Trypanosoma cruzi: Differentiation to metacyclic trypomastigotes in the presence of ADP-ribosyltransferase inhibitors

EXPERIMENTALPARASITOLOGY64,424-429(1987)

Trypanosoma cruzi: Differentiation to Metacyclic Trypomastigotes the Presence of ADP-ribosyltransferase Inhibitors E. L.D. IsoLA,E.M. Cdtedra de Microbiologia,

in

LAMMEL,ANDS. M. GONZALEZCAPPA

Parasitologia e Inmunologia Facultad de Medicina, Universidad Paraguay 2ISSIPiso 13”/1121, Buenos Aires, Argentina

de Buenos Aires,

(Accepted for publication 9 April 1987) ISOLA, E. L. D., LAMMEL, E. M., GON~,~LEZCAPPA, S. M. 1987. Trypanosoma cruzi: Differentiation to metacyclic trypomastigotes in the presence of ADP-ribosyltransferase inhibitors. Experimental Parasitology 64, 424-429. The participation of ADP-ribosyltransferase in Trypanosoma cruzi differentiation to the metacyclic stage was evaluated by analyzing morphogenesis blockage by specific enzyme inhibitors: benzamide, 3-aminobenzamide, theophylline, and nicotinamide. In vitro assays showed a statistically significant reduction in the number of metacyclic forms only when any one of the four inhibitors was added during the period of interaction between epimastigote and Triatoma infestans intestinal homogenate or when present throughout the subsequent culture period in Grace’s medium. When nicotinamide or benzamide was present during both interaction and culture period, morphogenesis was virtually abolished (SZ%). In the in vivo assays, mice inoculated with parasites obtained from the insect vectors fed with trypomastigote-infected blood containing one of the four enzyme inhibitors developed lower parasitemias and showed longer survival in every case, compared with the respective controls. These findings suggest ADP-ribosyltransferase participation in T. cruzi differentiation both in vitro and in vivo.

0 1987 Academic Press, Inc.

INDEX DESCRIPTORSAND ABBREVIATIONS: Trypanosoma cruzi; Differentiation; ADP-ribosyltransferase (EC 2.4.2.30); Enzymatic inhibitors, Grace’s medium (GM) supplemented with Triatoma infestans intestinal homogenate (GM/IH).

INTRODUCTION

The process of differentiation leads to changes in cell structure and function and, consequently, in the specific protein profile. It has been suggested that protein modification by ADP-ribosyltransferase, an enzyme reported to be present in protozoa (Tsopanakis et al. 1978; Okolie and Onyezili 1983), is required in this morphogenie process (Purnell et al. 1980; Caplan and Rosenberg 1975; Rastl and Swetly 1978). In Trypanosoma cruzi, differentiation results in a series of transitions through morphologically distinct stages: extracellularly, in the insect vector, from trypomastigote to amastigote (spheromastigote) and later to the metacyclic form (Brack 1968; Alvarenga 1977); and intracel-

lularly, in the mammalian host, from amastigote to trypomastigote (Brener 1979). Recently, it has been reported that competitive inhibitors of ADP-ribosyltransferase block the T. cruzi differentiation from the intracellular amastigote to trypomastigote and from the extracellular amastigote to flagellate stages in in vitro assays (Williams 1983). The aim of this study is to present evidence that these inhibitors can also block the epimastigote to metacyclic morphogenesis in an axenic culture as well as to interfere with the process of metacyclic trypomastigote production in the insect vector. MATERIALS

3-Aminobenzamide, nicotinamide, benzamide, and theophylline were used (Sigma Chemical Co., St. 424

0014-4894187$3.00 Copyright 0 1987 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

