Lipid composition of metacestodes of Taenia taeniaeformis and lipid changes during growth

Molecular and Biochemical Parasitology, 3 ( 1981) 301 - 318

301

Elsevier/North-Holland Biomedical Press

L I P I D C O M P O S I T I O N O F M E T A C E S T O D E S O F TAENIA T A E N I A E F O R M I S AND LIPID CHANGES DURING GROWTH

GARY L. MILLS, DANIEL C. TAYLOR and JEFFREY F. WILLIAMS

Department of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824, U.S.A. (Received 16 October 1980; accepted 20 January 1981)

A lipid analysis was performed on developing metacestodes of Taenia taeniaeformis removed from the livers of rats at times varying from 3 to 35 weeks post infection. Lipid accounted for 7 - 2 1 % of the dry weight of the parasites. The highest proportions were found at the earlier stages. The distribution was as follows; neutral lipid 2 7 - 4 5 % ; glycolipid 5-11%; and phospholipid 5 0 - 6 1 % . The major neutral lipid was cholesterol, and minor neutral lipids were sterol esters, triglycerides, diglycerides and monoglycerides. Hydrocarbons were present throughout development, but in the highest amounts at the earlier stages. Five different glycolipids were found, all of which were identified as glycosphingolipids. An increase in the proportion of more complex glycolipids was noted as parasites grew older. Ten different phospholipids were identified, with the major components being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Other phospholipids were: lysophosphatides, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, sphingomyelin, and an unknown phospholipid component. Changes in the relative amounts of the two major phospholipids were found when the early and late stages were compared. Two lipids found throughout development were identified as glycosylated dolichol phosphates, and they comprised between 1 and 3% of the total phospholipid fraction. Nineteen fatty acids were detected, and the fatty acid distribution for each lipid ctass at each stage was determined. Seven major fatty acids were common to each. These were: hexadecanoic, octadecanoic, oleic, linoleic, arachidonic, docosanoic, and docosahexaenoic. Key words: Taenia taeniaeformis, Neutral lipids, Glycosphingolipids, Phospholipids, Fatty acids, Lipid changes.

INTRODUCTION Studies o f lipids in h e l m i n t h parasites have dealt p r i m a r i l y w i t h e i t h e r c h e m i c a l iden-

Abbreviations: TLC, thin-layer chromatography; GLC, gas-liquid chromatography; HC, hydrocarbons; SE, sterol esters; TG, triglycerides; FS, free sterols; DG, diglycerides; MG, monoglycerides; GL 1 - 5 , glycosphingolipids; PC, phosphatidylcholine; SPH, sphingomyelin; LPC, lysophosphatidylcholine; PS, phosphatidylserine; DPG, diphosphatidylglycerol; PI, phosphafidylinositol; PA, phosphatidic acid; PE, phosphatidylethanolamine; LPE, lysophosphatidylethanolamine; X, unknown phospholipid; DOL-P-hexose, glycosylated dolichol phosphate. 0166-6851/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

302 tification or synthesis, and there is little information on lipid changes during growth and development. Functionally, lipids are important structural membrane constituents and may also serve as energy reserves. Furthermore, it is becoming increasingly apparent that lipids play important roles in enzyme regulation, cell surface recognition, cell interaction, glycoprotein synthesis, and in the expression of surface antigenic determinants [ 1 - 4 ] . Therefore, it seems reasonable to expect that shifts in lipid constituents may reflect changes in their functional roles, which may, in turn, be associated with key developmental processes. We have been studying hepatic metacestodes of Taenia taeniaeformis, with particular interest in the ability of this parasite to evade host immunological defenses during its establishment and growth in the liver. Post oncospheral stages are susceptible to antibodymediated attack for the first 5 - 7 days, but thereafter they become resistant and are able to live in the tissues of infected rats for many months [5]. Ultrastructurally, a number of distinct changes occur in the early stages, one of the most important of which is the establishment of a syncytial tegument interfacing with the host through microvilli, which are replaced after about 7 days by whip-like microtriches [6]. The initial surface changes coincide with the loss of susceptibility to antibody and complement, and it is probable that these and other modifications in the outer membrane are implicated in evasion of host defenses and prolonged survival. We have thus become interested in the biochemical composition of the tegument during different stages of development. This report deals with the total lipid composition of the larvae of T. taeniaeformis, and the changes these lipids undergo during growth from 21 days of age onwards. Since this paper was submitted for publication, a lipid analysis of developmental stages of another cestode, Spirometra mansonoides, has appeared [7] and the data in that report provide a useful basis for comparison of our results. MATERIALS AND METHODS

Procedures for obtaining Taenia taeniaeformis larvae. The strain of T. taeniaeformis used in these experiments has been propagated in our laboratory using the methods described by Leid and Williams [8]. Four-month-old cysticerci were dissected from the livers of infected rats and 10-15 larvae were given orally to parasite-free cats. Gravid segments generally appeared in the feces no less than 6 weeks later and were collected daily thereafter. Eggs were freed from the proglottids by teasing them apart in 0.85% saline with dissecting needles. Egg suspensions were given to 28-day-old Spartan (Spb: [SD], Spartan Research Co., Haslett, Michigan) female rats by gastric intubation. Viable larvae were removed from livers at intervals of 3, 4, 5, 6, 9, 21 and 35 weeks. The larvae were gently washed in 25 mM phosphate buffer, pH 7.0, and freeze-dried.

