the phospholipids. Fluorescent probes attached to phosphoglyceride molecules support observations seen with radiolabeled phosphoglycerides. Though trichomonads are able to transacylate phosphoglycerides, it is evident that the trichomonads lack a variety of enzymatic activities necessary for de novo synthesis of complex phosphoglycerides and must rely on environmental sources to supply them. Key words: Trichomonas vaginalis; Tritrichomonasfoetus; Fatty acid; Cholesterol; Cholesteryl ester; Triacylglycerol; Phospholipid; Sphingolipid; Glycolipid.
Introduction
The trichomonads Trichomonas vaginalis and Tritrichomonas foetus do not synthesize fatty acids de novo from acetate or acetate sources [1-3] when grown in vitro in a serum-based culture medium. Instead, they take up unesterifled and esterified, medium and long chain, saturated and unsaturated fatty acids from the medium [2,3]. Unesterified fatty acids are probably acCorrespondence address: Donald G. Lindmark, Department of Biology, Cleveland State University, Cleveland, OH 44115, U.S.A.
Abbreviations: TLC, thin-layer chromatography; UV, ultraviolet.
quired by non-specific binding of serum albuminfatty acid complexes to the trichomonad surface [4] while esterified and amide-linked fatty acids are obtained by non-specific binding of lipoproteins and by receptor-mediated endocytosis of lipoproteins [5]. Demonstrated sources of fatty acids are phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, triacylglycerols and cholesteryl esters [3]. The fatty acids are not employed as energy sources, nor are they structurally altered (e.g., elongated, shortened, saturated, or desaturated) [2]. Rather, they are used directly in the formation and in the turnover of phosphoglycerides and sphingolipids. They are not, however, incorporated into triacylglycerols or cholesteryl esters
0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
98 [3].
T. vaginalis and T. foetus are also unable to form cholesterol or other sterols from sources of acetate, from mevalonic acid or from squalene [1-3,6,7]. They do, however, incorporate environmental free cholesterol and cholesteryl esters, probably by non-specific lipoprotein binding and by receptor-mediated lipoprotein endocytosis [4,5]. Cholesteryl esters are deacylated and the fatty acyl groups are used to acylate other lipids; e.g., phosphoglycerides. Trichomonads do not, however, use internal or external cholesterol to form cholesteryl esters; nor do they metabolize cholesterol to form other sterols [1-3,7]. Since trichomonads were dependent upon environmental lipids, it became of particular interest to examine the metabolism of the most dynamic of the membrane lipids, the phospholipids. Accordingly, we have exposed T. vaginalis and T. foetus to radiolabeled phospholipid precursors and phospholipids and have defined the distribution of those labels among the phospholipids of the trichomonads.
Cl (82 Ci mmol-1); [1,2-14C]choline-C1 (55 Ci m o l - l ) ; phosphoryl_[methyl_14C]choline, NH4 salt (50 Ci m o l - 1); L_[methyl_3H]methionine (71 Ci mmol-l); [u-lac] glycerol (165 Ci mol-L); [1-14C]stearic acid (53 Ci mol-1); [l_14C]linoleic acid (57 Ci tool- 1); L-3-phosphatidylcholine, 1,2di[ 1-14C]palmitoyl (108 Ci m o l - l); L-3-phosphatidyl[N-methyl-3H]choline,l,2,-dipalmitoyl (76 Ci m o l - 1); L-lyso-3-phosphatidylcholine, 1-[ 1-14C]palmitoyl (55 Ci mol-1); [N-methyl- 14C]sphingomyelin (58 Ci mol-~); L-3-phosphatidylethanolamine,l,2-di[1-'4C]palmitoyl (110 Ci mol-1); L3-phosphatidyl- [2-14]ethanolamine, 1,2-dioleoyl (55 Ci m o l - ' ) ; L-3-phosphatidylinositol,l-palmitoyl2-[1-14C]linoleoyl (58 Ci mol-~); L-3-phosphatidylL-[3-'4C] serine, 1-2-dioleoyl (54 Ci m o l - 1). Purity of each radiolabeled compound was assessed by analytical thin-layer chromatography (TLC) procedures recommended by the commercial suppliers (Amersham Corp., New England Nuclear). Those with a radiochemical purity of
97
Elsevier MOLBIO 01436
Phospholipid metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus David H. Beach l, George G. Holz, Jr. l, Bibhuti N. Singh I and Donald G. Lindmark 2 1Department of Microbiology and Immunology, SUNY Health Science Center, Syracuse, NY, U.S.A. and 2Department of Biology, Cleveland State University, Cleveland, OH, U.S.A. (Received 30 March 1990; accepted 27 July 1990)
Trichomonas vaginalis and Tritrichomonasfoetus grown in a fetal calf serum-based culture medium were exposed to radiolabeled phospholipids and lipid precursors to determine the extent to which these organisms can incorporate complex lipids and/or de novo synthesize their major membrane phosphoglycerides. Phosphatidylethanolamine and phosphatidylcholine were the dominant phospholipids (40-50% of extractable phospholipids), with acidic lipids, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and O-acylphosphatidylglycerol accounting for the remaining phosphoglycerides. T. vaginalis was rich in sphingomyelin while T. foetus lacks significant amounts of this lipid. Incubation with [32p]orthophosphate resulted in only modest incorporation into extractable phospholipids; the most striking observation being the failure to label choline-containing lipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin). Phosphatidylethanolamine was heavily labeled with modest labeling observed in the acidic phosphoglycerides. [U-14C]Glucose failed to label choline-containing lipids in T. foetus but did so in T. vaginalis, with phosphatidylethanolamine again being heavily labeled. Choline, phosphorylcholine, ethanolamine, serine, inositol, glycerol and methionine were incorporated poorly or failed to label the expected phosphoglycerides in either of the trichomonads, demonstrating an impairment in synthesis. Intact phosphoglycerides, labeled in the fatty acyl groups, labeled most phospholipids indicating that turnover of membrane lipids can occur with respect to the acyl component of the phospholipids. Fluorescent probes attached to phosphoglyceride molecules support observations seen with radiolabeled phosphoglycerides. Though trichomonads are able to transacylate phosphoglycerides, it is evident that the trichomonads lack a variety of enzymatic activities necessary for de novo synthesis of complex phosphoglycerides and must rely on environmental sources to supply them. Key words: Trichomonas vaginalis; Tritrichomonasfoetus; Fatty acid; Cholesterol; Cholesteryl ester; Triacylglycerol; Phospholipid; Sphingolipid; Glycolipid.
