Protein synthesis during cercaria-schistosomulum transformation and early development of the Schistosoma mansoni larvae

Comp. Biochem. Physiol., 1977, Vol. 57B, pp. 27 to 30. Peroamon Press. Printed in Great Britain

PROTEIN SYNTHESIS DURING CERCARIASCHISTOSOMULUM TRANSFORMATION AND EARLY DEVELOPMENT OF THE SCHISTOSOMA MANSONI LARVAE* YOSHIE NAGAI I, G. GAZZINELLI1, G. W. G. DE MORAES1 AND J. PELLEGRINO2 1Departamento de Bioquimica e Imunologia; 2Centro de Pesquisas Ren6 Rachou (FIOCRUZ), Instituto de Ci~ncias Biol6gicas, Universidade Federal de Minas Gerais, 30,000 Belo Horizonte, Brasil (Received 6 September 1976) Abstract--1. Protein synthesis during early development of Schistosoma mansoni larva was investigated in vitro and assessed by labelled amino-acid incorporation. 2. The rate of protein synthesis was low at the first 6 hr, increasing gradually as the time elapsed. 3. The rate of 14C-leucine incorporation was strongly dependent on the enrichment of the incubation media with an amino-acid mixture. 4. The protein synthesis was not affected by the addition of 100 and 200 #g/ml of cycloheximide and chloroamphenicol to the incubation media. On the other hand, puromycin (500 #g/ml) and actinomycin (0.65-1.30 #g/ml) strongly inhibited protein synthesis. 5. Proteins of tegument incorporated 2-3 times more isotope than those of the larval body.

tubes and cooled in an ice bath for 10 min to reduce motility of the organisms. Following centrifugation at low speed for 1 min the supematant fluid was decanted. The packed cercariae were suspended in 2 ml of cold sterile Hanks'-BSS containing 1 mg/ml of streptomycin and 1000 units/ml of penicilin (HPSS) and whorled for 45~0 sec in a Vortex Jr. mixer (Scientific Industries Inc., Queens Village, NY). For isolation of cercarial bodies from tails a 2.0-ml suspension was transferred to another glass conical centrifuge tube containing 10ml of HPSS. After 10min at 5-10°C the tail-rich supernatant was decanted and the sedimented bodies resuspended again in 10 ml of the same solution. This procedure was repeated twice. The resulting bodies, containing 5% of tails, were washed 3-5 times with HPSS solution and suspended in the incubation media.

INTRODUCTION Penetration of Schistosoma mansoni cercariae through host skin is accompanied by pronounced changes o f the larval permeability to water (Stirewalt, 1963), changes of its ultrastructure (Smith et al., 1969; Hockley & MacLaren, 1973), and elimination of their acetabular glands (Stirewalt, 1963; Stirewalt et al., 1966). The resulting larva is called schistosomulum which further develop into mature worms in about 30 days in a susceptible host. The sequence of changes which are observed in vivo may be reproduced in vitro by cultivation of cercarial bodies obtained by physical method (Tiba et al., 1974; Colley & Wikel, 1974). Thus, it was possible to study, in a complete defined media, the rate of protein synthesis of S. mansoni larvae during and immediately after their transformation to schistosomulum. The results of this study are here presented.

Incubation procedures and radioactivity determination Half ml of cercarial body suspensions (10-15,000 organisms) were introduced in sterile 20-ml vials containing 2.4 ml of sterile Eagle's Minimum Essential Culture Medium (Eagle, 1949) without serum and leucine or valine and with a mixture streptomycin-penicillin (1 mg-1000 units/ml). After a pre-incubation period of 15 min, 2 #Ci in 0.1 ml of tgc-leucine or valine (270#Ci/mM) were added. The vials were then stoppered and incubated with gentle agitation at 37°C for the time stated in each experiment. For obtaining the enriched medium (medium II) the following amino-acids were added to the above medium (mg/ml): alanine 25, aspartic acid 30, cystein 0.1, glutamic acid 75, glycine 50, hydroxyproline 10, proline 40, and serine 25. Incubation was interrupted by addition of 3.0 ml of cold solution of unlabelled amino-acid (260/zg/ml) and centrifuged at 700 g for 2 min. The supernatant was discarded and the sediment was washed 2 times with the same solution. In two experiments, before homogenization, the tegument was removed from the larvae as later described. The washed bodies or bodies without tegument were suspended in 0.5 ml of cold Hanks' solution and homogenized in a