Lipid extraction. Freeze-dried larvae were weighed, suspended in CHC13/CH3OH/H20 (2:1:0.1, v/v/v), sonicated (50 W, 30 s), and extracted ovemight at 4°C. The ratio of solvent to material was 19:1. The homogenate was filtered through a coarse, fritted

303 glass Biichner funnel and the particulates re-extracted with CHCIa/CH3OH (2:1, v/v) and again filtered. Both filtrates were combined and concentrated under reduced pressure and dried with N2. Non-lipid contaminants were removed by Sephadex column chromatography [9]. The lipid fraction was concentrated as above and dissolved in CHC13/ CH3OH (2:1, v/v). Aliquots were removed, dried overnight at 60°C under vacuum, and the total lipid content determined gravimetrically.

Column, thin-layer, and paper chromatography. Lipids were fractionated into neutral, glycolipid and phospholipid classes by silicic acid (100 mesh, MaUinckrodt Chemical Co., St. Louis, Missouri) column chromatography. Neutral lipids were further separated on columns of silicic acid or on 7% hydrated Florisil [10]. Glycolipids were separated into individual components with Florisil [11] and phospholipids were fractionated on a diethylaminoethyl (DEAE)-cellulose column (12, elution sequence 5). The purity of column fractions was checked by thin-layer chromatography (TLC). Neutral lipids were separated on ITLC-SG chromatography medium (Gelman Instrument Co., Ann Arbor, Michigan) using hexane/diethyl ether (95:5, v/v) as the solvent. Glycolipids were chromatographed on ITLC-SA medium developed with 2-propanol]NH4OH (100:7, v/v), and phospholipids were separated with the same solvent but on ITLCSG chromatography medium. Lipid components were routinely visualized with UV light and by spraying TLC plates with 0.6% K2Cr207 (w/v) in 50% H2SO4 (v/v) followed by heating at 120°C for 10 min. Specific sprays included SbC13 and also H2SO4/CHaCOOH (1:1, v/v) for sterols and sterol esters, 0.2% ninhydrin in butanol for free amino groups, Dragendorff reagent for choline-containing compounds, and the reagent of Dittmer and Lester for phosphorus. Glycolipids were detected with orcinol and diphenylamine. All spray reagents were prepared and used according to the procedures given by Skipski and Barclay [13]. Glycerol phosphate esters were prepared by deacylating the phospholipids in 0.2 N methanol/NaOH for 15 min at room temperature [10]. The solution was neutralized with Dowex 50 [H÷] after partitioning with chloroform and water, and the water soluble hydrolysis products were concentrated with N2 and chromatographed, descending, on Whatman No. 1 paper. Clycerol phosphate esters were resolved with phenol/H20 (100: 38, w/v) and the phosphate groups detected with acid molybdate [14].

Analytical procedures. Individual lipid compounds were analyzed as follows. Lipid phosphorus was determined after digestion of samples with 10 N H2SO4 by the method of Bartlett [15]. Total nitrogen was assayed with the microprocedure of Sloane-Stanely [16], and acyl esters were estimated by the ferric hydroxamate method [17] with tripalmitin as a standard. Glycerol was assayed by the method of Hanahan and Olley [18] using a-glycerol phosphate and a synthetic lecithin as standards. Total sugars were determined with phenol/H2SO4 [19], and total and free sterols by digltonin precipitation and the ferric chloride procedure, as outlined by Kates [10].

