Comp. Biochem. Physiol., 1977, Vol. 58B, pp. 195 to 199. Pergamon Press. Printed in Great Britain
LIPID COMPOSITION OF TRYPANOSOMA CRUZI M. M. OLIVEIRA,S. L. TIMM,* AND S. C. G. COSTA* Instituto de Biofisica de Universidade Federal do Rio de Janeiro and *Funda(;Ao Oswaldo Cruz, Rio de Janeiro, Brazil
(Received 19 January 1977) Abstract--1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35~o of phospholipids and 65~o of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4~o), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32p showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.
INTRODUCTION
20% heated (58°C) horse serum and 10Yo rabbit hemoglobin. Organisms from 7 days culture were collected by centrifugation at 4°C, at 1000 g for 10 min, resuspended in buffered saline and the procedure repeated twice. The final suspension in saline was immediately used for lipid extraction.
Trypanosoma cruzi, a pathogenic flagellate, has been the subject of much recent research (Brener, 1973) but its basic biology is not ~¢et fully understood, Lipids account for about 20yo of its dry weight, but, as with proteins, there is little knowledge of their functioning and metabolism (Gutteridge, 1975). Most "of the studies on its lipids dealt with neutral lipids (von Brand, 1972). The phospholipids as natural components of biological membranes may play an important role in the host-parasite relationship, since the membranes are in contact when the infection takes place. There is no detailed account on the phospholipid composition of this organism, only a qualitative study on several monocellular organisms (Hack et al., 1962). The aim of this paper is to study the lipid composition of T. cruzi, with special attention to the quantitation and metabolism of phospholipids.
Lipid extraction Lipids were extracted with chloroform-methanol essentially according to the method of Folch et al. (1957). An aliquot of the washed parasite suspension in saline (usually 5-10 ml) was extracted at room temperature with 20 vols of chloroform-methanol (2:1) containing 10mg/1 2,6-ditert-butyl-p-cresol (BHT) as antioxidant, homogenizing it with a Potter homogenizer driven manually. Nonlipid material was removed by washing the filtered extract with 0.05~o CaCI2 0.2 vol). The chloroform extract was dried under vacuum in a Biichi Rotavapor and total lipid measured gravimetrically.
MATERIALS AND METHODS All solvents were reagent grade, purchased from Merck Darmstadt. Phospholipids standards were obtained from Lipid Products, South Nutfield, England. Cholesterol, cholesterol esters, ergosterol, triolein, and oleic acid were purchased from Merck Darmstadt. The same source was used to obtain precoated silica gel thin-layer chromatographic plates. Trypanosoma strain Trypanosoma cruzi strain Y was maintained in mice by weekly passages at Instituto Oswaldo Cruz. Blood forms were obtained from infected mice between the 7th and 12th day of infection by collecting the blood in 3,8~o sodium citrate. The strain was then isolated in N N N media containing 100 units Penicillin G and 100g of streptomycin sulphate/ml.
Harvesting of organisms The flagellates were cultivated in LIT medium (Camargo, 1964) which consisted of a solution 68mM NaCI; 5 mM KCI; 56 mM Na2HPO4; 22 mM glucose and 1~o tryptose, pH 7.3. After autoclaving for 15 min at 110°C, following was added through Seitz filter: 0.5~o Bacto liver,
Thin-layer chromatography Precoated thin-layer plates were activated at 100~o prior to use. Solvent systems used for thin-layer chromatography are designated as follows: System A, chloroform-meth~ anol-acetic acid-water (65:28:8:4, v/v) a modification of a previous solvent described by Skipspi et al. (1962); System B, chloroform-methanol-acetic acid-water (38:65:8:4, v/v); System C, petroleum ether-diethyl ether-acetic acid (70:30:1, v/v) (Selztman et al., 1975); System D, chloroform-methanol-ammonium hydroxide 7N (60:35:5) Skidmore & Entenman (1962). Isolation and analysis of phospholipids An aliquot of total lipid extract (a volume containing 20-30/~g of phosphorus) was applied as a single spot to a thin-layer chromatography plate and developed in the first dimension with Solvent System A and the second dimension with System B. Phospholipids were identified using group-specific spray reagents and by co-chromatography with authentic reference lipids. For quantitative estimation of individual phospholipids the thin-layer plates were exposed to iodine vapor, the stained areas were marked, the iodine was allowed to evaporate, and appropriate areas were removed by suction. The phospholipids were eluted with three 5 ml washes of Solvent System D. The eluates were evaporated to dryness
195
196
M . M . OLIVEIRA, S. L.
under vacuum. Phosphorus determinations were performed according to the method of Bartlett (1959). Total phosphorus represents the phosphorus contained in the original lipid extract. The phospholipid content was taken as the phosphorus content multiplied by a factor of 25. Neutral lipid content was estimated by subtracting the phospholipid from the total lipid content. Isolation and analysis of neutral lipids Neutral lipids were isolated by chromatography of the lipid extracts on silicic acid columns according to the procedure of Hirsch & Ahrens (1958). Prior to chromatography the silicic acid was activated by heating to 120°C for 24 hr. The separation of neutral lipids was acomplished by single-dimension thin-layer chromatography, using System C Solvent. The classes of neutral lipids were identified by comparison with authentic reference standards. Visualization of the lipids was achieved by exposure to iodine vapours. Protein Protein was determined by the biuret method. In dried samples a previous dilution in 10% (w/v) NaOH was done before the determination. All chemical determinations were done in duplicates.