MATERIALS AND METHODS Cercariae of S. mansoni (LE strain, Belo Horizonte, Brazil) were obtained from laboratory reared and infected Biomphalaria glabrata and concentrated to about 1000 organisms/ml as described by Gazzinelli et al. (1973). Cercariae were used within 3-5 hr after emergence from the snails. Cercarial bodies Cercarial bodies were prepared as follows (RamalhoPinto et al., 1974): aliquots of 10 ml of concentrated intact cercariae were pipetted into 15 ml glass conical centrifuge *This work was supported, in part, by "Conselho Nacional de Pesquisas', and the "Banco Nacional para o Desenvolvimento Econrmico (FUNTEC)'. Contribution No. 88 from the Schistosomiasis Research Unit. 27

28

YOSHIE NAGAI et al.

Potter-Elvejhem homogenizer for 5 min. The homogenization was repeated for 5 rain after addition of 0.5 ml of 10% trichloroacetic acid (TCA). The precipitate collected in a glass fiber paper (GF/A, 24mm ~b) was thoroughly washed with 5% TCA and dried under infrared light. The glass fiber paper containing the dried protein was placed in an appropriate vial and 10 ml of a scintillation mixture consisting of toluene (11.), 2,5-diphenyloxazole (5 g) (PPO), and 1,4-bis-2(4 methyl-5-phenyloxazolyl)benzene (0.1 g) (DMPOPOP) was then added. Radioactivity was measured in a Beckman liquid scintillation spectrophotometer (LS-150). The appropriate quenching correction was obtained by channel or external ratio methods.

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Separation of the tegument from cercarial bodies Tegument was recovered from the bodies by the technique reported by Kusel (1972). Shortly, after the incubation period the larvae were washed as described previously and treated for 7 min with 3.0ml of 5% saponin in 3% calcium chloride. The larval surface lifted away from the bodies, could be isolated by whorling the treated bodies in a vortex for 15 sec. The vortex treatment sheared off the surface from the bodies which could be spun off as a pellet at 300 g for 1 min to leave the surface fragments in the supernatant. The protein from the supernatant was recovered in a glass fiber by addition of 0.3 ml of 50% TCA, thoroughly washed with 5% TCA and dried under an infra-red lamp. For radioactivity determination it was proceeded as previously described.

Protein determination Protein in the glass-fiber papers were assayed by the method of Lowry et al. (1951) modified as follows (Moraes, 1975): after radioactivity measurements, the glass-fiber papers were removed from the vials, dried under i.r. for 60 rain, and transferred to glass tubes (16 × 160mm). To each tube were added: 0.5 ml of 1 N NaOH, 0.1 ml of 5% sodium desoxycholate (DOC) plus 0.4 ml of distilled water. The tubes were placed in boiling water for 30 min and after cooling 5.0 ml of a freshly prepared cupric-alkaline solution consisting of 1.0 ml of 2% copper sulfate, 1.0 ml of 4% sodium and potassium tartarate and 3% sodium bicarbonate to 100 ml was then added. After 10 min, 0.5 ml of a half-diluted Folin reagent was mixed. Thirty min later the tubes were centrifuged at 14000 for 5min and the supernatant was read at 600 pm in a Coleman Jr. spectrophotometer.

