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Trypanosoma cruzi: Comparative fatty acid metabolism of the epimastigotes and trypomastigotes in vitro
EXPERIhfENTAL
PARASITOLOGY
38, 202-207
(1975)
Trypanosoma cruzi: Comparative Fatty Acid Metabolism Epimastigotes and Trypomastigotes in Vitro l DUELL Department Hopkins
of the
E. WOOD" AND EVERETT L. SCHILLER
of Pathobiology, School of Hygiene Univemity, 615 North Wolfe Street,
(Accepted
for publication
January
and Public Baltimore,
Health, Maryland
The Johns 21205
20, 1975)
WOOD, D. E. AND SCHILLER, E. L. 1975. Trypanosoma cruzi: comparative fatty acid metabolism of the epimastigotes and trypanomastigotes in vitro. Experimental PaTusitology 38, 202-207. In vitro studies on the fatty acid metabolism of the epimastigotes and trypomastigotes of Trypanosoma cruzi showed the following: (1) Trypomastigotes demonstrated the ability to convert labeled palmitic acid to CO,. Epimastigotes either did not convert this fatty acid to CO, or did so at a very low rate. (2) Trypomastigotes incorporated palmitic acid into neutral lipids, but, at a rate less than that of the epimastigotes. (3) While epimastigotes readily incorporated palmitic acid into phospholipid lipids, trypomastigote forms seemed to lack this ability. INDEX DESCRIPTORS: Trypanosoma crud; Metabolism; Fatty acid; Epimastigotes; Trypomastigotes; Carbon dioxide; Palmitic acid.
INTRODUCTION Studies on the metabolism of Trypanosoma cruzi require the availability of large quantities of organisms. In order to fulfill this requirement most investigators have relied on in &TO cultivation (cf. von Brand 1966; Honigberg 1967). Consequently, much of the knowledge about the metabolism of these protozoans is based on the culture (epimastigote) forms. Within the past few years, in vitro culture systems which yield substantial quantities of the trypomastigote forms of T. cruzi have been devised (Castellani Ed al. 1967; Wood and Pipkin 1969). The culture method utilized by Wood and Pipkin 1 The opinions or assertions contained herein are those of the authors and are not to be construed as reflecting the views of the Navy Department or the Naval Service at large. 2 Present Address: Gorgas Memorial Laboratory, Box 2016, Balboa Heights, Canal Zone, Republic of Panam;i.
Copyright All rights
Q 1975 by Academic Press, Inc. of reproduction in any form reserved
(1969) employed the insect-derived culture system of Grace (1962). In that system, dividing epimastigotes differentiated into trypomastigotes in a manner simlar to that described as occurring in the gut of reduviid vectors (Brumpt 1912; Petana 1971) . Trypanosoma cruzi harvested from the insect-derived culture system has the ability to utilize long chain fatty acids as a source of energy (Wood 1975). The results obtained in that study indicated that the ability to convert fatty acids to CO, might possibly be attributable only to the trypomastigotes in the experimental pop"lation. In order to investigate that possibility further, the following study was undertaken. METHODS
AND
MATERIALS
A4ost of the methodology has already been described (Wood 1975). Since T.
FATTY ACID METABOLISM
cruzi from the insect-derived culture system readily converts palmitic acid to COZ (Wood 1975)) this fatty ‘acid was used exclusively for these experiments. Stock solutions of labeled palmitic acid were prepared as previously described. Secondary in vitro passages of the Goble strain of T. cruzi were established in 50 ml of medium, prepared as described by Wood ( 1975), and incubated at 28 C in 250-ml plastic tissue culture flasks (Falcon Plastics, Inc., Culver City, California). When differentiation of epimastigote to trypomastigote forms was first noted, aliquots of the culture populations were removed and the parasites were harvested. Four additional havests were made after subsequent incubation times as differentiation of the parasites in the culture population proceeded. The percentage of epimastigotes as compared to trypomastigotes in succeeding harvests decreased as illustrated in Table I. On the day of harvest, each experimental population was treated according to the following protocol: Sets of four reaction flasks, each containing approximately 9.5 X lo7 parasites in 5 ml of Grace’s Medium (GMA) (Grace 1962) and one flask containing GMA alone were included in each experiment. All flasks were sealed, as described by Wood ( 1975), and placed in a 37 C shaking water bath for 5 min to allow for temperature equilibration. One-tenth milliliter of a plus [1-l’C]bovine plasma albumin palmitic acid stock solution, having approximately 1 &i of activity, was then injected into each flask. One flask containing parasites was immediately injected with 0.25 ml of 100% trichloroacetic acid (TCA) and served as the zero-time control. At the end of 60 min, all flasks were brought to 5% TCA and the CO, was collected. The contents of each flask were then harvested and the lipids were extracted. Collection of CO2 and extraction of the lipids were accomplished using