T]MM
AND S. C. G. COSTA
Table 1. Phospholipid percentual composition of T. cruzi. The values are the average _+ S.E.M. of five different preparations Phospholipid
%
Origin Lysolecithin Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylethanolamine Phosphatidic acid Cardiolipin Unidentified
1.9 0.7 3.8 44.2 12.3 27.8 4.2 2.1 2.9
+ + + + + + + + +
0.6 0.2 0.9 1.5 1.0 0.9 0.9 1.1 1.2
Incubations with 3zp a2pi obtained from the Brazilian Institute of Atomic Energy was purified by extraction as phosphomolybdate with isobutyl alcohol/benzene, re-extraction to the aqueous phase with ammonia, and precipitation as MgNH4PO4 (Kanazawa & Boyer, 1973). To each bottle of 200 ml of culture medium was added 0.1 ml of 32Pi (Na salt) 0.1 mCi. Every 2 days the trypanosomes were harvested, four bottles
PEA
o
--. PI
U
SM -. L L
tO O
Origen b
sgt ye.ta Fig. 1. Phospholipids pattern of T. cruzi. Two-dimensional thin-layer chromatography stained by iodine vapour. LL-lysolecithin, SM-sphingomyelin, PC-phosphatidylcholine, U-unidentified, PI-phosphatidylinositol, PEA-phosphatidylethanolamine, PA-phosphatidic acid, CLP-cardiolipin and NL-neutral lipids.
Lipids of T. cruzi in the 2nd day of culture and two bottles in the 4th and 7th days. The collected mass was divided in half and lipid extracted as the usual procedure, and chromatographed in system A and system B. Three chromatographic plates were done for every lipid extract. One of them was used for radioautography using short time exposure (3 days) of the plate to Kodak Medical X-ray film PP/S54. The radioactive spots were eluted as the usual procedure, evaporated to dryness, dissolved in 0.4 ml of chloroform and half of the amount was used for phosphorus determination, and half was used for radioactive counting on a Beckman LS-150 Liquid scintillator counter, with a mixture of 50 mg of 1,4-bis-(5-phenyloxazol-2-yl) benzene plus 3.5g of 2,5-dipbenytoxazole in 11. of toluene as the scintillant.
RESULTS
Total lipids
The total lipid content of the epimastigote form of T. cruzi in the log phase of growth was 0.18 mg/mg protein as the average of five different preparations. This corresponded to 15% of its dry weight. Phospholipids
Phospholipids (PL)~comprised 34.7% (+0.6) of total lipids extractable from T. cruzi. This value represents the average of duplicate determinations made on five different preparations, all in the same log phase of growth. The differential distribution of phospholipids is shown on Table 1. Phosphatidylcholine (PC) was the most abundant phosphoglyceride with 44% of the total found. Phosphatidylethanolamine (PEA) was the second with 27.8% but phosphatidylserine (PS) was found in trace amounts and not always present. Phosphatidylinositol (PI) was present in 12.3% being the third major component of the PL. Cardiolipin was found close to the solvent front as 2% of the total. Sphingomyelin was consistently present in 3.8% of all phospholipids. An unidentified spot, probably plasmalogen, ninhydrin negative, was found in 2.9%. Lysolecithin had a variable appearance and made only 0.7% of the total. Phosphatidic acid corresponded to 4.2% of the PL. At the origin of the chromatograms was always found a phosphorus-containing material which amounted to 1.9%. These percentages were calculated from phosphorus determinations in eluted spots from duplicate chromatograms. 20.000
--
PEA
197
Figure 1 shows a typical two-dimensional chromatogram of total lipids, showing the phospholipids stained with iodine vapors. Labeling with 32p
In order to better understand the role of phospholipids during the growth of trypanosome in culture, in a preliminary experiment we have added 32pi to the culture medium, harvested the flagellates in the 2nd, 4th and 7th day, extracted the lipids and separated the phospholipids for quantitation and radioactive counting. The net amount of the individual phospholipids did not vary significantly, but the specific labeling of each one was different. The time-course of the incorporation of Pi into the individual phospholipids pools is depicted in Fig. 2. As can be seen the highest specific activity was found in phosphatidylethanolamine, followed by phosphatidylinositol, and phosphatidylcholine. In these three phospholipids a maximum incorporation was observed in the 4th day of growth, declining in the 7th day. Lysolecithin did not incorporate 32p. Sphingomyelin incorporated very little radioactivity, and it was difficult to quantitare it without error. The same can be said for phosphatidic acid and cardiolipin due to the small amount of phosphorus in the spot. The unidentified phospholipid (plasmalogen?) had a time-course similar to the three major ones, having a specific activity of 3400 in the 2nd day, 13,000 in the 4th day, and 3000 in the 7th day. All values were corrected for the phosphorus decay. Radioautography performed in these experiments (not shown) did not reveal any further spot that had not been detected by chemical means. Neutral lipids
Neutral lipids of trypanosomatids, including T. cruzi have been studied in detail (for a review see von Brand, 1973) so we have only'made a qualitative observation. Figure 3 shows a chromatogram of lipids from T. cruzi developed in solvent system C and stained with iodine vapors. At the origin are phospholipids that did not move with the solvent. Diglycerides are seen at RI 0.02, cholesterol is seen at Rj. 0.21 ergosterol moving just ahead, traces of free fatty acid can be observed at R~ 0.47, triglycerides as the major component are detected at RI 0.77 and cholesterol esters can also be seen at Rs 0.85. Neutral lipids are the major part of total lipids from T. cruzi in culture making up to 65% of its total.