Chromatography of the larval extract Forty to fifty thousand larvae were incubated in 5 ml of medium II with 14C-leucine (1 #Ci/ml) for 18 hr. The experiment was stopped by addition of unlabelled leucine and the larvae were washed several times with 0.14M NaC1. The washed larvae were suspended in 2 ml of 0.02 M EDTA in 0.05 M Tris pH 7.5 and sonicated in an ice bath 10 times for 30 sec, with 1-min intervals, in a sonifier cell disruptor (W-185 D, Heat Systems Ultrasonics Inc., Plainville, NY) with the output control adjusted to No. 4 and using a microtip. Cellular debris were centrifuged off. The supernatant was dialysed against the same buffer overnight and applied to a 0.9 x 30cm DEAE sephadex A-50 column previously equilibrated with the same buffer. Elution was performed by establishing a NaC1 gradient from 0 to 0.5 M at a flow rate of 1.(~1.5 ml/hr. For radioactivity measurements, eluated samples of 0.5 ml were transferred to appropriate vials and 10 ml of scintillation mixture was then added.

Digestion of the extract The extracts of S. mansoni larvae obtained as described above were incubated with trypsin for 1 hr at 37°C and dialysed against 0.05 M Tris~.02 M EDTA buffer for

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Fig. 1. Rate of protein synthesis during early development of Schistosoma mansoni larvae. Ten thousand larvae were incubated at 37°C in 3.0 ml of medium II containing 2 #Ci of U-X4C-leucine (270mCi/mM) under agitation. At the time stated, a sample of 0.05 ml was taken to determine the total number of larvae and the reaction was then stopped by addition of unlabelled leucine (264 mg/l). For protein and radioactivity measurements see Materials and Methods.

24 hr. Radioactivity of the non-dialysed material was determined as described. RESULTS

1. Variation of the protein level in S. mansoni larva during transformation to schistosomulum and early development To establish the protein variation in the larva during the incubation period, the n u m b e r of larvae was estimated a n d the protein determined in the extract prepared from the same samples. Figure 1 shows that the a m o u n t of protein/larva decreased in the first 6 hr x~emaining constant during the completion of the experiment. After 24 hr of incubation 95% of the larvae presented n o r m a l motility.

2. Rate of protein synthesis in S. mansoni larva during transformation to schistosomulum and early development The time-curve of x4C-leucine incorporation by S.

mansoni larvae is shown in Fig. 1. After a lag phase in which the protein synthesis was very low, there

Table 1. Stimulation of protein synthesis by S. mansonf larvae by addition of amino-acid mixture Incubation media I II

(dis/min)/mg protein 14C.leucine. 14C.valinet 3450 + 896 10,148 + 663

4366 + 203 11,502 + 2522

* U- 14C.leucin e (270 mCi/mM)--2 #Ci/3 ml. t U -14C-valine (260 mCi/mM)--2.2/tCi/3 ml. Medium II contained, in addition, the following aminoacids: alanine, aspartic acid, cystein, glutamic acid, glycine, hydroxyproline, proline, and serine. Experimental conditions as in Fig. 1.

Protein synthesis during early development of S. mansoni Table 2. Effect of various antibiotics in the protein synthesis of Schistosoma mansoni larvae

Antibiotics Penicillin* Cycloheximide Chloroamphenicol Puromycin

3 1 2 5

x x × x

10 102 102 102

-9 0 71

0 2 0 76

Ten thousand cercarial bodies were incubated at 37°C in 2 ml of medium II containing antibiotics at the above concentration and 2 pCi of U-14C-leucine (270mCi/mM). After 5 and 18 hr the reaction was stopped and the material processed as described in Fig. 1. * D(--) aminobenzylpenicillin. was a substantial increase in ~4C-leucine incorporation. The pattern of the time curve with 14C-valine did not differ significantly from that shown in Fig. 1. The rate of incorporation is strongly dependent on the enrichment of the incorporation medium with an amino-acid mixture. Thus, the addition of alanine, aspartic acid, cystein, glutamic acid, glycine, hydroxyproline, proline and serine to media I stimulates the incorporation of both amino-acids used (Table 1). At the concentrations indicated in Table 2 the incorporation of the labelled amino-acid was inhibited by puromycin, but was not affected by addition of penicillin, cycloheximide, and chloroamphenicol to the incubation mixture.