OF Trypanosoma
cruzi TABLE
203 I
Ratio of Epimastiqotes to Trypomastiqotrs in Experimental Populations Harvested from the Insect Oriented Culture System After the Indicated Hours of Incubation Experimental POPUlation 1 2 3 4 5
Hours of incubation 120 I A0 1x0 192 240
Epimastigotes: trypomastigotes
9R:l x0:20 .X:42 48:52 2537.5
previously described methods (Wood 1975). The data are presented as the nanomoles of labeled substrate converted per 10s parasites per hour and are expressed as the mean -t the standard error of three replicate determinations. Conversions of the data, from counts per minute of the radioactive labeled conversion products to the nanomoles of fatty acid substrate converted, were calculated as previously described (Wood 1975). Linear regression analyses, least squares fits, and exponential analysis were made with a Wang 700 B computer (Wang Laboratories, Inc., Tewksbury, Massachusetts) using their Regression Analyses programs. RESULTS
The results, compiled in Table II, revealed that for every increase in the percentage of trypomastigotes in an experimental population there was a corresponding increase in the rate of conversion of palmitic acid to COZ. Conversely, there were decreases in the rates of phospholipid, lipid, and neutral lipid synthesis from palmitic acid under these same conditions. The results indicated: (1) that there was a positive relationship between the percentage of trypomastigotes in the experimental population and the rates of conversion of fatty acid to CO,; and (2) that
204
WOOD
AND ‘I’ARLF:
Ii&s
11
of ~~onv~rsion of Paltrsztic :lcid to CO p, iVrutra1 I,ipid und I’hospholipill Lipids by Five II’. cruzi Populations According to the Ratio of Epimastigotes: Trypomastigotes in Each ____~ CoIlversion
Ilatio 01 rplmsst.igot es: I,~~ypomnst,igol,es 99:l 80:20 r,8 : 42 48:52 25:75
SCHILLER
CO2 0.005 0.016 0.055 0.110 0.239
+ zt * f f
0.0003 0.0006 0.0041 0.0035 0.0068
p~.odu~ls~~~h
ITe~Itral lipid 0.664 0.585 0.480 0.434 0.41%
3~ 0.0032 f 0.0260 f 0.0220 f 0.0020 31 0.0232
Phclspholipitl 1.914 1.619 1.117 0.876 0.704
f f f f zk
0.0333 0.0103 0.0540 0.0179 0.0051 -
a Expressed as nanomoles of labeled palmitic acid converted/l08 parasites/hr. * Each value is the mean f standard error of three replicate determinat,iorl.
centage of trypomastigotes in the experithere was a negative relationship between mental population was found to be expothe percentage of trypomastigotes in the experimental populations and the rates of nential. The line of least squares fit is shown in Fig. 1C. The T value of these conversion of palmitic acid into phosphodata was 0.90023, indicating this relationlipids and neutral lipids. ship also was statistically significant (P < In order to investigate these relation0.01) , ships further, scatter plots were made of the data from 21 individual experiments. Linear regression analyses were carried DISCUSSION out on these data. Figure 1A shows the line In populations that contained 99% epiof least squares fit plotted from data on the mastigotes, the rates of conversion of palconversion rates of fatty acid to phosphomitic acid to COZ were very low, indicating lipids by populations of T. cruzi containing that the utilization of fatty acids as a different ratios of epimastigotes to tryposource of energy by these forms of T. mastigotes. A comparison of the correlation cruzi was, at most, very limited. This obcoefficien ( r ) , -0.9877, with a table of T servation agrees with earlier reports of the values (Snedecor 1956) indicated that the failure of fatty acids to stimulate manonegative relationship between the convermetrically measurable increases in the rates sion of fatty acid to phospholipid lipids of respiration of the culture forms of T. and the number of trypomastigotes in the cmxi (‘cf. von Brand 1966). experimental population was statistically In contrast to the results on utilization of significant (P < 0.01). Figure 1B shows the fatty acid as a source of energy, the rates line of least square fit for data on the of incorporation of palmitic acid into rates of conversion of fatty acid to neutral neutral li’pids and phospholipid lipids lipids in the different populations of T. showed that long chain fatty acids actively cruxi. The T value of this correlation was participate in the biosynthetic processes of -0.9131, indicating that the negative relathe epimastigotes. Since epimastigotes are tionship between the conversion of fatty the forms of T. cruxi which undergo rapid acid to neutral lipids and the number of division and are responsible for the intrypomastigotes in the experimental popucrease in the density of the culture population was statistically significant (P < lations, the occurrence of high rates of in0.01). corporation of palmitic acid into neutral The relationship between the conversion rates of fatty acids to CO, and the perlipids and phospholipid lipids in these
