Fatty acid composition of amastigote and trypomastigote forms of Trypanosoma cruzi

Acta Tropt~a 46(1989)131-136

131

Elsewer A T P 00017

Fatty acid composition of amastigote and trypomastigote forms of Trypanosoma cruzi Wilson Leon, Antonio M. Montelro, Celuta S Alwano, J.G. Esteves and Jayme Angluster

Manuel

~'nlver~Made Federal do Ru~ de Janetro, CMade Untver~ltarla, Rto de Janetro Braztl

(Received 8 June 1988, accepted 6 October 1988)

The fatty acid compos~tlon of total hplds from trypomast~gote and amastlgote forms of Tr~panosoma ~ruzt and of Vero cells before and after parasite lnfectmn were analyzed by gas-hqmd chromatography and mass spectrometry Even-numbered, saturated, monoenolc and polyenolc acids ranging from C-12 to C-18 were characterized m both T cruzt development stages Significant changes m the fatty acid composition occurred d u r m g the T ~ruzt hfe cycle Olelc and hnolelc acids were prominent m trypomastlgote forms whereas palmlllC acid was the major fatty acid of amastlgotes Other differences include hzgher steanc acid and lower palmltolelc and hnolemc acid levels as well as the absence of laurie acid m amastlgotes as compared with trypomastlgote forms The fatty acid pattern of Vero cells before T cruzl refection as compared with that after refection showed mostly quahtatwe differences Lmole~c and hnolenlc acids were observed only in T cruzl infected cells Key words T r ~ p a n o w m a ~ruzt Fatty acid Lipid, Vero cell

Introduction T r v p a n o s o m a cruz~ is the agent of Chagas' disease, a widespread protozoan mfectlon in South America The parasite exhibits different developmental stages m the mammalian host and In the trlatomlne vector Metacychc trypomastlgotes in the insect feces transform into amastlgotes inside cells of the mammalian host, multiply as such and transform again into blood trypomastlgotes before penetrating new cells or infecting trlatomlnes during their blood meal Trypomastlgotes mgested by the insect develop as replicating epxmastlgotes within the invertebrate host and then transform into metacychc trypomastlgotes before being released in the trlatomlne excreta (Brener, 1973, De Souza, 1984) These dtfferent morphological and functional cell stages can be obtained m reasonable amounts by using a cloned T cruzt Y strain (MortattI et al, 1985) An understanding, at the molecular level, of the mechanisms involved in differentiation of these stages may provide effective means of interfering in the life cycle of the parasite to control the spread of the disease Morphologically defined forms of T cruzl have dtfferent functions in the trypanosome life cycle and the molecular basis defining distinct cell characteristics are poorly known Prof Wdson Leon, lnstltuto de Mlcroblologla, Centro de Clenclas da Saude, Umvers~dade Federal do Rio de Janelro, Cldade Umversltana, 21 941 Rio de JaneJro, Brazd C o r r e w o n d e n c e address

0001-706X,,89/$03 50 ,~ 1989 Elsevier Science Pubhshers B V (Biomedical Dwlslon)

132 A comparison of the specific trypanosome hplds present in each stage of the hfe cycle could be a useful approach for understanding some of the mechanisms of the host-parasite relationship, Including lipid exchanges with the host Llplds may also serve as biochemical markers of cell differentiation In this report a systematic analysis of fatty acid composition in trypomast~gotes and amastlgotes of T cruzz cultured in Vero cells was undertaken A comparison of the fatty acid pattern of Vero cells before and after parasite mfectmn was also determined

Materials and Methods

Parasite strain

A cloned Y strain of T cru2l was maintained by weekly passage m mice and Vero tissue culture cells Vero cells were lmtlally infected with trypomastlgotes obtained from the blood of mice 7 days post-inoculation Thereafter, trypomastlgotes were transferred every 3 days to fresh tissue culture cells In vitro cell cultlvatton

Vero cells were cultivated in 70ml flasks using 199 medium (Mlcroblologica Manufactors) supplemented with 10% fetal bovine serum (FBS), 300 mg/ml glutamine and 1 25 g of sodium bicarbonate, pH 6 8 Cultures were maintained at 37°C and the m e d m m changed every 2 days The initial infection of cells was made with blood trypomastlgotes at a parasite/cell ratio of 10/1, using the above medium with 2% FBS added Parasites not entering the host cells were removed during medium change Trypomastlgotes were obtained from cultured cells after 3 to 4 days incubation at 37°C whereas amastigotes were harvested only on the fifth day Both life cycle stages were washed 3 times in cold phosphate-buffered saline (PBS), pH 7 2, 001 M Isolatton oJ para~tte~