~\,,,\ \\\,
I
5,000
DISCUSSION
--
PI
,~
-
I0.000
--
Pe
,~\~ ~ q
~,~
-
o
\ \ \
,\\,
-
sooo
\\\\
,~\~
- -
_
--
\\\ \\\
\\~ ~ ~ 2
4
7
2
4
7
2
4
7
DAYS
Fig. 2. 32p uptake by T. cruzi at different phases of growth in culture media.
Meaningful lipid data on trypanosomes depend on the homogeneity and lack of contamination of the preparation. Our data was obtained from organisms in log phase of growth, between 7 and 8 days of culture. The method chosen for lipid extraction, i.e. Folch's procedure gave better results for this particular kind of preparation than the method of Bligh & Dyer (1959). The experimental procedure was carried out in freshly harvested organisms, because either freezing or freeze-drying would induce the formation of lysophosphatides. Only two preparations--the lipid extract and the protein determination--were done in
198
M . M . OLIVEIRA, S. L. TIMM AND S. C. G. COSTA
Cholesterol ester Triglycerides
g
Free Fatty Acids
t ~
8
,
Mixture
~
I
I
Diglyceride PL
8
I
Ergosterol Cholesterol
I
Oleic acid Ergosterol Prep B Triolein Cholesterol Prep A
Fig. 3. Neutral lipids pattern of T. cruzi. One-dimensional chromatography developed in system C solvent and stained by iodine vapours. the dried material in order to correlate our findings with the dry weight of the protozoans. The value found for total lipids is within the range found for other trypanosomes (Godfrey, 1967) and for the previous report on T. cruzi (von Brand, 1973). Phospholipids This class of compounds have not been studied in detail in T. cruzi. Hack et aL (1962) made a comparative investigation among flagellates, but did not report the percentage of phospholipids in the total lipids fraction, later Williamson et al. (1965) reported a percentage of 17.2~ of phospholipids. In our preparations we found considerably more than that, i.e. 34.7~o, which may be due to different phase of growth of the organisms, although both results were obtained with flagellates in culture. The total amount of phospholipids in 7". cruzi seem to be much lower that the 80~o found for T. brucei rhodesiense (Dixon & Williamson, 1970) and 60~o by Godfrey (1967) and Venkatesan & Ormerod (1976). Dixon & Williamson (1970) also found values ranging from 72 to 79~o of phospholipids in 7". lewisi B, T. lewisi C, and T. rhodesiense C, in culture form. Godfrey (1967) found from 54 to 65~o of phospholipids in T. lewisi. T. vivax, T. congolense, all in blood form. Although small differences may be due to technical procedures, such great differences shown in our results seem to indicate a very distinct distribution of lipids in the epimastigote form of T. cruzi as compared with the other trypanosomes. It can be seen in Table 1 that lecithin is the major
phospholipid in T. cruzi, as it is in the other trypanosomatids studied so far. It is interesting to note, however, that it incorporated comparatively little 32pi that was added in the culture medium (Fig. 2). For that matter, phosphatidylethanolamine was much more metabolically active, since it had the highest specific activity. This phosphoglyceride the second more abundant phospholipid, shows the same type of distribution among some other trypanosomatids such as 7", rhodesiense and T. lewisi, which increase the percentage of PEA when grown in culture (Dixon & Williamson, 1970). In the extensive work of Godfrey (1967) he showed that the blood forms of T. vivax, T. congelense, and T. brucei had either equal or slightly higher values for sphyngomyelin than phosphatidylethanolamine. In our preparations sphingomyelin had a very low percentage, only about 4~/o of the total, but it was constant in all preparations studied. Phosphatidylinositol was the third more frequent phospholipid, and also was metabolically active, as judged by 3zp uptake. It amounted to 12~o of the total, a much higher value than what was found for T. brucei rhodesiense (1~,~o). No other data was reported for this phospholipid. Cardiolipin was found in 2~o of total phospholipids. Hack et al. (1962) did not report cardiolipin in his studies on T. cruzi lipids, but his methods of detection might have been insufficient. In T. brucei rhodesiense only trace amounts of cardiolipin was found, and about 4 ~ of phosphatidylserine, the opposite o r " cruzi where only trace amounts of this phospholipid