FATTY
ACLD
METABOLISM
forms, as compared with trypomastigotes, was not surprising. Data obtained from these experiments clearly showed that as the number of trypomastigotes in each successive population increased, there were corresponding increases in the rates of ‘conversion of palmitic acid to COX. These increases in the rate of CO2 production appeared to be exponential as compared with the increases in the percentage of trypomastigotes (Table II). At the same time, decreases in the rates of incorporation of tpalmitic acid into neutral lipids and phospholipid lipids corresponded with decreases in the number of epimastigotes in each successive population of parasites. The exponential relationship between the number of trypomastigotes in each po’pulation and the rates of conversion of pahnitic acid to COP was confirmed by a curvilinear analysis of the data from 21 separate experiments. The results of this analysis showed that the ability of these organisms to convert fatty acid to CO2 probably was limited to the trypomastigote component of each population and was not attributable to the epimastigotes. One can only speculate as to the cause of the exponential increase in the rates of conversion of palmitic acid to CO2 in populations undergoing conversion from predominately epimastigote to predominately trypomastigote forms. One possible explanation may be the differences in the sources of energy required by these two forms. The principle source of energy for the epimastigotes is glucose (von Brand 1966)) and the data ,presented here demonstrate that utilization of long chain fatty acids as an energy source by these forms is, at most, very limited. In contrast, the present data indicate that trypomastigotes readily utilize fatty acids as sources of energy. Since, in the culture system employed in this study, epimastigotes differ-
OF
Trypanosoma cruxi
205
entiate into trypomastigotes through progressive transformation, it seems reason(Al
I
k
PERCENT PERCENT
EPIMASTIGOTE TRYPOMASTIGOTE
FORMS: FORMS
PERCENT PERCENT
EPIVASTIGOTE TRYPOMASTIGOTE
FORMS: FORMS
PERCENT PERCENT
EPIMASTIGOTE TRY”OMASTIGOTE
FORMS: FORhIS
Y
FIG. 1. Scatter plots of the rates of pa&tic acid to (A) phospholipid neutral lipids, and (C) COZ, from experiments with populations of cruzi composed of different ratios of to trypomastigotes, showing lines of fit from linear regression analyses exponential analysis ( C ) .
of conversion lipids, (B) 21 separate T~ypanosoma epimastigotes least squares (A, B), and
206
WOOD
AND
able to assume that any ‘change in their energy metabolism probably follows a similarly predicable pattern. If this is true, in each given parasite population there would be forms that utilize glucose for energy, i.e., epimastigotes, and forms that utilize glucose and fatty acids iuterchangeably for energy, i.e., “mature” trypomastidescribed gotes. In the experiments herein, ,parasite populations harvested at sequential incubation times contained dccreasing numbers of cpimastigotes, a varying number of develosping “intermediate” forms and increasing numbers of “mature” trypomastigotes. It is believed that the cumulative effect of the intermediate forms and the increasing numbers of “mature” trypomastigotes in each successive harvest could account for the exponential increases observed. A regression analysis of the data on lipid biosynthesis from the 21 separate experiments indicated that there was a linear decrease in the rates of incorporation of palmitic acid into neutral lipids which could be correlated with increase in the percentage of trypomastigotes, and consequent decreases in the percent of epimastigotes, in each pompulation. There also was a linear relationship between the increases in the percentage of trypomastigotes in each population and the corresponding decreases in the rates of palmitic acid incorporation into phospholipid lipids. These observations show that the epimastigotes actively incorporated palmitic acid int’o neutral lipids and phospholipids. Incorporation of this fatty acid into neutral lipids also occurred in trypomastigotes, but at rates less than those in epimastigotes. Trypomastigotes either did not incorporate palmitic acid into phospholipid or they incorporated it at a very low rate. In addition to the discovery that long chain fatty acids were used in the energy metabolism of T. cmzi, it has been demonstrated that these parasites, especially the epimastigotes, have the ability to form
SCHILLER