Bloodstream trypomastigotes were obtained as described by Wrightsman et al (1986) Isolation of trypomastlgotes and amastigotes from tissue culture was carried out as described by Carvalho and De Souza (1983) Extractton and tdenttftcatton o f hplds

Llplds were extracted from washed protozoa and Vero cells at room temperature with 30 vol each o f ( a ) chloroform-methanol (C/M) 2 1, (b) C/M 1 I and (c) C/M 1 2 at room temperature The combined extracts were evaporated to dryness Absolute methanol-dlethyl ether (3 1, v/v) (50 ml) was added to the hpld extract, followed by addition of 1 ml of 5 N N a O H for alkaline saponification of llplds Saponification was performed by refluxlng for 5 h under chemically pure nitrogen Water was added and the unsaponlfiable fraction was extracted with n-hexane For liberation of fatty acids the water phase containing the sodium salts of the fatty acids was acidified to

133 p H 1 with c o n c e n t r a t e d p h o s p h o r i c acid F r e e fatty acids were e x t r a c t e d several times with n-hexane T h e c o m b i n e d h e x a n e phases were e v a p o r a t e d to dryness (Porschm a n n et a l , 1982) F a t t y acids were c o n v e r t e d to their c o r r e s p o n d i n g methyl esters by m i x i n g equal v o l u m e s o f freshly distilled e t h e r - d x a z o m e t h a n e a n d the fatty acid s o l u t i o n In m e t h a n o l / d l e t h y l e t h e r (1 9, v/v) The fatty acid m e t h y l esters were s e p a r a t e d by gas-liquid c h r o m a t o g r a p h y ( G L C ) on a V a r l a n A e r o g r a p h Series 2400 C h r o m a t o g r a p h e q u i p p e d with a flame i o n i z a t i o n d e t e c t o r A stainless steel c o l u m n (6 ft x 1/8 inch, a p p r o x 1 83 m x 3 2 mm), with 10% B D S ( b u t a n e - I , 4-dlol-succlnate) c o a t e d with 80 100 mesh G a s - C h r o m W at 190°C was used M e t h y l esters were identified by their r e t e n t i o n ttme relative to m e t h y l esters o f k n o w n fatty acid s t a n d a r d s T h e c h a m lengths o f u n s a t u r a t e d fatty acids were also identified by G L C o f the p r o d u c t s o f h y d r o g e n a t i o n o f the methyl esters, carried out at r o o m t e m p e r a t u r e for l h in ethyl acetate with 10% p a l l a d i u m on c h a r c o a l u n d e r a h y d r o g e n pressure o f 40 lb/lnch 2 ( a p p r o x 117 7 k g / c m 2) C o m b i n e d G L C - M S (mass s p e c t r o m e t r y ) o f fatty acid m e t h y l esters, was carried o u t on an H P (Hewlett P a c k a r d 5992 A G C / M S System) I n s t r u m e n t at an i o n i z a t i o n energy o f 70 eV, with a stainless steel c o l u m n (6 f t x 1/8 inch), p a c k e d with 0 7 M OV-101 (methyl-silicone), a n d p r o g r a m m e d for a t e m p e r a t u r e range from 130 to 220°C at 8°C/mln

Results T a b l e 1 lists the p e r c e n t a g e o f fatty acids f r o m the total hplds fraction o f t r y p o m a s t l g o t e a n d a m a s t t g o t e forms a n d from Vero cells before a n d after T c r u z t infection In general e v e n - n u m b e r e d , s a t u r a t e d , m o n o e n o l c a n d p o l y e n o l c acids r a n g i n g from C - 1 2 to C-18 were f o u n d In t r y p o m a s t t g o t e a n d a m a s t l g o t e forms Also, in b o t h d e v e l o p m e n t a l stages a b o u t 70% o f the total fatty acids consisted o f 18c a r b o n fatty acids T h e m a j o r fatty acids in t r y p o m a s t l g o t e forms were olelc (C 18 1) TABLE 1 Fatty aod composition (%)a of total hplds from trypomastlgote and amastlgote forms of Trypano~oma gruzt and from Vero cells, before and after parasite mfechon Fatty acid

Launc Mynstlc Palmlhc Palmltolelc Steanc Olelc Lmoloc Lmolenlc

C db b

C C C C C C C C

12 0 14 0 16 0 16 1 18 0 18 1 18 2 18 3

stage

Vero cells

TrypoAmastlmast~gote gote

Before mfectmn

After mfectmn

4 4 12 11 6 20 36 7

4 17 7 14 58 -

1 3 18 6 17 36 17 2

T cruzt

4 22 7 14 25 24 4

"Values are the means of three independent experiments The standard dewatmns were about 5% of the mean value (range 2 6%) bNumber of carbon atoms number of double bonds absent

