Trypanosoma brucei: Biochemical and morphological changes during in vitro transformation of bloodstreamto procyclic-trypomastigotes

EXPERIMENTAL

PARASITOLOGY

51,

408-417 (1981)

Trypanosoma brucei: Biochemical and Morphological Changes during in Vitro Transformation of Bloodstreamto Procyclic-Trypomastigotes E. JAY BIENEN, Depurtment

of Pathology,

ETTIMAD

HAMMADI,

AND

GEORGE C. HILL

Graduate Program in Cellular and Molecular Biology, University, Fort Collins, Colorado 80523, U.S.A.

Colorado

State

(Accepted for publication 29 May 1980) BIENEN, E. J., HAMMADI, E., AND HILL, G. C. 1981. Trypanosoma brucei: Biochemical and morphological changes during in vitro transformation of bloodstream- to procyclictrypomastigotes. Experimental Parasitology 51, 408-417. Trypanosoma brucei brucei in whole rat blood inoculated into a semidefined medium undergoes complete morphological transformation (observed by light microscopy) in 72 hr. This reproducible system permits the biochemical and physiological study of transformation from bloodstream to procyclic trypomastigotes and mitochondrial biogenesis in these organisms. Infectivity for mice is lost after 6 days. Proline stimulates cell growth after transformation. High levels of glucose adversely affect the growth of transforming cells. Respiration during transformation is by an ol-glycerophosphate oxidase although a cyanide-sensitive pathway is present after 24-48 hr but does not become fully functional with respect to procyclic trypomastigotes until 20-24 days in culture. The success of this system will permit the biochemical characterization of African trypanosomes as the development of the cytochrome system occurs. INDEX DESCRIPTORS: Trypanosomu brucei brucei; Protozoa, parasitic; Hemoflagellate; Rat blood; Transformation; cu-Glycerophosphate oxidase; Mitochondrial biogenesis; Nutrition; Cytochrome aaB,

INTRODUCTION

The life cycle of African trypanosomes involves a vertebrate host and an insect (Glossina spp.) vector. The bloodstream trypomastigotes differ vastly from the insect forms in both morphology and ultrastructure (Steiger 1973), and biochemistry (Bowman 1974). The repressed mitochondrion of bloodstream trypanosomes, in the form of a single cristate canal, lacks a functional citric acid cycle and shows no spectral evidence for cytochromes (Fulton and Spooner 1959; Ryley 1956). Energy is produced via glycolysis with NADH reoxidized by an L-a-glycerophosphate oxidase (Grant and Sargent 1960) sensitive to hydroxamic acids (Evans and Brown 1973). Vector midgut forms (established procyclic trypomastigotes) possess a fully functional mitochondrion complete with Krebs cycle intermediates and cytochrome electron

transport system (Hill 1976) as well as an La-glycerophosphate oxidase. The transformation from bloodstream to procyclic trypomastigotes may be accomplished in vitro (Bowman et al. 1972; Brown et al. 1973; Cunningham 1977; Evans and Brown 1971; Ghiotto et al. 1979; Hill 1976; Srivastava and Bowman 1971; Srivastava and Bowman 1972; Steiger et al. 1977; Stuart 1975) and has been used to study development of cyanide sensitivity of trypanosome respiration (Bowman et al. 1972; Brown et al. 1973; Evans and Brown 1971; Srivastava and Bowman 1971; Steiger et al. 1977). These experiments, performed in various types of media, have provided somewhat conflicting results with regard to changes in the terminal oxidases. Further, only two time points have been used to obtain absolute values for the parameters studied. It is the purpose of this paper to define a repro-

408

0014-4894/81/030408-10$02.00/O Copyright All rights

0 1981 by Academic of reproduction in “y

Press, Inc. form reserved.