neutral lipids and phospholipid lipids from long chain fatty acid substrates. Various investigators have found that other kinetoplastid parasites also have lipid biosynthetic capabilities. Leishmania enriettii, Leishnmnia tarentolae, and Trypunosoma. lewisi synthesize a variety of fatty acids from a long chain fatty acid substrate without prior degradation of the substrate to shorter chain length fatty acids (Korn and Greenblatt 1963; Korn et al. 1965). Meyer and Holz (1966) have reported this same type of biosynthetic activity in a variety of other kinetoplastid flagellates. The data in the present study were not sufficient to determine whether interconversion and incorporation of fatty acids takes place in T. cruzi. However, the very low production of labeled CO, from labeled palmitic acid by Ihe epimastigotes indicates that degradation of this fatty acid to acetyl-CoA did not occur to any great extent in these parasites. Since degradation would have to take place in order for these parasites to produce lipids by way of the malonyl-CoA pathway, it is thought that direct interconversion of palmitic acid to other long chain fatty acids and esterificalion of these fatty acids to glycerol probably occurs in T. cruxi. The exact nature of this aspect of the fatty acid metabolism of these parasites was beyond the scope of this study but deserves further investigation. The data from the present study indicate that the fatty acid metabolism of the trypomastigotes of T. cruzi may be similar to that reported for striated muscle (Neptune et al. 1959), and for heart muscle (cf. Fritz 1961). This suggested similarity is very interesting and might, upon further investigation, help to explain the affinity of T. crusi for these tissues in the mammalian host. Carnitine, an organic compound which facilitates the transfer of fatty acids across mitochondrial membranes, is distributed throughout the mammalian body with highest concentration being found in skeletal muscle, heart
FATTY
ACID METABOLISM
muscle, kidney, and liver, in that order (cf. Fritz 1963). If fatty acid metabolism is as important in the energy metabolism of trypomastigotes in viva as it is in the trypomastigotes from in vitro culture, the concentration of carnitine in the above tissues may be more than circumstantially related to the factors that influence localization of these parasites in those areas of the body. Studies of the role of carnitine in the fatty acid metabolism of the trypomastigotes of T. cruzi and of the ‘possible role of this compound in the process of differentiation of these parasites from one morphological form to another, are presently being completed. The results of these and other studies dealing with the fatty acid metabolism of T. c~uzi will be presented in subsequent publications. ACKNOWLEDGMENTS This work was financed by the Bureau of Medicine and Surgery, Navy Department, Research Task No. MR005.05.01.0016 BZXE. REFERENCES BRUMPT, E. 1912. Le Trypanosoma cruzi Evolve chez Conorhinus magistus, Cimex lectularius, Cimex boueti et Ornithodorus moubata cycle evolutif de ce parasite. Bulletin Sociedad Pathologia Exotica 5, 36&367. CASTELLANI, O., RUBIERO, L. V., AND FERNANDEZ, J. F. 1967. Differentiation of Trypanosomu cruzi in culture. Journal of Protozoology 14, 447451. FRITZ, I. B. 1961. Factors influencing the rates of long chain fatty acid oxidation and synthesis in mammalian systems. Physiological Review 41, 52-129. FRITZ, I. B. 1963. Carnitine and its role in fatty acid metabolism. Advances in Lipid Research 1, 286332.
OF
Trypanosoma
cruzi
207
GRACE, T. D. C. 1962. Establishment of four strains of cells from inset tissues grown in vitro. Nature (London) 195, 788-789. HONINGBERG, B. M. 1967. Chemistry of parasitism among some protozoa. In “Chemical Zoology” (M. Florkin and B. T. Sheer, eds.) Vol. 1, 695-802 Academic Press, New York. KORN, E. D., AND GREENBLATT, C. L. 1963. Synthesis of a-linoleic acid by Leishmaniu enriettii, Science 142, 1301-1302. KORN, E. D., GREENBLATT, C. L., AND LEES, A. M. 1965. Synthesis of unsaturated fatty acids in slime mold. Physarum polyphalum and zooflagellates, Leishmania tarentolae, Trypanosonar lewisi and Crithidia sp.-A comparative study. Lipid Research 6, 43-50. MEYER, H., AND HOLZ, G. G. JR. 1966. Byosynthesis of lipids by kinetoplastid flagellates. Journal of Biological Chemistry 241, 50005007. NEPTUNE, E. M., JR., SUDDUTCH, H. C., FASH, F. J., AND FOREMAN, D. R. 1959. Quantitative participation of fatty acid and glucose substrates in the oxidation metabolism of exised rat diaphragm. American Journal of Physiology, 196, 269-272. PETANA, W. B. 1971. American Trypanosomiasis in British Honduras: IX. Development of Trypanosomu ( Schizotrypanum) cruzi in Triatomu dimidiata ( Hemiptera Reduviidae) and a note on the occurrence of dividing trypomastigote forms in the gut of some of the naturally infected bugs. Annals of TTOPical Medicine and Parasitology 65, 25-30. SNEDECOR, G. W. 1956. “Statistical Methods” the Iowa State College Press, Ames, Iowa. VON BRAND, T. 0. 1966. “Biochemistry of Parasites.” Academic Press, Inc. New York, New York. WOOD, D. E., AND PIPKIN, A. C. 1969. Multiplication and differentiation of Trypanosoma crmzi in an insect cell culture system. Experimental Parasitology 24, 176-183. WOOD, D. E. 1975. Trypanosoma cruzi: Fatty acid metabolism in vitro. Experimental Parasitology 37, 60-66.