3. Demonstration of amino-acid incorporation into protein To demonstrate the incorporation of a4C-leucine into protein two different experiments were performed. An extract of larvae incubated for 18 hr in medium II containing 1 #Ci/ml of 14C-leucine was

3.(-

Extract

(dis/min)/mg protein

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11,260 8357

-26

Alone Plus trypsin

Labelled extract prepared according to Materials and Methods. chromatographed in a DEAE-sephadex column. A control in which the labelled amino-acid was added to the extract prepared from larvae incubated in the same conditions was also run. Figure 2 shows that, besides the peak correspondent to the free 14C-leucine, other superponing fractions with A2a0 absorbancy were observed. In other experiment, the extract incubated with 14C-leucine was treated with trypsin and dialyzed overnight. As indicated in Table 3, 2670 of the original counts were released after proteolysis.

4. Comparison of the rates of incorporation of 14C-leucine into proteins of tegument and cercarial bodies Tegument was isolated by the saponin-calcium chloride treatment (Kusel, 1972). After the incubation period as stated in Fig. 3, the tegument was isolated from the bodies and the radioactivity in both tegument and bodies were measured as described. It is clearly shown (Fig. 3) that proteins of the tegument incorporate about twice as much counts than that of the bodies. During the period of observation there was a slight increase in the ratio of tegument/body protein synthesis (Fig. 3). Leucine incorporation in both tegument and body was inhibited by actinomycin at approximately the same rate (Table 4). On the contrary of puromycin, the level of actinomycin inhibition was dependent on the incubation period (Tables 2 and 4).

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Fig. 2. Fraction of labelled Schistosoma mansoni larval extract by column chromatography. Experimental conditions as described in Material and Methods. H A280; O O radioactivity; - - - NaC1 gradient.

Fig. 3. Rate of protein synthesis in the body ( H ) and tegument (O O) of Schistosoma rnansoni larvae. Twenty thousand cercarial bodies were incubated in 3.0ml of medium II containing 4 pCi of U-14C-leucine (333mCi/ mM). At the time stated the reaction was stopped by addition of unlabeled leucine (264mg/L) and the tegument separated from the larval bodies for protein and radioactivity measurements as described in Materials and Methods. Ratio tegument/body (A A).

30

YOSHIE NAGAI et al.

Table 4. Effect of actinomycin D on the incorporation of 14C-leucine into proteins of tegument and body of Schistosoma mansoni larvae Incubation (hr)

Actinomycin D concentration (#g/ml)

5 15 15 19

0.65 0.65 1.30 0.65

% of inhibition in: Tegument Body 12 32 49 59

15 26 45 63

Experimental conditions as described in Fig. 2. * In relation to controls without antibiotic. DISCUSSION A typical 3-hr schistosomulum is strikingly different to both cercaria and 30-rain schistosomulum in that parts of the tegumental outer membrane were multilaminated. On the other hand, the outer surface of the 24-hr schistosomulum is more folded and pitted than the surface of the younger worms (Hockley & McLaren. 1973). Thus, in early development there is in the schistosomulum a replacement of the trilaminate outer membrane by a heptalaminate membrane and an increase in its surface area. Of course, the inclusion into the tegument of additional pieces of membrane, originated from the subtegumental cells as suggested by Hockley & McLaren (1973), should be preceeded by protein synthesis unless the proteins which were inserted into the membrane during this period were already synthesized and stored. Supporting the data of Kusel et al. (1975), our results indicate that, immediately after transformation, the incorporation of labelled amino-acid into protein was not very high. However, a substantial increase in the rate of protein synthesis was observed starting after 6 hr and continuing within the period of observation (Fig. 1). This suggests that, soon after transformation, most of material incorporated in the outer membrane preexist in cercarial body. The utilization of the preformed material during this period stimulate the synthesis of new membranous bodies by the subtegumental cells. The above explanation is supported by the data showed in Table 2 demonstrating an increase of actinomycin inhibition as the incubation time elapse, and suggesting a progressive requirement for DNA dependent RNA synthesis. This was not observed with puromycin whose rate of inhibition did not change with the incubation time (Table 1). A similar inhibition rate with the same concentration of puromycin was reported by Kusel (1972). Furthermore, the results presented here pointed out to a preferential synthesis of the tegumental proteins at the stage of development studied (Fig. 3). In fact, a rapid turnover of surface membrane was demonstrated in earlier stages of development (Kusel et al., 1975b). The detection of radioactivity in body proteins may represent a mere reflexion of the presence of many membranous bodies in the subtegumental cells. During the period of 6-8 hr of incubation it was observed a decrease in the protein of the larvae (Fig. 1). This is probably due to the elimination of acetabular glands as demonstrated by Gazzinelli et al. (1973), and Oliveira et al. (1975) at this experimental condition.