Trypanosoma brucei: BIOCHEMICAL ducible in vitro system for the study of trypanosome transformation and to use this system to study the biochemical parameters involved during this process as cell differentiation and synthesis of the cytochrome system occur. MATERIALS

AND METHODS

TRANSFORMATION

CHANGES

409

medium (BSM), modified as above, was added. To each flask was added a maximum of 20 ~1 of whole blood (based on cell count) containing 4 x lo6 cells giving a final concentration of 2 x IO6cells/ml. The cells were counted daily using a Neubauer hemacytometer and the percentage of forms displaying morphological transformation was calculated. The initial criteria for transformation were based on morphological characteristics as observed by light microscopy (i.e., size and shape, motility, presence or absence of undulating membrane). Transformed cells were larger than bloodstream trypomastigotes, lacked an undulating membrane, and had motility characteristics of procyclic cells. Permanent slides were Giemsa stained to confirm and permanently record these results. Largescale transformation experiments for the purpose of studying cell respiration were carried out in screwcap 500-ml Erlenmeyer flasks (Bellco) to which 200 ml of BSM medium was added. For these experiments, rats were sacrificed when the parasitemia reached 8 x lo8 cells/ml blood, and 0.5 ml of whole blood was added to each flask. The flasks were counted every 24 hr to determine cell growth and percentage of cells transformed. For electron microscopy, cells were dehydrated, fixed, and stained by procedures which we have previously described (Anderson and Hill 1969).

Organisms Trypanosoma (Trypanozoon) brucei brucei (LUMP 1026) was obtained from Dr. David Evans at the London School of Tropical Medicine and Hygiene, London, England, and maintained as stabilates from a rat sublethally irradiated with 800 rad from a 6oCo source. The stabilates were kept at -196 C and were thawed and counted prior to inoculation. Wistar rats weighing 200-300 g were inoculated intraperitoneally with 2 x lo6 cells. The parasitemia was checked daily until a minimum level of 2 x IO8cells/ml of blood was reached, at which point the rat was anesthetized and the parasites were obtained by exsanguination via cardiac puncture. Sodium citrate (0.5%) was used as an anticoagulant. The established procyclic trypomastigotes used for respiratory studies were from an early transformation experiment (August 1978) and had been maintained in culture at 25 C for over 5 months prior to use. The cells were subcultured every 4 days in a semidetined medium (Cunning- Nutritional Studies ham 1977) modified by addition of 25.2 For these studies, 2x concentrated memM N-2-hydroxyethylpiperazine-N’-2dium was made which lacked proline, gluethanesulfonic acid (Hepes) and 23.8 mM of cose, sucrose, and fructose. These subsodium bicarbonate, reduction of glutamine strates were made up separately as 10x soconcentration by 90%, and supplemented lutions in sterile distilled water and filtered by addition of 10% fetal bovine serum through a Swinnex Millipore filter with a (FBS) (Sterile Systems, Logan, Utah). Metrigard superfine pretilter. Five percent glycerin was also tried as a possible subTransformation Procedure strate and was added to the deficient medium in the same manner as the other Initial experiments were carried out at 25 substrates. One milliliter of medium and C in lo-ml capped Micro-Fernbach flasks 200 ~1 of substrate, either singly or in comwith DeLong necks (Bellco, Vineland, N.J.) to which 2 ml of Cunningham’s (1977) bination were added to each tube. The vol-

410

BIENEN,

HAMMADI.