PARASITOLOGY
38, 202-207
(1975)
Trypanosoma cruzi: Comparative Fatty Acid Metabolism Epimastigotes and Trypomastigotes in Vitro l DUELL Department Hopkins
of the
E. WOOD" AND EVERETT L. SCHILLER
of Pathobiology, School of Hygiene Univemity, 615 North Wolfe Street,
(Accepted
for publication
January
and Public Baltimore,
Health, Maryland
The Johns 21205
20, 1975)
WOOD, D. E. AND SCHILLER, E. L. 1975. Trypanosoma cruzi: comparative fatty acid metabolism of the epimastigotes and trypanomastigotes in vitro. Experimental PaTusitology 38, 202-207. In vitro studies on the fatty acid metabolism of the epimastigotes and trypomastigotes of Trypanosoma cruzi showed the following: (1) Trypomastigotes demonstrated the ability to convert labeled palmitic acid to CO,. Epimastigotes either did not convert this fatty acid to CO, or did so at a very low rate. (2) Trypomastigotes incorporated palmitic acid into neutral lipids, but, at a rate less than that of the epimastigotes. (3) While epimastigotes readily incorporated palmitic acid into phospholipid lipids, trypomastigote forms seemed to lack this ability. INDEX DESCRIPTORS: Trypanosoma crud; Metabolism; Fatty acid; Epimastigotes; Trypomastigotes; Carbon dioxide; Palmitic acid.
INTRODUCTION Studies on the metabolism of Trypanosoma cruzi require the availability of large quantities of organisms. In order to fulfill this requirement most investigators have relied on in &TO cultivation (cf. von Brand 1966; Honigberg 1967). Consequently, much of the knowledge about the metabolism of these protozoans is based on the culture (epimastigote) forms. Within the past few years, in vitro culture systems which yield substantial quantities of the trypomastigote forms of T. cruzi have been devised (Castellani Ed al. 1967; Wood and Pipkin 1969). The culture method utilized by Wood and Pipkin 1 The opinions or assertions contained herein are those of the authors and are not to be construed as reflecting the views of the Navy Department or the Naval Service at large. 2 Present Address: Gorgas Memorial Laboratory, Box 2016, Balboa Heights, Canal Zone, Republic of Panam;i.
Copyright All rights
Q 1975 by Academic Press, Inc. of reproduction in any form reserved
(1969) employed the insect-derived culture system of Grace (1962). In that system, dividing epimastigotes differentiated into trypomastigotes in a manner simlar to that described as occurring in the gut of reduviid vectors (Brumpt 1912; Petana 1971) . Trypanosoma cruzi harvested from the insect-derived culture system has the ability to utilize long chain fatty acids as a source of energy (Wood 1975). The results obtained in that study indicated that the ability to convert fatty acids to CO, might possibly be attributable only to the trypomastigotes in the experimental pop"lation. In order to investigate that possibility further, the following study was undertaken. METHODS
AND
MATERIALS
A4ost of the methodology has already been described (Wood 1975). Since T.
FATTY ACID METABOLISM
cruzi from the insect-derived culture system readily converts palmitic acid to COZ (Wood 1975)) this fatty ‘acid was used exclusively for these experiments. Stock solutions of labeled palmitic acid were prepared as previously described. Secondary in vitro passages of the Goble strain of T. cruzi were established in 50 ml of medium, prepared as described by Wood ( 1975), and incubated at 28 C in 250-ml plastic tissue culture flasks (Falcon Plastics, Inc., Culver City, California). When differentiation of epimastigote to trypomastigote forms was first noted, aliquots of the culture populations were removed and the parasites were harvested. Four additional havests were made after subsequent incubation times as differentiation of the parasites in the culture population proceeded. The percentage of epimastigotes as compared to trypomastigotes in succeeding harvests decreased as illustrated in Table I. On the day of harvest, each experimental population was treated according to the following protocol: Sets of four reaction flasks, each containing approximately 9.5 X lo7 parasites in 5 ml of Grace’s Medium (GMA) (Grace 1962) and one flask containing GMA alone were included in each experiment. All flasks were sealed, as described by Wood ( 1975), and placed in a 37 C shaking water bath for 5 min to allow for temperature equilibration. One-tenth milliliter of a plus [1-l’C]bovine plasma albumin palmitic acid stock solution, having approximately 1 &i of activity, was then injected into each flask. One flask containing parasites was immediately injected with 0.25 ml of 100% trichloroacetic acid (TCA) and served as the zero-time control. At the end of 60 min, all flasks were brought to 5% TCA and the CO, was collected. The contents of each flask were then harvested and the lipids were extracted. Collection of CO2 and extraction of the lipids were accomplished using
OF Trypanosoma
cruzi TABLE
203 I