304

Gas-liquid chromatography. Fatty acid methyl esters of the total, neutral, glycolipid, and phospholipid fractions were prepared as follows. The total lipid and the glycolipid fractions were hydrolyzed in 1.0 ml of 1 N methanolic HC1 for 18 h at 70°C. The hydrolysates were cooled and extracted three times with chloroform. The chloroform layers were dried and saponified with 1.0 ml of 10% methanolic KOH, for 2 h at 70°C. Neutral and phospholipid fractions were also subjected to the same alkaline methanolysis. Nonsaponifiable materials for all four samples were extracted with petroleum ether, the aqueous layers acidified, and the free fatty acids extracted with petroleum ether and methylated with BF3 [20]. Methyl esters of fatty acids were analyzed with a Dohrmann model 15C-3 gas chromatograph, equipped with a stainless steel column (6 ft X 1/8 inch) packed with 5% DEGS-PS on Supelcoport (100-200 mesh) and operated isothermally at 150°C. The injector port and detector temperatures were 190 and 165°C, respectively, the detector current was 110 mA, and the carrier gas was helium with a flow rate of 20 ml/min. Methyl esters were identified by their retention time relative to fatty acid methyl ester standards obtained from Supelco, Inc., Bellafonte, Pennsylvania. Alditol acetate derivatives of the sugars present in the glycolipids were prepared as follows. Total and individual glycolipids were subjected to acid methanolysis as above. Fatty acid methyl esters were removed with petroleum ether, the methanolysate dried with N2, and the residue dissolved in 1.0 ml of 2 N NaOH. Long chain bases were extracted with diethyl ether and the alkaline-aqueous phase was treated with Dowex 50 [H÷]. The neutralized aqueous phase was dried under N2, redissolved in 100 #1 distilled water, reduced with sodium borohydride, and acetylated with acetic anhydride, all according to Kannan et al. [21]. Alditol acetates were analyzed using the same gas chromatograph as above, equipped with a glass column (6 ft X 2 mm, inner diameter) packed with 3% SP-2340 on Supelcoport (100-120 mesh) and run isothermally at 170°C. The injector port temperature was 190°C, the detector temperature 180°C, and the detector current 110 mA. The carrier gas was helium with a flow rate of 20 ml/min. The retention times of the unknown sugar alditol acetates were compared with those of alditol acetate derivatives prepared from high purity sugar standards. RESULTS

Lipid composition. Lipid extracted from T. taeniaeformis larvae accounted for between 7 and 21% of their total dry weight (Fig. 1). The lipid content shifted substantially during growth. Lipid content, calculated as a percent of the dry weight, was approximately three times higher at week 3 than at the later stages. The percent lipid decreased sharply from the third to the fifth week, rose slightly at week 6, and then leveled at about 8% between week 9 and week 35. Total lipid was separated into neutral, glyco-, and phospholipid classes (Table I). At all stages studied, phospholipids were the most abundant lipid class, while glycolipids were the least abundant. The distribution of neutral versus polar lipids was fairly constant throughout growth, except at week 5 where there was a noticeable decrease and increase in neutral and polar lipids, respectively.

305

25 2O

K 15 ,-,,I

10

I

I

I

I

I

I

I

3

4

5

6

7

8

9

/,i

I

I

21

35

WEEKS Fig. 1. Lipid content of Taenia taeniaeformis larvae during growth. Values are given as percent of the total dry weight of the larvae.

ldenafication and characterization oflipids. Each lipid class was fractionated into either individual compounds, or else into groups of similar compounds by column chromatography. Each fraction was checked for purity by TLC and where necessary individual components were further purified by preparative TLC. Twenty-eight different lipids were detected. Identification of each was based upon column and TLC behavior as compared to lipid standards. Thirty different lipid standards (obtained from either Supelco, Inc., Calbiochem, or Sigma Chemical Co.) were used. When possible, lipid identification was confirmed by direct chemical analysis, the reactions of lipid to spray reagents, molar ratios, and by the identification of specific hydrolysis products. Eleven different neutral lipids were found. Three of these components yielded positive results when sprayed and chemically assayed for sterols. One compound, R f 0.30 -+ 0.03", TABLEI Distribution of the total lipid between lipid classes. Age in weeks Lipid class

Neutral Glycolipid Phospholipid

3

4

5

6

9

40.1 4.6 55.3

34.1 9.1 56.8

27.3 11.3 61.4

34.8 9.3 55.9

43.5 6.2 50.3

21

35

39.8 5.4 54.8

44.5 6.0 49.5

Total lipid was separated by silicic acid chromatography (2 x 8 cm); neutral lipids were eluted with 100 ml CHCla, glycolipids with 100 ml CH3COCHa, and phospholipids with 100 ml CH3OH. Values shown are percentages and were determined after gravimetric analysis of each fraction. All values are averages of two or more experiments. * All Rf values listed in this paper are averages of 10 or more separate TLC runs; standard deviations are given to show the variability.

306 was digitonin precipitable and co-chromatographed with cholesterol. The other two, Rf values of 0.76 -+ 0.04 and 0.59 + 0.03, were sterol esters. Two of the neutral lipids, Rf values of 0.49 -+ 0.03 and 0.41 -+ 0.01, were identified as triglycerides. The least polar neutral lipids, as determined by TLC (Rf 0.97 -+ 0.01) and by column elution on both silicic acid and Florisil, were hydrocarbons. These lipids had no distinct color and are thought to be polyisoprenols. The total lipid extract did, however, have a distinct reddish color that was due to one neutral lipid component which eluted as a tight band before the triglyceride fraction. When an absorption spectrum was run (in CHC13) a broad peak with an absorption maximum at 495 nm was obtained. This compound was not identified. The last four, more polar, neutral lipids were identified as diglycerides (Rf values of 0.17 +--0.02 and 0.14 + 0.01) and monoglycerides (Rf values of 0.10 + 0.01 and 0.05