AND METHODS

Trypanosoma cruzi: ADP-RIBOSYLTRANSFERASEINHIBITION Louis, MO, U.S.A.). Neutralized nicotinic acid, a related compound, though not an inhibitor, was used as a control. Trypanosoma cruzi trypomastigotes of the Ildahuen strain were obtained by bleeding outbred Rockland mice from the orbital sinus at the peak of parasitemia. Dilutions to reach suitable parasite concentrations for insect feeding were performed using whole normal mouse blood. Epimastigotes of the Tulahuen strain were maintained by weekly transfers in biphasic medium and harvested during the exponential growth period when there were less than 1% metacyclic forms; they were used to start differentiation assays in vitro. GM modified by Junker ef al. (1967) (Gibco) and GM/IH (Isola er al. 1981) were used for morphogenesis. Third instar larvae of Triatoma infestans, reared in our laboratory at 28 C and 60% humidity, were used throughout. A modification of Pipkin and Connors’ feeding apparatus was employed to infect the insects with T. cruzi trypomastigotes (Isola et a/. 1981). In a first experimental step, 4 X IO6epimastigotesl ml were preincubated for 15 min in GM&H and then centrifuged for 15 min at 3500 rpm (Isola ef al. 1986a). In a second step, the sedimented parasites were resuspended in GM. Each inhibitor (2.5 mM 3-aminobenzamide, 10 mM nicotinamide, 2.5 mM benzamide, or 5 mM theophylline) was added separately to the first or both steps. Samples without inhibitor and with 10 mM of neutralized nicotinic acid were used as controls. Cultures were examined every 48 hr for 15 days. Morphogenesis was evaluated according to parasite motility, shape, and relative kinetoplast-nucleus position (Isola et al. 1981). The percentage of differentiation to metacyclic forms was determined in at least 200 forms in wet and stained (May-Griinwald-Giemsa) preparations. Growth curves were plotted by counting parasites in a Neubauer chamber. Each experimental variable was assayed in quintuplicate. Four batches of 20 insects each were artificially fed with normal mouse blood containing 1 x IO6 trypomastigotes/ml supplemented with the respective inhibitor. Each inhibitor was used in a final concentration equal to that employed in the in vitro morphogenic experiments. A control batch of 20 insects was likewise fed but without inhibitor. Insects were weighed before and after feeding, discarding and replacing those not feeding. Mid- and hind-guts were removed 30 days after infection and pools from 10 insects were each ground manually in 0.5 ml phosphate-buffered saline (pH 7.2) in a glass tissue grinder. An indirect measure of morphogenesis was used to evaluate metacyclics’ differentiation in the vector. This was necessary since under experimental conditions in the in vivo assays the number of metacyclics observed in the insect feces was usually very low (Lammel er al. 1981). In addition, it has been proved that parasites showing an epimastigotal shape may al-

425

ready possess proteins on their surface which are characteristic of the metacyclic stage (Contreras et al. 1985). Therefore, parasites recovered from the insects were counted in a Neubauer chamber and their morphogenesis was indirectly evaluated by injecting 21 -C 1 day-old male Rockland mice, IO per group, subcutaneously with 1 x 103parasites per mouse. Parasitemia and mortality were recorded for 45 days after infection (Lammel et a/. 1981). Statistical significance was determined by the Student’s r test.

RESULTS

The percentages of Trypanosoma cruzi morphogenesis from epimastigote to metacyclic reached after treatment with either ADP-ribosyltransferase inhibitors and neutralized nicotinic acid during the first or both experimental steps, as well as differentiation control batches cultivated without any inhibitor, are shown in Fig. 1. The four antagonists assayed patently interfered with the morphogenic process, since under all experimental conditions, metacyclics failed to exceed 25%, whereas controls and neutralized nicotinic acid batches exhibited 73.5 + 6.5 and 69.0 ? 7.2% morphogenesis by Day 7 (P < O.OOl), respectively. Similar values were recorded up to the 15th day. Even when inhibition was effective in every case, some differences in its efficacy should be pointed out. When benzamide was used, the presence of the enzyme antagonist in both experimental steps induced a morphogenic inhibition significantly higher than during the first step alone (P < 0.001) (Fig. 1C). In fact, when nicotinamide and benzamide were added in both steps, morphogenesis was almost entirely abolished (~2% metacyclics) (Figs. 1B and C). However, in the case of theophylline or 3aminobenzamide, differentiation blockage failed to increase when inhibitor was added during both experimental steps (P < 0.3) (Figs. 1A and D). The absolute number of parasites present in each culture batch on Day 7 is shown in Table I. By this time, the exponential phase