Lipids of T, cruzi was found. Trace amounts of cardiolipin was also found in T. rhodesiense and T. lewisi (Dixon & Williamson, 1970). Lysolecithin was found in very small amounts and it seems to be present in the organisms and not a product of degradation during the extraction procedure, because it did not incorporate radioactive phosphate while lecithin did. Our results indicate that the phospholipid distribution of T. cruzi epimastygote form is quite distinct of other trypanosomatids studied so far. It will be interesting to see if this particular distribution will keep the same in the other forms of T. cruzi. Neutral lipids
They are the major part of total lipids of T. cruzi in culture, comprising 65% of the total. This value is in accordance with previous report by Williamson et al. (1965). We have made only a qualitative observation among the various classes of neutral lipids, since it has been studied in detail by other groups (Von Brand, 1972). One interesting feature is the large amount of triglyceride, the major component found (Fig. 3). It may suggest as a storage lipid that is accumulated when the flagellate is in culture. In the excellent work of Dixon & Williamson (1970) they have shown a spectacular increase in the content of triglyceride in T. rhodesiense and in T. lewisi in the 7th day of culture as compared with the amount found in blood form, pointing out the same type of phenomenon. Acknowledgements--We are grateful to Professor Carlos Chagas for the use of his laboratory and to Dr Leopoldo de Meis for the gift of purified radioactive phosphorus. This investigation was partially supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico grant No 2222.0032/75 and grant No 3521/75.
199
BRENSER Z. (1973) Biology' of Trypanosoma cruzi. Ann. Rev. Mierobiol. 27, 347-381. CAMARGOE. C. (1964) Growth and differentiation in Trypanosoma cruzi. Origin of metacyclic trypanosomes in liquid media. Rev. Inst. Med. Trop. S&~ Paulo 6, 93-100. Dixon H. & WlLLIAMSONJ. (1970) The lipid composition of Trypanosoma lewisi and Trypanosoma rhodesiense. Comp. Biochem. Physiol. 33, 11-128. DIXON H., GINGERC. D. & WILL1AMSONJ. (1972) Trypanosome sterols and their metabolic origins. Comp. Biochem. Physiol. 41B, 1-18. FOLCH J., LEES M. & SLOANE STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. GODFREYD. G. (1967) Phospholipids of Trypanosoma lewisi, T. vivax, T. congolense and T. brucei. Expl Parasit. 20, 106-118. GUTTERIDGEW. F. (1975) Biochemistry of Trypanosoma cruzi, Proceedings of the International Symposium on New Approaches in American Trypanosomiases Research. Pan American Sanitary Bureau Scientific Publications No. 318. HACK M. H., YAEGERR. G. & MACCAFFERVT. D. (1962) Comparative lipid biochemistry. II Lipids of plants and animal flagellates, a non-motile alga, an ameba and a cilliate. Comp. Biochem. Physiol. 6, 247-252. HIRSCH J. & AHRENSJR. E. H. (1958) The separation of complex lipide mixtures by the use of silicic acid chromatography. J. biol. Chem. 233, 311-320. KANAZAWA T. & BOYER P. D. (1973) Occurrence and characteristics of a rapid exchange of phosphate oxygen catalyzed by sarcoplasmic reticulum vesicles. J. biol. Chem. 248, 3163-3172. SELZTMANT. P., FINN F. M., WIDNELL C. C. 8/; HOFMANN K. (1975) Lipids of bovine adrenal plasma membranes. J. biol. Chem. 250, 1193 1196. SKIDMOREW. D. 8L ENTENMAN C. (1962) Two-dimensional
thin-layer chromatography of rat liver phosphatides. J. Lipid Res. 3, 471-475. SKIPSKIV. P., PETERSONR. F. & BARCLAYM. (1962) Separation of phosphatidylethanolamine, phosphatidylserine and other phospholipids by thin-layer chromatography. J. Lipid Res. 3, 467-470. VENKATESAN S. t~ ORMEROD W. E. (1976) Lipid content
REFERENCES
BARTLETT G. R. (1959) Phosphorous assay in column chromatography. J. biol. Chem. 234, 466-468. BLIGH E. G. & DYER W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.
of the slender and stumpy forms of Trypanosoma brucei rhodesiense: A comparative study. Comp. Biochem. Physiol. 53B, 481-487. VON BRANDT. (1973) Biochemistry of Parasites. 2nd edn. Academic Press, New York. WILLIAMSON J. & GINGER C. D. (1965) Lipid constitution of some protozoa, spirochaetes and bacteria. Trans. R. Soc. trop. Med. Hyg. 59, 366-367.