Ratio of Epimastiqotes to Trypomastiqotrs in Experimental Populations Harvested from the Insect Oriented Culture System After the Indicated Hours of Incubation Experimental POPUlation 1 2 3 4 5
Hours of incubation 120 I A0 1x0 192 240
Epimastigotes: trypomastigotes
9R:l x0:20 .X:42 48:52 2537.5
previously described methods (Wood 1975). The data are presented as the nanomoles of labeled substrate converted per 10s parasites per hour and are expressed as the mean -t the standard error of three replicate determinations. Conversions of the data, from counts per minute of the radioactive labeled conversion products to the nanomoles of fatty acid substrate converted, were calculated as previously described (Wood 1975). Linear regression analyses, least squares fits, and exponential analysis were made with a Wang 700 B computer (Wang Laboratories, Inc., Tewksbury, Massachusetts) using their Regression Analyses programs. RESULTS
The results, compiled in Table II, revealed that for every increase in the percentage of trypomastigotes in an experimental population there was a corresponding increase in the rate of conversion of palmitic acid to COZ. Conversely, there were decreases in the rates of phospholipid, lipid, and neutral lipid synthesis from palmitic acid under these same conditions. The results indicated: (1) that there was a positive relationship between the percentage of trypomastigotes in the experimental population and the rates of conversion of fatty acid to CO,; and (2) that
204
WOOD
AND ‘I’ARLF:
Ii&s
11
of ~~onv~rsion of Paltrsztic :lcid to CO p, iVrutra1 I,ipid und I’hospholipill Lipids by Five II’. cruzi Populations According to the Ratio of Epimastigotes: Trypomastigotes in Each ____~ CoIlversion
Ilatio 01 rplmsst.igot es: I,~~ypomnst,igol,es 99:l 80:20 r,8 : 42 48:52 25:75
SCHILLER
CO2 0.005 0.016 0.055 0.110 0.239
+ zt * f f
0.0003 0.0006 0.0041 0.0035 0.0068
p~.odu~ls~~~h
ITe~Itral lipid 0.664 0.585 0.480 0.434 0.41%
3~ 0.0032 f 0.0260 f 0.0220 f 0.0020 31 0.0232
Phclspholipitl 1.914 1.619 1.117 0.876 0.704
f f f f zk
0.0333 0.0103 0.0540 0.0179 0.0051 -
a Expressed as nanomoles of labeled palmitic acid converted/l08 parasites/hr. * Each value is the mean f standard error of three replicate determinat,iorl.
centage of trypomastigotes in the experithere was a negative relationship between mental population was found to be expothe percentage of trypomastigotes in the experimental populations and the rates of nential. The line of least squares fit is shown in Fig. 1C. The T value of these conversion of palmitic acid into phosphodata was 0.90023, indicating this relationlipids and neutral lipids. ship also was statistically significant (P < In order to investigate these relation0.01) , ships further, scatter plots were made of the data from 21 individual experiments. Linear regression analyses were carried DISCUSSION out on these data. Figure 1A shows the line In populations that contained 99% epiof least squares fit plotted from data on the mastigotes, the rates of conversion of palconversion rates of fatty acid to phosphomitic acid to COZ were very low, indicating lipids by populations of T. cruzi containing that the utilization of fatty acids as a different ratios of epimastigotes to tryposource of energy by these forms of T. mastigotes. A comparison of the correlation cruzi was, at most, very limited. This obcoefficien ( r ) , -0.9877, with a table of T servation agrees with earlier reports of the values (Snedecor 1956) indicated that the failure of fatty acids to stimulate manonegative relationship between the convermetrically measurable increases in the rates sion of fatty acid to phospholipid lipids of respiration of the culture forms of T. and the number of trypomastigotes in the cmxi (‘cf. von Brand 1966). experimental population was statistically In contrast to the results on utilization of significant (P < 0.01). Figure 1B shows the fatty acid as a source of energy, the rates line of least square fit for data on the of incorporation of palmitic acid into rates of conversion of fatty acid to neutral neutral li’pids and phospholipid lipids lipids in the different populations of T. showed that long chain fatty acids actively cruxi. The T value of this correlation was participate in the biosynthetic processes of -0.9131, indicating that the negative relathe epimastigotes. Since epimastigotes are tionship between the conversion of fatty the forms of T. cruxi which undergo rapid acid to neutral lipids and the number of division and are responsible for the intrypomastigotes in the experimental popucrease in the density of the culture population was statistically significant (P < lations, the occurrence of high rates of in0.01). corporation of palmitic acid into neutral The relationship between the conversion rates of fatty acids to CO, and the perlipids and phospholipid lipids in these
FATTY
ACLD
METABOLISM
