Molecular and Biochemical Parasitology, 1 ( 198 O) 143 - 149 © Elsevier/North-Holland Biomedic~ Press

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PRODUCTION AND SECRETION OF LEISHMANIA BRAZILIENSIS PROTEINS

e, NGEL G. HERNANDEZ t , NORIS RODRIGUEZ j , FRACEHULi DAGGER j and CHARLES L. GREENBLATT3,*

Department of Cell Biology, Faculty of Sciences, Universidad Central de Venezuela, P.O. Box 10098, Caracas, Venezuela; 2 Department o f Protozoology and The Kuvin Centre for the study o f lnfectious and Tropical Diseases, Hebrew University.Hadassah Medical School, P.O. Box 1172, Jerusalem, Israel

(Received 23 October 1979; accepted in revised form 8 January 1980)

The kinetics of secretion of proteins by Leishmania braziliensis was followed by incorporation of [3H'~:,'ucine into macmmolecules produced by the cells which are released into the growth medium. About 10% of the total protein synthesized by actively growing cells is secreted. Cycloheximide (100 oug/mD and puromycin (0.5 raM) inhibited the incoiporat~on of l~heiled leucine by 85 and 99%, respectively. The secreted proteins do not seem to result from cell lysis since, first, the kinetics of production are linoar and, secondly, less than 1% of thymidine or uridine incorporated by the cells is found in the medium. Cells grown with 13H|leucine and then transferred to fresh medium show two phases of secretion. During the first six hours, it is slow and reaches a plateau. The release in- creases about ten-fold during the next six hours. An analysis of the ~creted material showed that following precipitation ~ith methanol and sodium acetate, three isotopically labelled peaks were eluted from Sephadex G-120-150. The first of these, containing 50% of the radioactivity, did not react with anti-leishmanial serum, while the last two did. Since the last two fractions could be label- led with [3H]glucosamine as well as PHlleucine it is suggested that they are glycoprotein in nature and are similar to the products released by other species of Leishmania.

Key words: Leishmania braziliensis, Leishmanial proteins, Protein synthesis and secretion.

INTRODUCTION

Complex protein-carbohydrate structures are known to be components of the surface coat of different types of cells [1] and these macromolecules have been shown to be associated with leishmania cell surfaces [2, 3]. Leishmania promastigotes in culture are known to excrete species specific, immunologicaUy active substances during the log and stationary phase of culture growth [4, 5]. In recent reports [6, 7] these compounds have been characterized biochemically as carbohydrate-rich compounds [6] or as glycoproteins [7]. In all of these studies, secreted products were identified by their reactivity towards antibodies raised by different methods against leishmania cells. The present study was

* Author to whom correspondence should be sent.

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designed to investigate the kinetics of production and secretion of the leishmania derived materials during cell growth and then to detect the release of proteins or polypeptides into the environment under non-growth conditions.

It has been suggested [8] that materials released during cell growth could act as condi- tioning agents for the parasite, promoting their survival within the host organism. The use of radioactively-labelled leucine and glucosamine shows that not all the products released by the parasites react with antisera raised in rabbits and that these immune. logically non-active components must also be considered as possible conditioning agents. Furthermore, the production of these metabolites starts at the earliest stages of growth and, as seen in electron microscopic studies, seems to be associated with a mechanism operating at the leishmania cell surface.

MATERIALS AND METHODS

The strain of Leishmania braziliensis used in this study was initially isolated from a truman patient and was obtained from the National Institute of Dermatology, Caracas, Venezuela. It is serologically identical to strain LRC-L94, serotype A3 B2, isolated from a human cutaneous case and the two strains display similar enzyme electrophoretic mobilities (L. Schnur, pers. commun.). Due to the clinical manifestations of the patient from whom the strain was isolated, it will be referred to as L. braziliensis.