LIPID COMPOSITION OF TRYPANOSOMA CRUZI M. M. OLIVEIRA,S. L. TIMM,* AND S. C. G. COSTA* Instituto de Biofisica de Universidade Federal do Rio de Janeiro and *Funda(;Ao Oswaldo Cruz, Rio de Janeiro, Brazil
(Received 19 January 1977) Abstract--1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35~o of phospholipids and 65~o of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4~o), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32p showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.
INTRODUCTION
20% heated (58°C) horse serum and 10Yo rabbit hemoglobin. Organisms from 7 days culture were collected by centrifugation at 4°C, at 1000 g for 10 min, resuspended in buffered saline and the procedure repeated twice. The final suspension in saline was immediately used for lipid extraction.
Trypanosoma cruzi, a pathogenic flagellate, has been the subject of much recent research (Brener, 1973) but its basic biology is not ~¢et fully understood, Lipids account for about 20yo of its dry weight, but, as with proteins, there is little knowledge of their functioning and metabolism (Gutteridge, 1975). Most "of the studies on its lipids dealt with neutral lipids (von Brand, 1972). The phospholipids as natural components of biological membranes may play an important role in the host-parasite relationship, since the membranes are in contact when the infection takes place. There is no detailed account on the phospholipid composition of this organism, only a qualitative study on several monocellular organisms (Hack et al., 1962). The aim of this paper is to study the lipid composition of T. cruzi, with special attention to the quantitation and metabolism of phospholipids.
Lipid extraction Lipids were extracted with chloroform-methanol essentially according to the method of Folch et al. (1957). An aliquot of the washed parasite suspension in saline (usually 5-10 ml) was extracted at room temperature with 20 vols of chloroform-methanol (2:1) containing 10mg/1 2,6-ditert-butyl-p-cresol (BHT) as antioxidant, homogenizing it with a Potter homogenizer driven manually. Nonlipid material was removed by washing the filtered extract with 0.05~o CaCI2 0.2 vol). The chloroform extract was dried under vacuum in a Biichi Rotavapor and total lipid measured gravimetrically.
MATERIALS AND METHODS All solvents were reagent grade, purchased from Merck Darmstadt. Phospholipids standards were obtained from Lipid Products, South Nutfield, England. Cholesterol, cholesterol esters, ergosterol, triolein, and oleic acid were purchased from Merck Darmstadt. The same source was used to obtain precoated silica gel thin-layer chromatographic plates. Trypanosoma strain Trypanosoma cruzi strain Y was maintained in mice by weekly passages at Instituto Oswaldo Cruz. Blood forms were obtained from infected mice between the 7th and 12th day of infection by collecting the blood in 3,8~o sodium citrate. The strain was then isolated in N N N media containing 100 units Penicillin G and 100g of streptomycin sulphate/ml.
Harvesting of organisms The flagellates were cultivated in LIT medium (Camargo, 1964) which consisted of a solution 68mM NaCI; 5 mM KCI; 56 mM Na2HPO4; 22 mM glucose and 1~o tryptose, pH 7.3. After autoclaving for 15 min at 110°C, following was added through Seitz filter: 0.5~o Bacto liver,
Thin-layer chromatography Precoated thin-layer plates were activated at 100~o prior to use. Solvent systems used for thin-layer chromatography are designated as follows: System A, chloroform-meth~ anol-acetic acid-water (65:28:8:4, v/v) a modification of a previous solvent described by Skipspi et al. (1962); System B, chloroform-methanol-acetic acid-water (38:65:8:4, v/v); System C, petroleum ether-diethyl ether-acetic acid (70:30:1, v/v) (Selztman et al., 1975); System D, chloroform-methanol-ammonium hydroxide 7N (60:35:5) Skidmore & Entenman (1962). Isolation and analysis of phospholipids An aliquot of total lipid extract (a volume containing 20-30/~g of phosphorus) was applied as a single spot to a thin-layer chromatography plate and developed in the first dimension with Solvent System A and the second dimension with System B. Phospholipids were identified using group-specific spray reagents and by co-chromatography with authentic reference lipids. For quantitative estimation of individual phospholipids the thin-layer plates were exposed to iodine vapor, the stained areas were marked, the iodine was allowed to evaporate, and appropriate areas were removed by suction. The phospholipids were eluted with three 5 ml washes of Solvent System D. The eluates were evaporated to dryness
195
196
M . M . OLIVEIRA, S. L.