forms, as compared with trypomastigotes, was not surprising. Data obtained from these experiments clearly showed that as the number of trypomastigotes in each successive population increased, there were corresponding increases in the rates of ‘conversion of palmitic acid to COX. These increases in the rate of CO2 production appeared to be exponential as compared with the increases in the percentage of trypomastigotes (Table II). At the same time, decreases in the rates of incorporation of tpalmitic acid into neutral lipids and phospholipid lipids corresponded with decreases in the number of epimastigotes in each successive population of parasites. The exponential relationship between the number of trypomastigotes in each po’pulation and the rates of conversion of pahnitic acid to COP was confirmed by a curvilinear analysis of the data from 21 separate experiments. The results of this analysis showed that the ability of these organisms to convert fatty acid to CO2 probably was limited to the trypomastigote component of each population and was not attributable to the epimastigotes. One can only speculate as to the cause of the exponential increase in the rates of conversion of palmitic acid to CO2 in populations undergoing conversion from predominately epimastigote to predominately trypomastigote forms. One possible explanation may be the differences in the sources of energy required by these two forms. The principle source of energy for the epimastigotes is glucose (von Brand 1966)) and the data ,presented here demonstrate that utilization of long chain fatty acids as an energy source by these forms is, at most, very limited. In contrast, the present data indicate that trypomastigotes readily utilize fatty acids as sources of energy. Since, in the culture system employed in this study, epimastigotes differ-
OF
Trypanosoma cruxi
205
entiate into trypomastigotes through progressive transformation, it seems reason(Al
I
k
PERCENT PERCENT
EPIMASTIGOTE TRYPOMASTIGOTE
FORMS: FORMS
PERCENT PERCENT
EPIVASTIGOTE TRYPOMASTIGOTE
FORMS: FORMS
PERCENT PERCENT
EPIMASTIGOTE TRY”OMASTIGOTE
FORMS: FORhIS
Y
FIG. 1. Scatter plots of the rates of pa&tic acid to (A) phospholipid neutral lipids, and (C) COZ, from experiments with populations of cruzi composed of different ratios of to trypomastigotes, showing lines of fit from linear regression analyses exponential analysis ( C ) .
of conversion lipids, (B) 21 separate T~ypanosoma epimastigotes least squares (A, B), and
206
WOOD
AND
able to assume that any ‘change in their energy metabolism probably follows a similarly predicable pattern. If this is true, in each given parasite population there would be forms that utilize glucose for energy, i.e., epimastigotes, and forms that utilize glucose and fatty acids iuterchangeably for energy, i.e., “mature” trypomastidescribed gotes. In the experiments herein, ,parasite populations harvested at sequential incubation times contained dccreasing numbers of cpimastigotes, a varying number of develosping “intermediate” forms and increasing numbers of “mature” trypomastigotes. It is believed that the cumulative effect of the intermediate forms and the increasing numbers of “mature” trypomastigotes in each successive harvest could account for the exponential increases observed. A regression analysis of the data on lipid biosynthesis from the 21 separate experiments indicated that there was a linear decrease in the rates of incorporation of palmitic acid into neutral lipids which could be correlated with increase in the percentage of trypomastigotes, and consequent decreases in the percent of epimastigotes, in each pompulation. There also was a linear relationship between the increases in the percentage of trypomastigotes in each population and the corresponding decreases in the rates of palmitic acid incorporation into phospholipid lipids. These observations show that the epimastigotes actively incorporated palmitic acid int’o neutral lipids and phospholipids. Incorporation of this fatty acid into neutral lipids also occurred in trypomastigotes, but at rates less than those in epimastigotes. Trypomastigotes either did not incorporate palmitic acid into phospholipid or they incorporated it at a very low rate. In addition to the discovery that long chain fatty acids were used in the energy metabolism of T. cmzi, it has been demonstrated that these parasites, especially the epimastigotes, have the ability to form
SCHILLER