Promasti~ote cultures were grown in LIT or Davies medium [2] supplemented with 5% foetal calf serum. After one day of growth the appropriate precursor was added and samples we~c then taken at various times for cell counts and for the measurement of radioactivity inco~orated into macromolecules. Cell counts were carried out in a Neu- bauer chamber. Amino acid incorporation into proteins was stopped by transferring the sample culture to crushed ice followed by the addition of unlabelled leucine 0 5 retool). "Organisms were removed from the medium by centrifuging at 2000 X g for 10 rain at 4°C. The cells were washed twice with 7 mM phosphate buffered 0.15 M saline; pH 7.~. Measurements of radioactivity in cell and supernatant proteins were carried out as de- scribed by Hernandez [9] except that in some experhnents heating of the samples was done immediately after separation of cells from medium. Radioactivity was meas~,red in a Packard Tricarb scintillation counter with 10 ml of Aquasol (New England Nuclear) with an efficiency of 50% for 3H. Allowance was made for a sample taken at zero time. These counts were generally very low. The statistical accuracy of counting was between +2 and+5%.

When further purification of the supematant fluid was performed in order to charac- terize the different components secreted by ~the leishmania cells, the procedure described by Slutzky and Greenbla~t [6] was applied, except that the sample was not boiled, since boiling did not precipitate protein from the medium under the conditions of our experi- ments. Also, before carrying out the methanol-acetate procedure, the supernatant was dialyzed against frequent changes of d~stilled water containing 7.5 mmol leucine until the [3H]leucine counts in the dialysate reached background level.

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Anti-sera (type As B2) to leishmania were prepared by six intravenous injections of living promastigotes into rabbits as described by Sclmur et al. [4] and immunological activity was determined by double diffusion on agar plate~. Polyacrylamide gel electro- phoresis was carried out as described by Fa~rbanks et al. [10] on gels containing 7.5% acrylamide and 0.1% SDS. Protein concentration was determined by the method of

Lowry et al. [ 11 ] with bovine serum albumin as standard.

RESULTS AND DISCUSSION

Release of materials by leishmania into the medium were analyzed in the following types of experiments.

Cells which had been grown without radioactive precursors were washed and then suspended in Davies modified medium [2] at an initial concentration of 2.0 × 106 cells/ ml. They were incubated in sterile stoppered glass flasks with air as the gas phase at 28°C for a given tL, ne in the presence of [3H]leucine (2.6 ttCi/ml). At the time intervals shown in Figure 1, samples of the medimn were removed, cells counted and the incorporation of [3 H]leucine measured. Figure 1 shows that cells incorporated amino acids linearly up to 24 h during the late lag phase of growth, and then the amount of [3H]leucine taken in by the cells tends to remain const~t during the log phase (24-72 h) and at the early stationary phase. These results suggest that when [3H]leucine is used as a precursoz, leishmania proteins become saturated with label during the first 24 h and then the cells incorporate amino acids into protein in parallel with cell growth. Cycloheximide (100

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146

~g/ml) and puromycin (0.5 raM), both specific inhibitors of protein synthesis, inhibited incorporation of [aH]leucine into the leishmania proteins by 85 and 99%, respectively. At these concentrations of il~tibilors~ inhibition is likely to be due to specific effects on the protein synthetic mechanisms [9].

Figure 1 also shows that 10% of total protch'~ synthesized for the whole cells is se- creted into the incubation medium. It is possible to conclude from the results shown in Figure 1 that an active process of secretion or shedding of ceil protein exists. Since the amounts of label incorporated into both retained and released proteins remain approx- imately the same during the entire growth curve, damage to the leishmania cells during growth, with the subsequent leakage of cell components to the medium, is probably not the cause for the appearance of leishmanial proteins in the culture fluid. Two addition- al observations support the active secretion proposal: cells grown in the presence of [a H]thymidine or [a H]uridine release less than 1% of incorporateE label into the medium. Secondly, when the cells were harvested at the log and stationary phase of growth and stained with ruthenium red, a stain which Luft [ 12] has shown to precipitate a large range of acid proteins and polysaccharides, it was observed that cells from cultures in the log phase of growth possessed a gel-like mesh of material possibly aggregate glyeoproteins extending from the cell surface into the extracellular space [13]. This material almost disappears during the stationary phase. Whether it is 'shed' from the cell surface, or is secreted directly through the membrane at specific sites such as in the flagellar pocket [5] remains to be investigated. The exact process of release or secretion is still unclear. Some authors have sought to rule out cell leakage as a source [ 14] while others conclude that cell lysis could account for up to 20% of the material released by some leishmanial species [ 15 ]. In our system little if any of the secreted products arose through cell damage or leakiness during growth.