AND HILL

ume was brought to 2 ml by the addition of blood preparation does not contribute to sterile distilled water. Controls of deficient the 0, uptake by trypanosomes. medium, normal medium, and 2x normal RESULTS medium diluted 1:l with sterile distilled water, were prepared simultaneously. In The kinetics of a typical transformation experiments using excess glucose, the ap- experiment with Trypanosoma brucei are propriate amount of glucose was dissolved shown in Fig. 1. As may be seen, transforin 10 ml of medium and filtered through a mation from bloodstream trypomastigotes Swinnex Millipore filter. Two milliliters to procyclic trypomastigotes is complete within 72 hr after initiating the experiment. was added to each flask. All transformation experiments were performed in duplicate. By this time the cells have the characteristic motility of procyclic trypomastigotes, an Respiratory Studies elongated body, and loss of the undulating Oxygen consumption by trypanosomes membrane. Initially there is a brief lag was monitored polarographically using phase in growth as transformation begins. whole cells in a closed oxygen electrode Following the lag phase, the cells grow system with a Clark-type oxygen electrode logarithmically (sometimes as high as 3-4 (YSI model 53: Yellow Springs Instrument x lo7 cells/ml) for 2-4 days before there is Co.). Incubation was carried out at 25 C in 3 a decline in cell number. Cells are usually ml of the BSM medium. Potassium cyanide subcultured in late log phase, 4 or 5 days was dissolved in water and salicylhydroxpostinoculation, and every 4 or 5 days amic acid (SHAM) was dissolved in 95% thereafter. ethanol. Final concentrations added to the Although transformation is complete cuvette were 1 and 2.7 mM for cyanide and using morphological criteria as observed by SHAM, respectively. The final ethanol light microscopy within 72 hr, 2 x 10” cells concentration was less than l%, a level inoculated into mice will result in an infecwhich had no inhibitory effect on respira- tion up to 6 days after the transformation tion. Since these transformation studies were done in the presence of whole blood, it was necessary to eliminate the erythrocytes prior to the polarographic assay. This was accomplished by changing the osmolarity of an isotonic solution with trypanosome dilution buffer (TDB), using the method outlined by Rosen et al. (1979) for T. congolense. After centrifugation, the cells were washed once, repelleted, and suspended in BSM to a concentration of 2 x 10” cells/ml. One-tenth milliliter of this suspension containing 2 x IO8cells was then added to the cuvette for the assay. Established procyclic trypomastigotes were harvested by centrifugation, washed with TDB, and analyzed in the same manner as the transformFIG. 1. Kinetics of Trypanosomu brucei LUMP ing cells. Whole blood from uninfected rats 1026 transformation in BSM medium, initial inoculum 2 was treated with TDB and used for the X 10” cells/ml. (0) percentage transformation, (A) cell polarograph assay to show that the lysed growth.

Trypanosoma

brucei: BIOCHEMICAL

experiment was begun with a twofold increase in the prepatent period. The loss of the surface coat was followed using electron microscopy. At 0 hr, all cells had a complete surface coat. By 48 hr, cells were seen both with and without a surface coat (Fig. 2) and there was complete loss of the coat in all cells observed after 96 hr in culture . In a natural transformation situation, the trypanosomes transfer from a glucose-rich environment (bloodstream) to a proline-rich environment (tsetse fly) with residual glucose still being present from the bloodmeal. Thus, some of the nutritional requirements for transformation were investigated. When individual substrates were added to media deficient in glucose, sucrose, fructose, and

TRANSFORMATION

CHANGES

411

proline, the only substrate which supported a normal growth and transformation rate was proline (Figs. 3A and B). With the other substrates, transformation took longer than the controls (both normal BSM and deficient BSM reconstituted with the four substrates) and the cell growth was 50% less than the control values. Control medium deficient in all four substrates did not support growth. Glycerin, not normally present in the medium, was also used as a substrate with results similar to those for the other sugars. Growth of transforming cells in the presence of excess glucose is shown in Fig. 4. The rationale behind this approach was the idea that a possible influence on the repression-induction of trypanosome mito-

FIG. 2. Electron micrograph of two Trypano~o~a brucei LUMP 1026 cells 4%hr after transformation has begun. Note the presence of the surface coat on one cell (SC) and its absence on the other. By morphological criteria, the total population at 48 hr was 68% transformed. x 144,000

412

BIENEN,

HAMMADI,

AND

HILL

$-+GllKOSC m- -mGlycerin Proline . . ..rgek(

0

A

HoursI” Culture

B

FIG. 3. Kinetics of Ttypunosoma brucei LUMP 1026 transformation growth. (B) Percentage transformation (A. .A) BSM medium, (+--+) (m---m), glycerin.