-+ 0.02). The glycolipid fraction was separated into five different components. All five were resistant to mild alkaline deacylation and were identified as glycosphingolipids. The two least polar glycolipids, GL-1 (Rf 0.84 + 0.03) and GL-2 (Rf 0.76 -+ 0.03) were weakly positive to sugar spray reagents, while the other glycolipids, GL-3, GL-4, and GL-5 (with R f values of 0.47 + 0.04, 0.35 + 0.03, and 0.17 -+ 0.02, respectively) gave very strong reactions when sprayed with either orcinol or diphenylamine. None of the glycolipids reacted positively to ninhydrin. When the total glycolipid fraction was subjected to acid hydrolysis and the sugar components analyzed by GLC, fourteen different sugars were detected. The percent distribution of each carbohydrate and the distribution of the sugars associated with each of the individual glycolipids are given in Table II. Although 27 different sugar standards were used, not all of the peaks, including the two major ones, could be identified. Positive identification of these glycolipids will have to await a more complete chemical and structural analysis. Twelve different phospholipids were detected when thin-layer chromatograms of the phospholipid column fractions were sprayed for phosphorus. Three compounds were positive for the choline reagent, two of which yielded glycerylphosphorylcholine upon mild deacylation, while the other was non-saponifiable. The latter compound, R f 0.20 -+ 0.02, was identified as sphingomyelin. The other choline compounds were identified as PC (Rf 0.29 -+ 0.04, molar ratio* of 1:2.02:1.01:0.89) and LPC (R e 0.09 + 0.01). Phosphatidylserine (Rf 0.00) was ninhydrin positive and further identified based upon its column elution from DEAE with acetic acid, and upon its Rf value of 0.28 for glycerylphosphorylserine after deacylation and paper chromatography of its hydrolysis product. Three other phospholipids were strongly ninhydrin positive. Two of these yielded glycerylphosphorylethanolamine upon deacylation and were identified as PE (R e 0.76 -+ 0.04, molar ratio of 1:2.15:1.00:0.93) and LPE (Rf 0.56 -+ 0.04). The other compound (X) was not identified but had an Rf value of 0.34 -+ 0.05 and a molar ratio of 1:1.85: 1.89:1.16. Three phospholipids were found to contain no nitrogen. Mild deacylation of * Molar ratios are listed in the following order: Phosphorus to acyl esters to nitrogen to glycerol. If only three are listed then nitrogen is omitted.

8.7

9.8

12.4

13

14

glucose

galactose

mannose

N.I.

deoxyglucose

arabinose

N.I.

erythrose

1.7

2.7

0.8

23.3

1.5

4.8

52.0

0.4

0.9

2.0

1.9

2.6

0.7

4.7

Total

.

-

-

-

.

4.9

8.5

10.8

28.8

-

47.0

-

-

GL-1

Glycosphingolipids

-

.

.

6.6

18.8

10.7

4.3

-

3.5

17.7

4.8

45.1

2.5

1.4

7.1

-

17.9

-

-

GL-4

4.6

-

3.2

16.2

5.1

8.6

56.4

-

-

2.2

-

3.7

-

-

GL-5

are individual glycolipids after separation by Florisil

.

.

4.7

25.7

13.3

-

4.2

-

16.4

-

-

GL-3

a n d f u r t h e r p u r i f i c a t i o n b y p r e p a r a t i v e T L C . V a l u e s r e p r e s e n t p e r c e n t d i s t r i b u t i o n o f i n d i v i d u a l p e a k s a n d i d e n t i f i c a t i o n is b a s e d o n

while GL-1-5

.

.

-

-

-

2.6

1.1

6.7

6.2

78.7

GL-2

r e t e n t i o n t i m e s as c o m p a r e d t o s t a n d a r d s . P r o c e d u r e s f o r l i p i d h y d r o l y s i s a n d f o r m a t i o n o f t h e s u g a r d e r i v a t i v e s a r e d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . a N.I., not identified.

chromatography

T o t a l g l y c o l i p i d r e f e r s t o t h e g l y c o l i p i d f r a c t i o n a f t e r silicic a c i d c h r o m a t o g r a p h y ,

7.2

5.0

8

11

4.3

7

12

N.I.

3.8

6

5.7

3.0

5

6.3

N.I.

2.4

4

9

fucose

1.8

3

10

rhamnose

1.4

N.I.

0.9

N.I. a

Identification

1

time

Retention

2

Peak

D i s t r i b u t i o n o f g l y c o l i p i d c o n s t i t u e n t s u g a r c o m p o n e n t s f r o m a d u l t larvae.

T A B L E II

308 each and chromatography o f the hydrolysis products yielded glycerylphosphorylglycerylphosphorylglycerol (Rf 0.17), glycerylphosphorylinositol (Rf 0.12), and glycerophosphate (Rf 0.37). These compounds were identified as DPG (Rf 0.66 -+ 0.04, molar ratio of 1:1.36:1.28), PI (Rf 0.24-+ 0.06, molar ration o f 1:2.35:0.94), and PA (Rf 0.08-+ 0.01, molar ratio of 1:1.54:0.92). Two other phosphorus-containing lipids were present which had Rf values o f 0.87 -+ 0.04 and 0.81 -+ 0.02. Both were resistant to alkaline hydrolysis, gave positive reactions to sugar sprays, and were eluted from DEAE with ammonium salts (prepared according to Rouser et al. [12] general elution scheme 5). These lipids were identified as Dol-P-hexoses.