neutral lipids and phospholipid lipids from long chain fatty acid substrates. Various investigators have found that other kinetoplastid parasites also have lipid biosynthetic capabilities. Leishmania enriettii, Leishnmnia tarentolae, and Trypunosoma. lewisi synthesize a variety of fatty acids from a long chain fatty acid substrate without prior degradation of the substrate to shorter chain length fatty acids (Korn and Greenblatt 1963; Korn et al. 1965). Meyer and Holz (1966) have reported this same type of biosynthetic activity in a variety of other kinetoplastid flagellates. The data in the present study were not sufficient to determine whether interconversion and incorporation of fatty acids takes place in T. cruzi. However, the very low production of labeled CO, from labeled palmitic acid by Ihe epimastigotes indicates that degradation of this fatty acid to acetyl-CoA did not occur to any great extent in these parasites. Since degradation would have to take place in order for these parasites to produce lipids by way of the malonyl-CoA pathway, it is thought that direct interconversion of palmitic acid to other long chain fatty acids and esterificalion of these fatty acids to glycerol probably occurs in T. cruxi. The exact nature of this aspect of the fatty acid metabolism of these parasites was beyond the scope of this study but deserves further investigation. The data from the present study indicate that the fatty acid metabolism of the trypomastigotes of T. cruzi may be similar to that reported for striated muscle (Neptune et al. 1959), and for heart muscle (cf. Fritz 1961). This suggested similarity is very interesting and might, upon further investigation, help to explain the affinity of T. crusi for these tissues in the mammalian host. Carnitine, an organic compound which facilitates the transfer of fatty acids across mitochondrial membranes, is distributed throughout the mammalian body with highest concentration being found in skeletal muscle, heart
FATTY
ACID METABOLISM
muscle, kidney, and liver, in that order (cf. Fritz 1963). If fatty acid metabolism is as important in the energy metabolism of trypomastigotes in viva as it is in the trypomastigotes from in vitro culture, the concentration of carnitine in the above tissues may be more than circumstantially related to the factors that influence localization of these parasites in those areas of the body. Studies of the role of carnitine in the fatty acid metabolism of the trypomastigotes of T. cruzi and of the ‘possible role of this compound in the process of differentiation of these parasites from one morphological form to another, are presently being completed. The results of these and other studies dealing with the fatty acid metabolism of T. c~uzi will be presented in subsequent publications. ACKNOWLEDGMENTS This work was financed by the Bureau of Medicine and Surgery, Navy Department, Research Task No. MR005.05.01.0016 BZXE. REFERENCES BRUMPT, E. 1912. Le Trypanosoma cruzi Evolve chez Conorhinus magistus, Cimex lectularius, Cimex boueti et Ornithodorus moubata cycle evolutif de ce parasite. Bulletin Sociedad Pathologia Exotica 5, 36&367. CASTELLANI, O., RUBIERO, L. V., AND FERNANDEZ, J. F. 1967. Differentiation of Trypanosomu cruzi in culture. Journal of Protozoology 14, 447451. FRITZ, I. B. 1961. Factors influencing the rates of long chain fatty acid oxidation and synthesis in mammalian systems. Physiological Review 41, 52-129. FRITZ, I. B. 1963. Carnitine and its role in fatty acid metabolism. Advances in Lipid Research 1, 286332.
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GRACE, T. D. C. 1962. Establishment of four strains of cells from inset tissues grown in vitro. Nature (London) 195, 788-789. HONINGBERG, B. M. 1967. Chemistry of parasitism among some protozoa. In “Chemical Zoology” (M. Florkin and B. T. Sheer, eds.) Vol. 1, 695-802 Academic Press, New York. KORN, E. D., AND GREENBLATT, C. L. 1963. Synthesis of a-linoleic acid by Leishmaniu enriettii, Science 142, 1301-1302. KORN, E. D., GREENBLATT, C. L., AND LEES, A. M. 1965. Synthesis of unsaturated fatty acids in slime mold. Physarum polyphalum and zooflagellates, Leishmania tarentolae, Trypanosonar lewisi and Crithidia sp.-A comparative study. Lipid Research 6, 43-50. MEYER, H., AND HOLZ, G. G. JR. 1966. Byosynthesis of lipids by kinetoplastid flagellates. Journal of Biological Chemistry 241, 50005007. NEPTUNE, E. M., JR., SUDDUTCH, H. C., FASH, F. J., AND FOREMAN, D. R. 1959. Quantitative participation of fatty acid and glucose substrates in the oxidation metabolism of exised rat diaphragm. American Journal of Physiology, 196, 269-272. PETANA, W. B. 1971. American Trypanosomiasis in British Honduras: IX. Development of Trypanosomu ( Schizotrypanum) cruzi in Triatomu dimidiata ( Hemiptera Reduviidae) and a note on the occurrence of dividing trypomastigote forms in the gut of some of the naturally infected bugs. Annals of TTOPical Medicine and Parasitology 65, 25-30. SNEDECOR, G. W. 1956. “Statistical Methods” the Iowa State College Press, Ames, Iowa. VON BRAND, T. 0. 1966. “Biochemistry of Parasites.” Academic Press, Inc. New York, New York. WOOD, D. E., AND PIPKIN, A. C. 1969. Multiplication and differentiation of Trypanosoma crmzi in an insect cell culture system. Experimental Parasitology 24, 176-183. WOOD, D. E. 1975. Trypanosoma cruzi: Fatty acid metabolism in vitro. Experimental Parasitology 37, 60-66.