chondrial function might be the change in glucose concentration similar to mitochondrial glucose repression in yeast (Perlman and Mahler 1974), albeit the glucose levels used to repress yeast are much higher than blood glucose levels. Excess glucose, while having an inhibitory effect on cell growth, supported a normal transformation pattern. Significant results were obtained investigating trypanosome respiration. Bloodstream trypomastigotes have an L-a-glycerophosphate oxidase (L-(w-GP oxidase) which is cyanide insensitive but salicylhydroxamic acid (SHAM) sensitive, as the exclusive means of respiration (Fig. 5A). This pathway is also present to a lesser extent in established procyclic trypomastigotes, where the primary oxidase is the cyanidesensitive mitochondrial cytochrome electron transport system. In the closed polarograph system, this accounts for 70-80% of the total cell respiration, with the L-wGP oxidase responsible for 20-30% (Fig. 6).

24

40

72

96

Hours in Culture

in various substrates. (A) Cell glucose (O-O), proline

If these results are compared with the polarograph tracing of transformed cells at 72 hr (Fig. 5B), it may be seen that normal cyanide sensitivity with respect to established procyclic trypomastigotes is absent. At this point, inhibitor sensitivity seems to be dependent upon the sequence of addition of inhibitors with the SHAM-sensitive pathway still acting as the primary terminal oxidase. Levels of cyanide sensitivity approaching those of established procyclic trypomastigotes are not seen until 20-24 days (four subcultures) postinoculation into the transformation medium. The respiratory levels during this period are shown in Figs. 7A and B. Further subculturing does not show any significant changes in cyanide sensitivity, the level of inhibition being maintained at 70-80%. These experiments have been repeated several times with the same results. Concomitant with these changes in respiratory pathways, there is also a decrease in the total respiration rate of whole cells. During the first 10 days of

Trypanosoma

brucei: BIOCHEMICAL

TRANSFORMATION

CHANGES

413

oxygen consumption stabilized at this latter value. r&w -50

3.amm mM

DISCUSSION

l 278mM

The results reported here describe a system whereby the in vitro transformation of Trypanosoma brucei LUMP 1026 may be reproducibly obtained. This enables us to study several parameters including the nutritional requirements of transforming cells as well as the synthesis of the mitochondrial cytochrome system. In our system, morphological transformation, at the level of light microscopy, takes place within 72 hr. This is in good 2.5L \ agreement with results obtained with T. brucei and T. rhodesiense using other systems (Ghiotto et al. 1979; Stuart 1975), but half the value obtained with a different strain of T. brucei using SM medium (Cun1.0. 144 192 240 0 48 96 ningham 1977). One would expect that difHcun in Culhn ferent strains in different media would show FIG. 4. Growth of transforming Trypanosoma brucei LUMP 1026 cells in the presence of excess variability with respect to rates of transglucose. Morphological transformation complete by formation, since it has been our experience 72 hr. (A), BSM medium, 3.8 nnI4 (O), 50 mh4 (W), that not all strains of trypanosomes will 278 mA4. give consistent or even similar results in a given medium. culture, there is an approximate fourfold The sigmoidal type growth curve is typidecrease in oxygen utilization from 80 cal of predicted population growth. Previnmole O,/min/l x lOa cells to 22.5 nmol ous transformation studies using T. brucei Oz/min/l x lo8 cells. After 10 days, the LUMP 1026 (Hill 1976) resulted in a sharp 0 HOURS

72

HOURS cqlls

FIG. 5. Polarograph tracing of whole cell respiration of transforming Z’rypan~~oma brucei LUMP 1026(see text for assay conditions). (A) Zero-hour time point, equivalent to bloodstream trypomastigotes (B) Seventy-two hours after inoculation into culture, 100% morphological transformation.