under vacuum. Phosphorus determinations were performed according to the method of Bartlett (1959). Total phosphorus represents the phosphorus contained in the original lipid extract. The phospholipid content was taken as the phosphorus content multiplied by a factor of 25. Neutral lipid content was estimated by subtracting the phospholipid from the total lipid content. Isolation and analysis of neutral lipids Neutral lipids were isolated by chromatography of the lipid extracts on silicic acid columns according to the procedure of Hirsch & Ahrens (1958). Prior to chromatography the silicic acid was activated by heating to 120°C for 24 hr. The separation of neutral lipids was acomplished by single-dimension thin-layer chromatography, using System C Solvent. The classes of neutral lipids were identified by comparison with authentic reference standards. Visualization of the lipids was achieved by exposure to iodine vapours. Protein Protein was determined by the biuret method. In dried samples a previous dilution in 10% (w/v) NaOH was done before the determination. All chemical determinations were done in duplicates.
T]MM
AND S. C. G. COSTA
Table 1. Phospholipid percentual composition of T. cruzi. The values are the average _+ S.E.M. of five different preparations Phospholipid
%
Origin Lysolecithin Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylethanolamine Phosphatidic acid Cardiolipin Unidentified
1.9 0.7 3.8 44.2 12.3 27.8 4.2 2.1 2.9
+ + + + + + + + +
0.6 0.2 0.9 1.5 1.0 0.9 0.9 1.1 1.2
Incubations with 3zp a2pi obtained from the Brazilian Institute of Atomic Energy was purified by extraction as phosphomolybdate with isobutyl alcohol/benzene, re-extraction to the aqueous phase with ammonia, and precipitation as MgNH4PO4 (Kanazawa & Boyer, 1973). To each bottle of 200 ml of culture medium was added 0.1 ml of 32Pi (Na salt) 0.1 mCi. Every 2 days the trypanosomes were harvested, four bottles
PEA
o
--. PI
U
SM -. L L
tO O
Origen b
sgt ye.ta Fig. 1. Phospholipids pattern of T. cruzi. Two-dimensional thin-layer chromatography stained by iodine vapour. LL-lysolecithin, SM-sphingomyelin, PC-phosphatidylcholine, U-unidentified, PI-phosphatidylinositol, PEA-phosphatidylethanolamine, PA-phosphatidic acid, CLP-cardiolipin and NL-neutral lipids.
Lipids of T. cruzi in the 2nd day of culture and two bottles in the 4th and 7th days. The collected mass was divided in half and lipid extracted as the usual procedure, and chromatographed in system A and system B. Three chromatographic plates were done for every lipid extract. One of them was used for radioautography using short time exposure (3 days) of the plate to Kodak Medical X-ray film PP/S54. The radioactive spots were eluted as the usual procedure, evaporated to dryness, dissolved in 0.4 ml of chloroform and half of the amount was used for phosphorus determination, and half was used for radioactive counting on a Beckman LS-150 Liquid scintillator counter, with a mixture of 50 mg of 1,4-bis-(5-phenyloxazol-2-yl) benzene plus 3.5g of 2,5-dipbenytoxazole in 11. of toluene as the scintillant.
RESULTS
Total lipids
The total lipid content of the epimastigote form of T. cruzi in the log phase of growth was 0.18 mg/mg protein as the average of five different preparations. This corresponded to 15% of its dry weight. Phospholipids
Phospholipids (PL)~comprised 34.7% (+0.6) of total lipids extractable from T. cruzi. This value represents the average of duplicate determinations made on five different preparations, all in the same log phase of growth. The differential distribution of phospholipids is shown on Table 1. Phosphatidylcholine (PC) was the most abundant phosphoglyceride with 44% of the total found. Phosphatidylethanolamine (PEA) was the second with 27.8% but phosphatidylserine (PS) was found in trace amounts and not always present. Phosphatidylinositol (PI) was present in 12.3% being the third major component of the PL. Cardiolipin was found close to the solvent front as 2% of the total. Sphingomyelin was consistently present in 3.8% of all phospholipids. An unidentified spot, probably plasmalogen, ninhydrin negative, was found in 2.9%. Lysolecithin had a variable appearance and made only 0.7% of the total. Phosphatidic acid corresponded to 4.2% of the PL. At the origin of the chromatograms was always found a phosphorus-containing material which amounted to 1.9%. These percentages were calculated from phosphorus determinations in eluted spots from duplicate chromatograms. 20.000
--
PEA
197
Figure 1 shows a typical two-dimensional chromatogram of total lipids, showing the phospholipids stained with iodine vapors. Labeling with 32p