the presence of [aH]leucine (2/~Ci/ml) for three days were centrifuged, washe6 in phos- phate buffered saline and resuspended in fresh medium so that the number of cells/ml remained that in the original labelled medium. The amount of labelled material released into the medium was then delermined over a 12-h period. As can be seen in Figure 2, labelled trichloroacetic acid precipitable material was released from the cells steadily and slowly during the first 6 h o,f incubation in the fresh medium until almost 0.4% of the total label had been released. Then it rose rapidly in a linear faslfion up to ~ 2 h, the maximum period tested in this experiment, by which time about 3.0% of the total label had been released. It does not seem likely that the materials released during the first 12 h were altered by the action of proteases or other enzymes in the ~edium, a conclu- sion supported by the fact that the material released during this short period behaves as that characterized below and by the finding of the secreted material in cultures of other leishmanias [14]. The reasons for the sudden increase in the release of material after 6 h are unknown at present.

The third type of experiment was designed to chezk the reactivity of the secreted products against the anti-leishmania sera, type Aa B2, as described by Schnur et al. [4].

147

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Fig. 2. Kinetics of secretion of material from [3Hlleucine-labelled L. braziliensis cells when lrans-

ferred to fresh culture medium.

After having established the optimal conditions for radioactive labelling of the L. bra-

ziliensis secreted material with [3Hlleucine, the procedure described by Slutzky and Greenblatt [6] was applied. After precipitation with methanol and sodium acetate, the precipitate retained 97% of radioactive trichloroacetic acid precipitable material present in the original preparation. When this material was applied to a column of Sephadex G- 120-150 and eluted with distilled water, three main fractions were obtained as detect- ed by the absorbance at 280 nm (Fig. 3). Peak 1, which eluted with the void volume, contained 50% of the total counts applied to the column and did not precipitate with L , t ~ aa,-~]snmaraa sera raised against the whole parasite. The next two fractions, 2 and 3, contained the remaining 50% of the tdchloroacetic acid precipitable radioactivity applied to the column. Both of these reacted with the anti-leishmania sera. It should be stressed here that similar fractions obtained from unused medium do not react with the anti-leishmania sera.

Of the total protein applied to the column, 62% was recovered in Peak 1, and 36.7% in fractions 2 and 3. When fraction 1 was analyzed in SDS gels [10] it showed five po- lypeptide bands; fraction 2 showed two bands; and fraction 3 only one band. Because of this no claims can be made at this stage about the purity of fractions 1 and 2. Fraction 3 has a M r of approx. 18 000 as determined by SDS electrophoresis and gel f'dtration. When a similar experiment was carried out with [3H]glucosamine as the radioactive precursor, 75 ~ of the radioactivity was recovered in fractions 2 and 3 and 20% in fraction 1. These results are in accord with the findings of Slutzky and Greenblatt [6] and Decker-

Jackson and Honigberg [7]. Therefore we suggest that just as the immunoreactive products secreted by L. tropica

and L. donovam are probably glycoproteins so are those ofL. braziliensis. The similarity of the released products from the three strains so far examined has been further empha-