414

BIENEN,

HAMMADI,

AND

HILL

KCN

6. Polarograph tracing of whole cell respiration of established Try~~~nosorna brucei LUMP 1026 (see text for assay conditions). FIG.

~-4

SHAM

Sensitivity

in the Absence

W

SHAM

Sensitivity

in the Presence

Days

e--O -

CN‘

in

of

of CN-

Culture

Sensitivity

in the Absence

of SHAM

in the Presence

of SHAM

in

trypomastigotes

of CN-

CN‘ Sensitivity

Days

procyclic

Culture

FIG. 7. Development of CN--sensitive respiration during Trypanosomu brucei LUMP 1026 transformation. (A) changes in levels of SHAM sensitivity in the presence (0) and absence (a) of CN-. (B) Changes in levels of CN- sensitivity in the presence (0) and absence (0) of SHAM.

Trypanosoma brucei:

BIOCHEMICALTRANSFORMATION

decrease in cell numbers over the first 6 days in culture with a subsequent slight increase to a stationary phase at a cell density less than half the density of stationary phase cells in our system. However, these experiments were performed in a medium (F-13) which we have found unsuccessful in our current studies. The brief lag phase coinciding with the beginning of cell transformation indicates that cell division is not needed for transformation to occur. Supporting evidence in this study for this proposal are the results showing inhibition of growth at high levels of glucose in the transformation medium. This agrees with the results obtained by Ghiotto et al. (1979) which show transformation still occurs in the presence of the mitotic inhibitor hydroxyurea. These investigators also found that infectivity is lost after 72 hr, in contrast to 6 days in our study. In both cases, an increase in the prepatent period was observed. Although loss of surface coat is seen by 96 hr in the micrographs we have observed, this does not rule out the possibility that there are still some trypanosomes which have not lost their surface coat. Since in effect only one trypanosome is actually needed to initiate an infection, most probably there is still a very small population of nontransformed cells retaining their infectivity. The nutritional studies described here indicate a proline requirement for stimulation of cell growth after transformation has occurred. This is not too surprising since it has previously been shown that not only is proline a major component of tsetse fly haemolymph and energy source for the fly (Bursell 1966; Cunningham and Slater 1974) but also that proline is the preferred carbon source for T. brucei (Evans and Brown 1972), T. rhodesiense (Srivastava and Bowman 1972), and T. congolense (Steiger et al. 1977) grown in culture. The preferential utilization of amino acids is not unique to trypanosomes. Glutamine has also been shown to be of major importance as an oxidation substrate of rabbit reticulocytes (Rapoport et al. 1971) and.lymphoma cells

CHANGES

415

(Lavietes et al. 1974), and is the major energy source of cultured HeLa cells (Reitzer et al. 1979). Although the blood glucose level of mammals (5 mM 0.9%) is lower than the levels (i.e., l-5%) which repress yeast mitochondria (Ibrahim et al. 1973), excess glucose was used to find out if transformation could be inhibited or delayed. As shown in Fig. 4, the effect was not on morphologic transformation, but rather on cell growth. The normal transformation pattern in the presence of excess glucose cannot rule out the possibility that the glucose may also be having an effect at the mitochondrial level. Previous studies on respiration during transformation of T. brucei (Brown et al. 1973; Evans and Brown 1971) and T. rhodesiense (Bowman et al. 1972; Srivastava and Bowman 1971; Srivastava and Bowman 1972) have focused on respiratory inhibition by potassium cyanide (KCN) after substrate stimulation of cell lysates. In all these studies, succinate oxidation increased during transformation, but KCN inhibition varied. Using T. rhodesiense grown in a diphasic blood agar medium, complete inhibition of respiration was obtained with 3 mM KCN after 3 days (Srivastava and Bowman 1971; Srivastava and Bowman 1972). In the case of T. brucei grown in a blood lysate broth, no cyanide sensitivity was seen with concentrations as high as 10 mM until after 14 days in culture (21%) or until after subculture into a diphasic medium (Brown et al. 1973; Evans and Brown 1971). None of these experiments with T. brucei made use of SHAM as an inhibitor of the L-a-GP oxidase during transformation. Whole cell respiratory studies of transforming T. congolense using both SHAM and KCN showed that after Day 4 only 20-30% of total cell respiration is cyanide sensitive, with 60-70% SHAM sensitivity, but the reverse occurs after Day 10 (Steiger et al. 1977). The order of inhibitor addition is not mentioned, however. The results reported in this study indicate