In order to better understand the role of phospholipids during the growth of trypanosome in culture, in a preliminary experiment we have added 32pi to the culture medium, harvested the flagellates in the 2nd, 4th and 7th day, extracted the lipids and separated the phospholipids for quantitation and radioactive counting. The net amount of the individual phospholipids did not vary significantly, but the specific labeling of each one was different. The time-course of the incorporation of Pi into the individual phospholipids pools is depicted in Fig. 2. As can be seen the highest specific activity was found in phosphatidylethanolamine, followed by phosphatidylinositol, and phosphatidylcholine. In these three phospholipids a maximum incorporation was observed in the 4th day of growth, declining in the 7th day. Lysolecithin did not incorporate 32p. Sphingomyelin incorporated very little radioactivity, and it was difficult to quantitare it without error. The same can be said for phosphatidic acid and cardiolipin due to the small amount of phosphorus in the spot. The unidentified phospholipid (plasmalogen?) had a time-course similar to the three major ones, having a specific activity of 3400 in the 2nd day, 13,000 in the 4th day, and 3000 in the 7th day. All values were corrected for the phosphorus decay. Radioautography performed in these experiments (not shown) did not reveal any further spot that had not been detected by chemical means. Neutral lipids
Neutral lipids of trypanosomatids, including T. cruzi have been studied in detail (for a review see von Brand, 1973) so we have only'made a qualitative observation. Figure 3 shows a chromatogram of lipids from T. cruzi developed in solvent system C and stained with iodine vapors. At the origin are phospholipids that did not move with the solvent. Diglycerides are seen at RI 0.02, cholesterol is seen at Rj. 0.21 ergosterol moving just ahead, traces of free fatty acid can be observed at R~ 0.47, triglycerides as the major component are detected at RI 0.77 and cholesterol esters can also be seen at Rs 0.85. Neutral lipids are the major part of total lipids from T. cruzi in culture making up to 65% of its total.
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Fig. 2. 32p uptake by T. cruzi at different phases of growth in culture media.
Meaningful lipid data on trypanosomes depend on the homogeneity and lack of contamination of the preparation. Our data was obtained from organisms in log phase of growth, between 7 and 8 days of culture. The method chosen for lipid extraction, i.e. Folch's procedure gave better results for this particular kind of preparation than the method of Bligh & Dyer (1959). The experimental procedure was carried out in freshly harvested organisms, because either freezing or freeze-drying would induce the formation of lysophosphatides. Only two preparations--the lipid extract and the protein determination--were done in
198
M . M . OLIVEIRA, S. L. TIMM AND S. C. G. COSTA
Cholesterol ester Triglycerides
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8
,
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Fig. 3. Neutral lipids pattern of T. cruzi. One-dimensional chromatography developed in system C solvent and stained by iodine vapours. the dried material in order to correlate our findings with the dry weight of the protozoans. The value found for total lipids is within the range found for other trypanosomes (Godfrey, 1967) and for the previous report on T. cruzi (von Brand, 1973). Phospholipids This class of compounds have not been studied in detail in T. cruzi. Hack et aL (1962) made a comparative investigation among flagellates, but did not report the percentage of phospholipids in the total lipids fraction, later Williamson et al. (1965) reported a percentage of 17.2~ of phospholipids. In our preparations we found considerably more than that, i.e. 34.7~o, which may be due to different phase of growth of the organisms, although both results were obtained with flagellates in culture. The total amount of phospholipids in 7". cruzi seem to be much lower that the 80~o found for T. brucei rhodesiense (Dixon & Williamson, 1970) and 60~o by Godfrey (1967) and Venkatesan & Ormerod (1976). Dixon & Williamson (1970) also found values ranging from 72 to 79~o of phospholipids in 7". lewisi B, T. lewisi C, and T. rhodesiense C, in culture form. Godfrey (1967) found from 54 to 65~o of phospholipids in T. lewisi. T. vivax, T. congolense, all in blood form. Although small differences may be due to technical procedures, such great differences shown in our results seem to indicate a very distinct distribution of lipids in the epimastigote form of T. cruzi as compared with the other trypanosomes. It can be seen in Table 1 that lecithin is the major