Changes in neutral lipids. Neutral lipids were fractionated into their individual components at different growth stages (Table III). Cholesterol was the major component at all stages studied, accounting for between 57 and 84% o f the total neutral lipid fraction. At the earliest stage studied, a decrease in FS and an increase in SE and HC were noted During subsequent growth there was a steady decrease in SE and HC from the third to the ninth week. Triglycerides represented approx. 10% o f the total neutral lipid at all stages, except at week 3, where they were at a low of 2% and at week 21 where they were at a high of 20%. The MG and DG were fairly constant throughout growth, accounting for only 1 - 4 % of the neutral lipids. Small amounts of free fatty acids were also found at all stages but quantitative data for them were not obtained.

Changes in glycolipids. Quantitative data were collected for individual glycolipids throughout larval growth (Table IV). A noticeable shift in glycolipid complexity was observed during development. At the earlier stages the major glycolipids were the less polar GL-1 and GL-2. These glycolipids accounted for 61% o f the total glycolipid fraction at week 3, decreased steadily to week 9, then remained fairly constant thereafter. In contrast, TABLE III Distribution of neutral lipids. Age in weeks Lipid 3 Hydrocarbons (HC) Sterol esters (SE) Triglycerides (TG) Free sterols (FS) Diglycerides (DG) Monoglycerides (MG)

t

4

5

6

9

21

35

33.0 5.2 2.2 56.8

13.8 4.2 8.3 72.2

10.4 3.5 10.3 74.1

6.7 1.7 9.4 81.6

1.7 0.6 11.2 84.4

0.9 0.3 20.4 75.9

1.7 0.6 9.6 84.0

2.8

1.5

1.7

0.6

2.1

2.5

4.1

Values shown are percentages and were determined after gravimetric analysis of each fraction after separation of the neutral lipid class on either silicic acid or Florisil columns using the procedures referenced in Materials and Methods. All values are averages of two or more experiments.

309 TABLE IV Distribution of glycolipids. Age in weeks Lipid

GL-I GL-2 GL-3 GL-4 GL-5

t

3

4

6

9

21

35

61.2

30.7

27.3

20.5

11.7

19.4

16.5 11.7 10.6

16.3 21.6 31.4

20.4 18.0 34.3

13.4 35.8 30.3

9.5 15.5 63.3

21.9 19.3 39.4

Values represent percent distribution based on the total carbohydrate associated with each glycolipid. Carbohydrate was determined by the method of Dubois et al. [18]. Individual glycolipids were separated by column chromatography with Florisil and further purified by preparative TLC. GL-1 and GL-2 were difficult to separate completely, therefore values are given together. In all stages studied GL-1 was the major component and GL-2 the minor. All values are averages of two or more experiments. the more polar glycolipids, GL-4 and GL-5, contributed only 22% to the total glycolipids at week 3, rose to 50% in the next two weeks, and at the later stages accounted for 6 0 80% of the total glycolipid fraction. GL-3 remained fairly constant (16-20%) from the third to the sixth week, decreased to 10% by week 21, and then increased to its original level by week 35.

Changes in phospholipids. Table V shows the changes in phospholipids during growth of the larvae. At all stages, PC, PE, and PS were the major phospholipids. At week 3, PC was the main phospholipid component but it decreased subsequently, while PE increased. By the sixth week, PE was the major phospholipid, and remained so for the rest of the stages sampled. Phosphatidylserine stayed at a constant 10-17% throughout growth. The lysophosphatides of the two major components also did not fluctuate much, but were minor components representing only 2 - 5 % and 2 - 4 % of the total phospholipids for LPC and LPE, respectively. Phosphatidylinositol and PA both contributed substantially to the phospholipid composition at the earlier stages of growth, but decreased steadily for the later stages. Minor phospholipid components were DPG (1-3%) and SPH ( 0 . 1 5%). At week 35, SPH was barely detectable. The glycosylated dolichol phosphates were also minor components (1-3%), with higher concentrations being found at the younger stages. The unidentified phospholipid comprised between 2 - 8 % of the total phospholipid fraction and was most abundant at week 4.