41.6

BIENEN,

HAMMADI,

that during transformation the components of a functional cyanide sensitive terminal oxidase are present after 24 hr in culture, but inactive unless the SHAM-sensitive pathway is blocked. This agrees with other results indicating that it might be necessary for the cells to pass through an initially cyanide-insensitive stage before developing cyanide sensitivity (Evans and Brown 1971). The reason for this is uncertain, but may be linked to the fact that morphometric analysis by electron microscopy of transforming cells has shown that quantitatively, establishment of procyclic trypomastigotes is still incomplete after 11 days (Ghiotto et al. 1979). The length of time required for the development of cyanide sensitivity usually associated with established procyclic trypomastigotes is significant. The value of 20-24 days for development of cyanidesensitive respiration observed in this study using a polarographic assay approximates the value obtained by Balber (1972) of 21/-3 weeks required for visual localization of cytochrome uu3 by positive diaminobenzidine staining (Seligman et al. 1968). It would be extremely important to try and determine what are the factors controlling the rate of development of a full complement of cytochromes. Preliminary experiments suggest that no spectral evidence for cytochrome uu3 is present on Day 5 of a transformation experiment. Thus, a close monitoring of the cytochrome absorption spectra in conjunction with respiratory studies will allow us to determine when cytochrome au3 begins to be synthesized. The delay in the development of the cytochrome au3 terminal oxidase may permit biochemical investigation of the events occurring during the biosynthesis of this important respiratory enzyme in Trypunosomu brucei. ACKNOWLEDGMENTS

This research was supported by an NIH Research Career Development Award (1-KO-4, AI-708 13-05)to G.C.H., and NIH research Grant (1-AI-11622-05) and a contract from the U.S. Army Medical Research and Development Command (DAMD-17-74-4046). We

AND

HILL

thank J. Sugimoto, Carol Greenwell, and Cynthia J. Turner for help in preparing the prints for the manuscript and Kathy Rounds and Sandy Swets for typing the manuscript. REFERENCES

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RYLEY, J. F. 1956. Studies on the metabolism of the protozoa. VII. Comparative carbohydrate metabolism of 11 species of trypanosomes. Biochemical Journal 62, 215-224. SELIGMAN, A. M., KARNOVSKY,M. J., WASSERKRUG, H. L., AND HANKER, J. S. 1968. Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). Journul of Cell Biology 38, I-14. SRIVASTAVA, H. K., AND BOWMAN, I. B. R. 1971. Adaptation in oxidative metabolism of Trypanosoma rhodesiense during transformation in culture. Comparative Biochemistry and Physiology 40B, 973-981. SRIVASTAVA, H. K., AND BOWMAN, I. B. R. 1972. Metabolic transformation of Trypnnosoma rhodesiense in culture. Nature (New Biology) 235, 152- 153. STEIGER, R. F. 1973. On the ultrastructure of Trypanosoma (Trypanozoon) brucei in the course of its life cycle and some related aspects. Acfa Tropica 30, 64- 168. STEIGER, R. F., STEIGER, E., TRACER, W., AND SCHNEIDER, I. 1977. Trypanosoma congolense: Partial cyclic development in a Glossina cell system and oxygen consumption. Journal of Parasitology 68, 861-867. STUART, K. D. 1975. Mitochondrial protein synthesis and respiratory development in Trypanosoma brucei. Journal of Cell Biology 67,420a (Abstract).