phospholipid in T. cruzi, as it is in the other trypanosomatids studied so far. It is interesting to note, however, that it incorporated comparatively little 32pi that was added in the culture medium (Fig. 2). For that matter, phosphatidylethanolamine was much more metabolically active, since it had the highest specific activity. This phosphoglyceride the second more abundant phospholipid, shows the same type of distribution among some other trypanosomatids such as 7", rhodesiense and T. lewisi, which increase the percentage of PEA when grown in culture (Dixon & Williamson, 1970). In the extensive work of Godfrey (1967) he showed that the blood forms of T. vivax, T. congelense, and T. brucei had either equal or slightly higher values for sphyngomyelin than phosphatidylethanolamine. In our preparations sphingomyelin had a very low percentage, only about 4~/o of the total, but it was constant in all preparations studied. Phosphatidylinositol was the third more frequent phospholipid, and also was metabolically active, as judged by 3zp uptake. It amounted to 12~o of the total, a much higher value than what was found for T. brucei rhodesiense (1~,~o). No other data was reported for this phospholipid. Cardiolipin was found in 2~o of total phospholipids. Hack et al. (1962) did not report cardiolipin in his studies on T. cruzi lipids, but his methods of detection might have been insufficient. In T. brucei rhodesiense only trace amounts of cardiolipin was found, and about 4 ~ of phosphatidylserine, the opposite o r " cruzi where only trace amounts of this phospholipid
Lipids of T, cruzi was found. Trace amounts of cardiolipin was also found in T. rhodesiense and T. lewisi (Dixon & Williamson, 1970). Lysolecithin was found in very small amounts and it seems to be present in the organisms and not a product of degradation during the extraction procedure, because it did not incorporate radioactive phosphate while lecithin did. Our results indicate that the phospholipid distribution of T. cruzi epimastygote form is quite distinct of other trypanosomatids studied so far. It will be interesting to see if this particular distribution will keep the same in the other forms of T. cruzi. Neutral lipids
They are the major part of total lipids of T. cruzi in culture, comprising 65% of the total. This value is in accordance with previous report by Williamson et al. (1965). We have made only a qualitative observation among the various classes of neutral lipids, since it has been studied in detail by other groups (Von Brand, 1972). One interesting feature is the large amount of triglyceride, the major component found (Fig. 3). It may suggest as a storage lipid that is accumulated when the flagellate is in culture. In the excellent work of Dixon & Williamson (1970) they have shown a spectacular increase in the content of triglyceride in T. rhodesiense and in T. lewisi in the 7th day of culture as compared with the amount found in blood form, pointing out the same type of phenomenon. Acknowledgements--We are grateful to Professor Carlos Chagas for the use of his laboratory and to Dr Leopoldo de Meis for the gift of purified radioactive phosphorus. This investigation was partially supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico grant No 2222.0032/75 and grant No 3521/75.
199
BRENSER Z. (1973) Biology' of Trypanosoma cruzi. Ann. Rev. Mierobiol. 27, 347-381. CAMARGOE. C. (1964) Growth and differentiation in Trypanosoma cruzi. Origin of metacyclic trypanosomes in liquid media. Rev. Inst. Med. Trop. S&~ Paulo 6, 93-100. Dixon H. & WlLLIAMSONJ. (1970) The lipid composition of Trypanosoma lewisi and Trypanosoma rhodesiense. Comp. Biochem. Physiol. 33, 11-128. DIXON H., GINGERC. D. & WILL1AMSONJ. (1972) Trypanosome sterols and their metabolic origins. Comp. Biochem. Physiol. 41B, 1-18. FOLCH J., LEES M. & SLOANE STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. GODFREYD. G. (1967) Phospholipids of Trypanosoma lewisi, T. vivax, T. congolense and T. brucei. Expl Parasit. 20, 106-118. GUTTERIDGEW. F. (1975) Biochemistry of Trypanosoma cruzi, Proceedings of the International Symposium on New Approaches in American Trypanosomiases Research. Pan American Sanitary Bureau Scientific Publications No. 318. HACK M. H., YAEGERR. G. & MACCAFFERVT. D. (1962) Comparative lipid biochemistry. II Lipids of plants and animal flagellates, a non-motile alga, an ameba and a cilliate. Comp. Biochem. Physiol. 6, 247-252. HIRSCH J. & AHRENSJR. E. H. (1958) The separation of complex lipide mixtures by the use of silicic acid chromatography. J. biol. Chem. 233, 311-320. KANAZAWA T. & BOYER P. D. (1973) Occurrence and characteristics of a rapid exchange of phosphate oxygen catalyzed by sarcoplasmic reticulum vesicles. J. biol. Chem. 248, 3163-3172. SELZTMANT. P., FINN F. M., WIDNELL C. C. 8/; HOFMANN K. (1975) Lipids of bovine adrenal plasma membranes. J. biol. Chem. 250, 1193 1196. SKIDMOREW. D. 8L ENTENMAN C. (1962) Two-dimensional
thin-layer chromatography of rat liver phosphatides. J. Lipid Res. 3, 471-475. SKIPSKIV. P., PETERSONR. F. & BARCLAYM. (1962) Separation of phosphatidylethanolamine, phosphatidylserine and other phospholipids by thin-layer chromatography. J. Lipid Res. 3, 467-470. VENKATESAN S. t~ ORMEROD W. E. (1976) Lipid content
REFERENCES
BARTLETT G. R. (1959) Phosphorous assay in column chromatography. J. biol. Chem. 234, 466-468. BLIGH E. G. & DYER W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917.
of the slender and stumpy forms of Trypanosoma brucei rhodesiense: A comparative study. Comp. Biochem. Physiol. 53B, 481-487. VON BRANDT. (1973) Biochemistry of Parasites. 2nd edn. Academic Press, New York. WILLIAMSON J. & GINGER C. D. (1965) Lipid constitution of some protozoa, spirochaetes and bacteria. Trans. R. Soc. trop. Med. Hyg. 59, 366-367.