Fatty acid composition and changes in the major fatty acids. The fatty acid composition of the total lipid and the neutral, glyco-, and phospholipid fractions for adult larvae is shown in Table VI. Nineteen different fatty acids were found. The major fatty acid

310

TABLE V Percent distribution of phospholipids. Age in weeks Lipid

Phosphatidylcholine (PC) Sphingomyelin (SPH) Lysophosphatidylcholine (LPC) Unknown (X) Phosphatidylserine (PS) Diphosphatidylglycerol (DPG) Phosphatidylethanolamine (PE) Lysophosphatidylethanolamine (LPE) Phosphatidylinositol (PI) Phosphatidic acid (PA) Glycosylated dolichol phosphate (Dol-P-hexose)

3

4

6

9

50.1 0.7 2.2 1.9 9.5 1.1 15.2 2.0 8.0 6.0 3.3

30.1 1.9 3.5 8.1 16.4 3.0 17.5 2.2 7.6 6.9 2.8

24.4 0.5 5.1 4.8 16.7 0.9 26.7 4.3 9.7 4.4 2.5

23.8 4.9 1.6 3.9 15.5 2.7 30.3 3.8 8.2 3.8 1.5

21

35

25.7 4.4 3.9 2.6 11.7 2.0 38.7 3.5 4.7 1.6 1.2

27.3 0.1 2.1 5.9 16.2 1.9 35.9 2.6 5.2 1.8 1.0

Values are averages of two or more experiments and are based on total phosphorus of each individual phospholipid component after DEAE column chromatography and preparative TLC.

TABLE VI Percent distribution of fatty acids from adult larvae. Fatty acid

Total

Neutral

Glycolipid

Phospholipid

16:0 a 18:0 18:1 18:2 20:0 20:1 20:2 20:4 22:0 22:1 22:2 22:5 22:6 24:1 post 22:6 Degree of unsaturation b

12.0 18.8 17.2 9.9 1.4 4.7 0.4 7.7 12.2 1.2 0.5 1.1 5.2 0.9 6.8 1.14

13.6 8.5 9.2 15.3 0.9 2.8 0.8 7.1 24.4 0.3 0.9 1.3 13.4 0.9 0.6 1.62

15.1 8.3 7.4 20.1 5.4 4.3 0.5 3.4 17.2 1.2 1.3 1.1 8.1 1.0 5.6 1.27

17.7 12.1 12.1 18.9 1.9 4.4 0.8 4.3 18.6 1.5 1.2 6.0 0.5 1.18

Procedures for lipid hydrolysis, GLC, and methyl ester formation are given in Materials and Methods. a The first and second numbers represent length of carbon chain and double bonds, respectively. b Calculated as 1 x [(% monoene)/100] + 2 X [(% diene)/100] + etc.

311 components were octadecanoic and oleic acids for the total lipid extract, docosanoic acid for the neutral lipids, linoleic and docosanoic acids for the glycolipids, and palmitic, linoleic, and docosanoic acids for the phospholipids. The fatty acids of the total lipid extract exhibited a low degree ofunsaturation. The neutral lipids were found to have the highest degree of unsaturation for their fatty acid components, and the phospholipids the lowest value. When the fatty acids of the neutral, glyco- and phospholipid fractions were analyzed for all the stages studied, it was found that seven fatty acids contributed between 80 and 90% of the total fatty acid composition. These fatty acids were: hexadecanoic (16:0), octadecanoic (18:0), oleic (18:1), linoleic (18:2), arachidonic (20:4), docosanoic (22:0), and docosahexaenoic (22:6). The distribution of these fatty acids for each lipid fraction at the different growth stages is shown in Fig. 2. A number of distinct shifts in fatty acid composition during development were noted for each of the lipid classes. Most of the variation in fatty acid composition for the neutral lipids occurred from week 3-9. At weeks 21 and 35 very little difference could be seen in the fatty acid distribution. The main fatty acid component for the neutral lipids at all stages was either docosanoic or palmific. When the glycolipids were analyzed it was found that there was a similarity, with minor variations, in the distribution of the major fatty acids at the earlier stages (compare week 4 and week 5), and also at the later stages (compare week 21 with week 35). However, a major shift was noted for the intermediate stages (weeks 6 and 9), this being due to the large quantity of linoleic acid present at these times. A distinct shift in the fatty acid composition of the phospholipids was also apparent. During the earlier stages of growth (weeks 3, 4 and 5), the distribution of the major fatty acids was similar with oleic and linoleic acids comprising 60-70% of the total. At the later stages, weeks 21 and 35, five of the seven fatty acids were almost equal in distribution, with the other two varying only slightly. DISCUSSION The lipid composition of the larvae of T. taeniaeformis is qualitatively similar to that of other parasitic helminths in general [22-24], and also to that of parasitic and freeliving members of the Platyhelrninthes [25-30]. This similarity is not entirely a result of parasitic adaptation, since both free-living and parasitic forms are comparable, but is, however, pfimariy due to the fact that all have lost the ability for de novo synthesis of sterols and fatty acids, while retaining the capacity for the synthesis of other complex lipids [27-30]. Quantitative differences in lipid composition of these organisms reflect the particular environment in which they live, since all depend directly on exogenous components for lipid assembly [27-32]. Lipids comprise 7-21% of the total dry weight of the larvae of T. taeniaeformis with the average value after 5 weeks being approx. 9%. This agrees well with values reported for T. taeniaeformis larvae by McMahon [33; 6.9%] and Salisbury and Anderson [34; 6.3%]. Our values are quite similar to those found in the cestodes Spirometra man-