148

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Fig. 3. Sephadex G-120-150 profile of methanol-precipitated leishmania secreted products labelled with [SH]leucJne. The effluent was monitored for both As:S0nm (O---O) and radioactivity (o o~. Arrows (left to right) indicate the position of bo'Ane catala~e (M r 224 000), bovine plasma albumin (M r 67 000) and horse heart cytochrome C (M r 13 400)•

sized by the recent work of Slutzky et al. [15] who recovered a [3H]~ucosamine-labelled glycopeptide fragment and a [3H]leucine-labelled protein fragment from cultures of L. tropica. The glycopeptide reacted with anti-leishmania serum while the protein did not. The nature of the immunologically non-reactive materials which constitute the bulk n f the r~l~se d n m A l ~ o t e ,~i" L i , , , . , ;z:~,.~. .: , .. t . .~. ~,.~,,,~,,~,~ are now u~m~ chara~etized chemicaliy and examined for biological activities.

ACKNOWLEDGEMENTS

This work was supported by grants CONICIT DDCT-SAL 9 and of the UNDP/Wodd Bank/WHO Special Programme for Research and Training in Tropical Diseases. We wish to thank Mr. H. Roman for his skillful assistance and Dr. G.lVl. Slutzky for many help- ful discussions.

REFERENCES

[ 1 ] Parsons, D.F. and Subjeck, J.R. (1972) The morphology of the polysaccharide coat of mammalian cells. Biochim. Biophys. Acta 265, 85-113.

[2] Dawidowicz, K., Hemandez, A.G., Infante, R.B. and Convit, J. (1975) The surface membrane of Leishmania. I. The effects of lectins of different stages o f L e i s h m a n i a braz i l i ens i s . J. Parasitol. 61,950-953•

[3] Dwyer, D.M. (197¢) Lecth~ binding saccharides on a parasitic protozoan. Science 184, 471-473.

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[4] Schnur, L.F., Zuckerman, A. and GreenMatt, C.L. (1972) Leishmanial serotypes as distinguished by the gel diffusion of factors excreted in vitro and in vivo. lsr. J. Med. Sci. 7,932 - 942.

[5] Zuckerman, A. (1975) Current status of the immunology of blood and tissue protozoa. 1. Leith-

mania. Exp. Parasitol. 28, 370- 400. [6] Slutzky, G.M. and Greenblatt, C.L. (i977) Isolation of a carbohydrate-rich ;,mmunologically

active factor from cultures ofLeishmania tropica. FEBS Lett. 80, 401-404. [7] Decker-Jackson, J.E. and Honigberg, B.M. (1978) Glycoproteins released by Leishma1:ia dono.

vani: Immunologic relationships with F, ost and bacterial antigens and preliminary biochemical analysis. J. Protozool. 25, 514-525.

[8] Handman, E. and Greenblatt, C.L. (i977) Promotion of leishmanial infections in non-permissive host macrophages by conditioned medium. Z. Parasitenk. 53, 143.

[9] Hernandez, A.G. (1974) Protein synthesis by synaptosomes form rat brain. Contribution by the intraterminal mitochondria. Biochem. J. 142, 7-17 .

[10] Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606- 2616.

[11] Lowry, O.H., Rosebrough, H.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 - 275.

[12] Luft, J.H. (1971) Ruthenium red and violet. I. Chemistry, purifications, methods of use for electron microscopy and mechanism of action. Anat. Rec. 171,347-368.

[13]Hughes, R.C. (1976) Detection and dist,-ibution of membrane glycoproteins. In: Membrane Glycoproteins, pp. 6 - 27, Butterworths, London and Boston.

[14]El-On, J., Schnur, L.F. and Greenblatt, C.L. (1979) Leishmania donovani: Physiochemical, immunological and biological characterization of excreted factor from promastigotes~ Exp. Parasitol. 47, 254 - 269.

[15] Slutzky, G.M., El-On, J. and Greenblatt, C.L. (1979) The leishmanial excreted factor: protein- bound and free forms from promastigote cultures ofL. tropica and L. donovani. Infect. Immun.

26, 519 -524.