134

and hnolelc (C 18 2) acids This pattern slgmficantly differed from that detected m amastlgote forms, in which palmltlC acid (C 16 0) together with olelc and hnolelc acids were most prominent We also observed a markedly higher concentration of stearlc acid (C 18 0), lower concentrations of pamltolelc (C 16 1) and hnolenlc (C 18 3) acids, and the absence of launc acid (C 12 0) in amastlgote forms as compared with trypomastlgote forms A comparison of the Veto cells before and after T c r u z t infection also showed sharp differences m the fatty acid c o m p o s m o n Lmolelc and hnolenlc acids were not detected in Vero cells before Infection However, in Vero cells, after T ~ruz~ infection. both unsaturated fatty acids were found in high and low concentrations, respectively The results obtained by catalytic hydrogenation confirmed the presence of the unsaturated fatty acids, the characteristic peaks of palmltolelc, olelc, hnolelc, and hnolenlc acids were completely abohshed with a corresponding increase in the size of the peaks of palmltlC and stearlc acids The fatty acMs of both T c r u z t developmental stages, as well as of Vero cells before and after parasite infection, were also identified by G L C - M S according to their corresponding M + and characteristic fragment ions (Table 2)

Discussion Even-numbered, saturated, monoenolc and polyenolc acids ranging from C-12 to C18 were detected as components of the total hpld fraction of T c r u z t Significant differences in fatty acid concentrations were found between two stages in the T ~ruzt life cycle Olelc and hnoletc acids were most prominent m trypomastlgotes whereas palmlttC acid as well as olelc and llnolelc acids, were the major components In amastigote forms Possibly, such changes m the pattern of fatty acids may be associated with the process of trypanosome differentiation In another trypanosomatld, H e r p e t o m o n a s samuelpe~'~oat, differentiation from promastlgote to opisthomastlgote forms is also followed by marked changes in the fatty acid profile (Pinto et a l , 1982) Studies on T c r u z t hfe cycle stages showed that they differ in other components, such as exposed cell surface carbohydrates (Perelra et a l , 1980) Also, TABLE 2 GLC-MS of fatty acid methyl esters of total hpJds from trypomastlgote (Tp) and amasugote (Am) torms of Tr~panmoma ~ruzl and Vero cells (Vc) obtained under the condmons described m Table I Fatty acid

C db"

Source

M + ion (m,'e)

CharacterlstJc fragment ions (m,'e)

Launc Mynstlc Palmltlc Palmllolelc Steanc Olelc Lmolelc Lmolemc

C120 C140 Cl6 0 C16 1 C180 C18 1 C18 2 C183

Tp,Vc TpAm,Vc Tp Am,Vc Tp,Am,Vc Tp Am,Vc Tp,Am,Vc Tp,Am,Vc Tp,Am,Vc

214 242 270 268 298 296 294 292

185(M + - 29), 213(M ~ 29), 241(M + - 29), 237(M- 31), 269(M - - 2 9 ) , 265(M- 31), 263(M+ 31), 261(M+ 31),

~Number of carbon atoms number of double bond~

183(M + - 3 I ) , 211(M + 31), 239(M ~ - 31), 236(M + 32), 267(M+ - 31), 264(M + 32), 262(M* 32), 260(M+-32),

171(M- - 4 3 ) 199(M- 43) 227(M - 43) 194(M- 74) 255(M 43) 222(M- 74) 220(M- 74) lI8(M+ 74)