312 40

NEUTRAL LIPID

20 10

4O Z

0 Iz)

30

m

20

tr 1-

GLYCOLIPID

10 ,,N.D. 40

PHOSPHOLIPID

30 20 10

IlL 3wk

4wk

5wk

6wk

9wk

21wk

+6..0

18:0

m 18:1

18:2

I 22:0

~ 20:4

35wk [iu! 22:6

Fig. 2. Distribution of the major fatty acids for each lipid class at the different growth stages. Legend at bottom of figure identifies fatty acids. The identification of these fatty acids is based upon their retention times compared to standards and also upon co-chromatography of each with its respective known standard. No attempt was made to confirm the identities of the fatty acids by using other techniques.

sonoides [28; 16%] and Hymenolepis diminuta [25;5.8% wet weight], the nematodes Mecistocirrus digitatus [23; 10.4%] and Dirofilatia immitis [22; 2.1% wet weight], the acanthoeephalan, Macracanthorhynchus hirudinaceus [24; 5.8% wet weight], and the trematode Echinostoma revolutum [35; 15%] However, they differ markedly from values for the trematode Schistosoma mansoni [36; 34%] and the marine cestode Callibothtium verticillatum [32; 25%].

313 In T. taeniaeformis the major neutral and phospholipid components are FS, TG, PC, PE, and PS. This is also true for the organisms mentioned above. The information on glycolipids is incomplete, but they have been detected in other cestodes [25,26,28], nematodes [22,23], the trematode E. revolutum [37], and in the free-living turbeUaria Dugesia dorotocephala [29] and Convoluta roscoffensis [30]. The glycolipids in most cases were identified as cerebrosides and in general were minor lipid components, although high concentrations were found in the tegument ofH. diminuta [38]. The most frequently occurring fatty acids for all lipid classes of T. taeniaeformis are 16:0, 18:0, 18:1, 18:2, 20:4, 22:0, and 22:6. With the exception of 22:0, the other fatty acids, particularly those with 16 and 18 carbons, are the most common fatty acid components of the parasitic helminths mentioned above. The high concentration of 22:0 seems to be peculiar to T. taeniaeformis and we must caution that we have not precisely confirmed its structural identity. The reason for its abundance is presently unclear, but the mechanism for its accumulation probably involves either the direct absorption of 22:0 from the host (absorption of long chain fatty acids, usually polyunsaturates, is known to occur in the cestode C. verticillatum [32] ) or else by chain elongation of host-derived smaller fatty acids. The latter is the more plausible explanation, since chain elongation of fatty acids is known to occur in all classes of Platyhelminthes [ 2 8 - 3 1 ] . This high concentration of 22:0 has also directly influenced the degree of unsaturation for the lipids of T. taeniaeformis. In this parasite there is an almost equal distribution of saturated and unsaturated fatty acids for all lipid classes. This is not the case for other parasitic helminths. In these organisms the fatty acids are predominantly unsaturated, especially the more polar lipids. A summary of the lipid changes for all lipid components at the different growth stages is presented in Table VII. Most of the changes occur between the third and ninth week. By week 21 the lipid composition seems to be fairly stable. When week 3 is compared to the other stages a distinct difference in the quantitative distribution of lipids is found. This is primarily due to a difference in the distribution of the phospholipids, a slight decrease in FS, and a noticeable increase in HC. At week 3, PC is the major phospholipid component with PE, PS and the acidic phospholipids, PI and PA, being present in substantial amounts. After one week PC drops sharply and remains constant thereafter, PS and PE both increase during growth, and by week 21, PE is the major phospholipid, while the acidic phospholipids decrease to become minor components. These shifts in the distribution of phospholipids during growth indicate that distinct membrane changes are probably occurring in the developing larvae. At week 3, the free sterol content of the larvae is at a lower level than at later stages. During growth, FS becomes the major lipid component. The FS in T. taeniaeformis was identified as cholesterol. Cholesterol was also found to be the main unsaponifiable lipid in adult T. taeniaeforrnis [39] accounting for 30% of its total lipid. Cholesterol seems to be the main sterol component of the Platyhelminthes, even though the evidence indicates that each has lost the ability to synthesize sterol de novo [28-30,40]. All are dependent on obtaining sterol from an exogenous source. It has been demonstrated that parasitic

314 TABLE VII Summ~y of lipid changes during growth of T. taeniaeformislarvae. Age in weeks Lipid

HC SE TG FS DG-MG GL-1 -GL-2 GL-3 GL-4 GL-5 PC LPC SPH X PS DPG PI PA PE LPE Dol-P-hexose

3

4

6

9

12.6 2.0 1.0 21.7 1.1 1.8 0.5 0.3 0.3 29.7 1.3 0.4 1.1 5.6 0.6 4.7 3.5 9.1 1.2 1.5