Introduction
The trichomonads Trichomonas vaginalis and Tritrichomonas foetus do not synthesize fatty acids de novo from acetate or acetate sources [1-3] when grown in vitro in a serum-based culture medium. Instead, they take up unesterifled and esterified, medium and long chain, saturated and unsaturated fatty acids from the medium [2,3]. Unesterified fatty acids are probably acCorrespondence address: Donald G. Lindmark, Department of Biology, Cleveland State University, Cleveland, OH 44115, U.S.A.
Abbreviations: TLC, thin-layer chromatography; UV, ultraviolet.
quired by non-specific binding of serum albuminfatty acid complexes to the trichomonad surface [4] while esterified and amide-linked fatty acids are obtained by non-specific binding of lipoproteins and by receptor-mediated endocytosis of lipoproteins [5]. Demonstrated sources of fatty acids are phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, triacylglycerols and cholesteryl esters [3]. The fatty acids are not employed as energy sources, nor are they structurally altered (e.g., elongated, shortened, saturated, or desaturated) [2]. Rather, they are used directly in the formation and in the turnover of phosphoglycerides and sphingolipids. They are not, however, incorporated into triacylglycerols or cholesteryl esters
0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
98 [3].
T. vaginalis and T. foetus are also unable to form cholesterol or other sterols from sources of acetate, from mevalonic acid or from squalene [1-3,6,7]. They do, however, incorporate environmental free cholesterol and cholesteryl esters, probably by non-specific lipoprotein binding and by receptor-mediated lipoprotein endocytosis [4,5]. Cholesteryl esters are deacylated and the fatty acyl groups are used to acylate other lipids; e.g., phosphoglycerides. Trichomonads do not, however, use internal or external cholesterol to form cholesteryl esters; nor do they metabolize cholesterol to form other sterols [1-3,7]. Since trichomonads were dependent upon environmental lipids, it became of particular interest to examine the metabolism of the most dynamic of the membrane lipids, the phospholipids. Accordingly, we have exposed T. vaginalis and T. foetus to radiolabeled phospholipid precursors and phospholipids and have defined the distribution of those labels among the phospholipids of the trichomonads.
Cl (82 Ci mmol-1); [1,2-14C]choline-C1 (55 Ci m o l - l ) ; phosphoryl_[methyl_14C]choline, NH4 salt (50 Ci m o l - 1); L_[methyl_3H]methionine (71 Ci mmol-l); [u-lac] glycerol (165 Ci mol-L); [1-14C]stearic acid (53 Ci mol-1); [l_14C]linoleic acid (57 Ci tool- 1); L-3-phosphatidylcholine, 1,2di[ 1-14C]palmitoyl (108 Ci m o l - l); L-3-phosphatidyl[N-methyl-3H]choline,l,2,-dipalmitoyl (76 Ci m o l - 1); L-lyso-3-phosphatidylcholine, 1-[ 1-14C]palmitoyl (55 Ci mol-1); [N-methyl- 14C]sphingomyelin (58 Ci mol-~); L-3-phosphatidylethanolamine,l,2-di[1-'4C]palmitoyl (110 Ci mol-1); L3-phosphatidyl- [2-14]ethanolamine, 1,2-dioleoyl (55 Ci m o l - ' ) ; L-3-phosphatidylinositol,l-palmitoyl2-[1-14C]linoleoyl (58 Ci mol-~); L-3-phosphatidylL-[3-'4C] serine, 1-2-dioleoyl (54 Ci m o l - 1). Purity of each radiolabeled compound was assessed by analytical thin-layer chromatography (TLC) procedures recommended by the commercial suppliers (Amersham Corp., New England Nuclear). Those with a radiochemical purity of