135

the stage specific gene expression precedes morphological changes during T cruzl metacyclogenesls (Contreras et al, 1985) The fatty acid composition observed m both trypomastlgote and amastlgote stages quahtatlvely resembled that previously detected in T cruzl eplmastlgote forms (Aeberhard et al, 1981) However, some quantitative differences were noted in contrast to our results, hnolelc acid was the only prominent fatty acid found in eplmastlgotes and hnolenlc acid was not detected Again, such differences may be due in part to T cruzt cell differentiation It is also possible that there may be strain differences in fatty acid content In the present study we infected tissue culture cells with trypomastlgotes which developed into amastlgote forms As expected, hnolelc acid, which is absent in mammalian cells, was not found in Vero cells After infection with trypomastlgotes alterations in Vero cell fatty acid composition occurred For example, they acquired hnolelc and hnolemc acids, probably directly from the parasite or from some interaction w~th it We cannot rule out that some of this material came from amastlgote forms present in undlsrupted infected cells As noted above, amastlgotes, m comparison with trypomastlgote forms, showed an increase in palmltlC acid Moreover, the concentration of this saturated fatty acid in Vero cells increased after parasite infection This result suggests that the increased palmltlC acid m amastlgotes may represent a real synthetic abdlty rather than a simple uptake of hplds from the cell host In trypanosomatlds the de novo synthesis of fatty acids was described for Let~hmanta tarentolae by Korn et al (1965) They also found, by radlolabehng with [1-14C]acetate, that bloodstream forms of T r y p a n o s o m a lewlsL elongated existing short chain fatty acids rather than relying on de novo synthesis to produce C-20 to C22 polyunsaturated fatty acids Other studies demonstrated that eplmastigotes of T cruzt synthesize fatty acids by a de novo pathway and not by fatty acid chain elongation (Aeberhard et al, 1981) The cell surface of parasites may play an important role m the process of pathogenesis It is through the cell surface that most parasites interact with the host cells (Wallach, 1979) There is also ewdence that llplds affect the cell surface recognition and interaction (Bramhull and Wlsnleskl, 1981, Hoover et al, 1977, Horwitz et al, 1974) The present study suggests that further work is necessary to clarify whether the differences detected in the fatty acid composition between trypomastlgotes, amastigotes and in host cells before and after infection may play a role on cell interaction, penetration and parasite release In other unrelated models, some viruses display host-lipid specificity during their assembly, with their release from cells always involving an alteration of the host membrane composition or structure (Bramhull and Wasnleskl, 1981)

Acknowledgments We thank Prof Lmz Rodolpho Travassos for his help during manuscript preparation Th~s work is supported by CNPq, Fmep and C E P G - U F R J References Aeberhard, E E , Lema M G and Broma, D l H (1981) Biosynthesis of fatty acids by Trypanosoma cruzl Llplds 16, 623 625

136 Bramhull, J and Wlsmeskl, B (1981) Virus receptors, part 2 In K Lomberg-Holm and L Phlhpson (Eds), The Role of Llplds in Virus-Cell Interaction, Vol 8, Chapman and Hall, New York, NY, p 141 Brener, Z (1973)Biology of Trypano~oma cruzt Annu Rev Mlcroblol 27, 347 382 Carvalho, T U and de Souza, W (1983) Separation of amastlgotes and trypomastlgotes of Trvpano~oma cruzt from cultured cells Parasltenkunde 69, 571-575 Contreras, V T , Morel, C M and Goldenberg, S (1985) Stage specific gene expression precedes morphological changes during Trvpanosoma ~ruzz metacyclogenesls Mol Blochem Parasltol 14 83 95 De Souza, W (1984) Cell Biology of Trvpano~oma cruzt lnt Rev Cytol 86, 197 283 Hoover, R L , Lynch R D and Karnovsky, M J (1977) Decrease in adhesion of cells cultured in polyunsaturated fatty acids Cell 12, 295 300 Horwltz, A F , Hatten, M E and Burger, M M (1974) Membrane fatty acid replacements and their effect on growthandlectm-lnducedagglutmablhty Proc Natl Acad Scl U S A 71, 3115 3119 Korn, E D , Greenblat, C L and Lees A M (1965) Synthesis of unsaturated fatty acid in the slime mold Phy~arum polycephalum and the zooflagellates Lelshmama tarentolae, Trypano~oma lewis1 and CrzthMta sp A comparative study, J L~p~d Res 6, 43-50 Mortattl, R C and Munk, M E (1985) Separation of bloodstream trypomastlgotes of trvpanosorna (ruzt by density gradient centrlfugatlon J Parasltol 71, 520 521 Perelra, M E A , Loures, M A , Vlllalta, F and Andrade, A F B (1980) Lectm receptor as markers for Trypanosoma cruz1 Developmental stages and a study of the interaction of wheat germ agglutlnln with slahc acid residues on eplmastlgotes cells J Exp Med 152, 1375 1392 Pinto, A S, Pinto, A C , de Souza, A and Angluster, J (1982) Fatty acid composition In Herpetomonas samuelpessoat influence of growth conditions Comp Blochem Physlol 73, 351 356 Porschmann, J , Welsch, T , Herzschuh, R , Engelwald, W and Muller, K (1982) Analys~s of fatty acids by combined apphcatlon of chemical chromatographic and spectroscopic methods J Chromatogr 241, 73 87 Wallach, D F H (1979) The membrane pathoblology of tropical diseases In Plasma Membrane of Eucaryotlc Cells Some General Principles, Schwabe, Basel, p 1 Wnghtsman, R A , Leon, W and Manning, J E (1986) Variation m antigenic determinants specific to the infective stage of Trvpano~oma ~ruzl Infect Immunol 53, 235 239