5.4 1.6 3.2 27.7 0.5 2.7 1.4 1.9 2.7 14.5 2.3 1.3 4.2 11.1 1.2 4.5 4.3 7.5 0.9 1.1

2.3 0.6 3.3 28.3 0.2 2.5 1.9 1.7 3.2 13.8 2.9 0.3 2.7 9.3 0.6 5.4 2.4 15.2 2.4 1.0

0.7 0.2 4.5 34.0 0.9 0.8 0.5 1.4 1.2 13.3 0.9 2.9 2.1 8.7 1.5 4.6 2.1 16.9 2.0 0.8

21

35

0.3 0.1 8.0 29.6 1.0 0.6 0.5 0.8 3.4 14.4 2.2 2.4 1.5 6.5 1.1 2.6 0.9 21.6 2.0 0.5

0.8 0.3 4.5 37.6 1.8 1.1 1.3 1.2 2.4 13.4 1.0 0.1 2.5 8.0 0.9 2.6 0.9 17.8 1.3 0.5

Values represent the percent distribution of the lipid compounds. Abbreviations are given at the beginning of the paper. helminths are capable of absorbing either cholesterol or an esterified form of cholesterol from their host [ 4 1 - 4 3 ] . The cholesterol ester is then subsequently hydrolyzed to cholesterol by the parasite [40]. Even though these organisms are n o t capable of synthesizing sterols, they are able to incorporate label from acetate, hydroxymethylglutarate, and mevalonate into sterol precursors [44,45]. The end product is famesol, a Cls isoprenoid alcohol [44]. When farnesol, obtained from lipid extracts of Echinococcus granulosus, was added to in vitro cultures o f H . diminuta, it was found that growth of encysted cysticercoids was stimulated as much as 100% [46]. Famesol has been found in lipid extracts of E. granulosus, [45,46], H. diminuta [44], and T. hydatigena [45]. It is assumed that free farnesol is a hydrolysis product of its corresponding pyrophosphate, farnesyl pyrophosphate [44], a precursor for the synthesis of polyisoprenols. Lipid extracts of T. taeniaeformis larvae contain hydrocarbons which we believe are polyisoprenols. These polyisoprenols are in exceptionally high concentrations at week 3 (12.6%) and decrease during growth. Polyisoprenols are k n o w n lipid intermediates involved in glycoprotein synthesis [5,47]. Usually the lipid intermediate is dolichol

315 phosphate, a phosphorylated a-saturated polyprenol that varies in chain length from Cso to Cn0 [48]. Dolichol phosphate, a lipid coenzyme, functions as a cyclic intermediate, accepting sugars from sugar nucleotides and then transferring these to a growing polysacchafide chain. It has also recently been found that other polyprenol phosphates, ranging in chain length from 20 to 95 carbon atoms, can also function as lipid intermediates [49]. Glycosylated polyprenol phosphates were found in the lipid extracts of larvae of T. taeniaeformis. These glycosylated lipids were in their highest concentration at the earlier stages sampled and decreased continuously from 1.5% at week 3 to 0.5% at week 21 where they then remained constant. We believe that the high concentration of polyisoprenols and the increased occurrence of the glycosylated polyprenol phosphates at earlier stages of growth is an indication that rapid glycoprotein synthesis is occurring. It is also during these stages that the larvae are growing most rapidly [50]. It should be possible to use the naturally occurring polyisoprenols of T. taeniaeformis for monitoring in vitro glycoprotein synthesis. This may provide important insight into the mechanisms that are involved in the synthesis of glycoproteins by these parasites, and should also be useful in determining the types of glycoproteins that are being formed during the growth of T. taeniaeformis larvae. There is evidence that sulfated glycoproteins released from the surface of T. taeniaeformis can frx complement non-specifically [51,52], and this may be a contributing factor in avoidance of complement-mediated membrane attack. Whether this mechanism, or others perhaps comparable to the lipid composition changes which accompany resistance of tumor membranes to host defenses [53,54], underly the ability of developing metacestodes to survive in tissues remains to be seen. It is clear, however, that further biochemical studies of the parasite and its limiting membrane, especially in the crucial early stages of development, have an important part to play in our understanding of the immunology of this host-parasite relationship. ACKNOWLEDGEMENTS We are especially grateful to Alma Shearer for technical assistance. This work was supported by NIH grant AI-10842. This is journal article 9577 from the Michigan Agricultural Experiment Station. REFERENCES 1 Cullis, P.R. and de Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta 559,399-420. 2 McMurchie, E.J. and Raison, J.K. (1979) Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes. Biochim. Biophys. Acta 554,364-374. 3 Yamakawa,T. and Nagai, Y. (1978) Glycolipids at the cell surface and their biological functions. Trends Biochem. Sci. 3, 128-131. 4 Elbein, A.D. (1979) The role of lipid-linked saccharides in the biosynthesis of complex carbohydrates. Ann. Rev. Plant Physiol. 30, 239-272.

316 5

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