The stimulation of leucine incorporation observed (Table 2) after addition of an amino-acid mixture to the incubation medium might be indicative of the existence of a small amino-acid pool in the transforming larvae. This is easily explained by the fact that the cercaria does not take up amino-acids from the media until permeability changes are established in the larvae during transformation (Ramalho-Pinto et al., 1974).

REFERENCES

COLLEY D. G. & W1KELS. K. (1974) Schistosoma mansoni: simplified method for the production of schistosomules. Exp. Parasit. 35, 44-51. EAGLE H. (1949) Amino acid metabolism in mammalian cell culture. Science, N.Y. 130, 432437. GAZZINELLI G., OLIVEIRAC. C., FIGUEIREDOE. A., PEREIRA L. H., COELHOP. M. Z. & PELLEGRINOJ. (1973) Schistosoma mansoni: Biochemical evidence for morphogenetic change of cercaria to schistosomule. Exp. Parasit. 34, 181-188. HOCKLEYD. J. & MCLAREND. G. (1973) Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. Int. J. Parasit. 3, 13-25. KUSEL J. R. (1972) Protein composition and protein synthesis in the surface membranes of Schistosoma mansoni. Parasitolooy 65, 55-69. KUSEL J. R., SnER A., PEREZ H., CLEGGJ. A. & SMITHERS S. R. (1975a) The use of radioactive isotopes in the study of specific schistosome membrane antigens. In Isotopes and Radiation in Parasitology IV, pp. 127-43. International Atomic Energy Agency, Vienna. KUSELJ. R. & MAC~ENZXEP. E. (1975b) The measurement of the relative turnover rates of proteins of the surface membranes and other fractions of Schistosoma mansoni in culture. Parasitolooy 71, 261-265. LOWRYO. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265 275. MORALSG. W. G. (1975) Caracterizaq~o morfoltgica e funcional das regites da glfindula salivar de Rhynchosciara angelae. D.Sc. Thesis, UFRJ, Rio de Janeiro. OLIVEIRA C. C., F'IGUEIREDOE. A., GAZZ1NELLI G., HowELLS R. E. & PELLEGRINOJ. (1975) Biochemical changes in the transformation of Schistosoma mansoni cercariae to schistosomules. Comp. Biochem. Physiol. 51B, 417420. RAMALHO-PINTO F. J., GAZZINELLI G., HOWELLS R. E., MOTA-SANTOST. A., F1GUEIREDOE. A. & PELLEGRINO J. (1974) Sehistosoma mansoni: defined system for stepwise transformation of cercaria to schistosomule in vitro. Exp. Parasit. 36, 360-372. SMITHJ. H., REYNOLDSE. S. t~z VON LICHTENBERGF. (1969) The integument of Schistosoma mansoni. Am. J. Trop. Med. Hyg. 18, 2849. STIREWALTM. A. (1963) Cercariae vs schistosomule (Schistosoma mansoni). Absence of the pericercarial envelope in vivo and the early physiological and histological metamorphosis of the parasite. Exp. Parasit. 13, 395406. STIREWALTM. A., MINNICK D. R. R. & FREGEAUW. A. (1966) Definition and collection in quantity of schistosomules of Schistosoma mansoni. Trans. Roy. Soc. Trop. Med. Hyg.. 60, 352-360. TmA Y., HOLANDAJ. C., RAMALHO-PINTOF. J., GAZZINELLI G. & PELLEGRINOJ. (1974) Schistosomula (Schistosoma mansoni) obtained in vitro: viability in culture and infectivity for mice. Trans. Roy. Soc. Trop. Med. Hyg. 68, 72.