426

ISOLA, LAMMEL,

AND GONZALEZ CAPPA TABLE I Effect of ADP-ribosyltransferase Inhibitors on Trypanosoma cruzi Epimastigote Growth Inhibitor

Proliferation rate parasites x 106/m] k SD

Benzamide 3-Aminobenzamide Theophylline Nicotinamide

FIG. 1. Effect of ADP-ribosyltransferase inhibitors on Trypanosoma cruzi differentiation from epimastigote to trypomastigote. Epimastigotes were inoculated into GM/IH for 15 min (first step) and then transferred to Grace’s medium (second step). Each inhibitor: A-5 mM theophylline, B-10 mM nicotinamide, C-2.5 mM benzamide, or D-2.5 mM 3aminobenzamide, was added in the first step (---) or in both steps (- * -). Samples without inhibitor served as control (-). In Fig. lB, the differentiation pattern reached in the presence of nicotinic acid (---) in both steps was also included. Results for when nicotinic acid was added only during the first step were similar. At the time of maximum differentiation, morphogenesis was inhibited significantly by any of the four enzymatic antagonists (P < 0.001 on Day 7). Morphogenie inhibition persisted up to the end of the experimental period on Day 15 (data not shown). When nicotinamide (B) or benzamide (C) was present throughout the experimental period, morphogenesis was practically abolished. The results are the means of five experiments k SD.

of the growth curve had been reached in every case. Except for theophylline, the epimastigote duplication rate was significantly diminished by nicotinamide (0.01 < P < O.OOl), 3-aminobenzamide and benzamide (P < 0.001). Motility was preserved despite the inhibitor employed and under the specified experimental conditions. Mice inoculated with T. crud parasites from insects fed with infected blood containing any of the enzyme antagonists developed lower parasitemia patterns and had longer survival than the controls injected

82.4 47.8 40.3 65.6 56.8

f * f f f

3.57 4.9* 1.5* 8.9 11.6**

Note. TUahuCn epimastigotes were sown in GraceIH medium for 15 min, and then transferred to Grace’s medium and incubated at 28 C, with or without 2.5 mM benzamide, 2.5 mM 3-aminobenzamide, 5 mM theophylline, or 10 mM nicotinamide. Values recorded were obtained at Day 7 and are the means and standard deviations of five samples each. * P < 0.001. ** 0.01 < P < 0.001.

with parasites without inhibitor treatment (Fig. 2). Theophylline proved to possess the highest capacity to prevent morphogenesis in these assays, as shown by the 100% survival of this group of mice and the lowest parasitemia curves (Fig. 2A). Benzamide was the second in efficacy, since mice injected with parasites obtained from insects fed with this inhibitor showed lower parasitemia (Fig. 2B) than those infected with parasites treated with 3-aminobenzamide (Fig. 2C) or nicotinamide (Fig. 2D). DISCUSSION

Differentiation mechanisms in the mammalian and insect host are essential for Trypanosoma cruzi perpetuation in nature. Interference at any step of this process would lead to the interruption of its life cycle. We have recently standardized the conditions for the epimastigote differentiation to metacyclic form: after 15 min of parasite and Triatoma infestans intestinal homogenate interaction, a morphogenic mechanism is triggered which conditions the parasites in GM to differentiate to 70-80% metacyclics (Isola et al. 1986b). This mechanism seems quite different from the one postulated by Contreras et al. (1985) in

Trypanosoma

35

cruzi:

ADP-RIBOSYLTRANSFERASEINHIBITION

L5

25

15

35

L5

15

25 35

45 15 25 35 DAYS

427

L5 p,

FIG. 2. Parasitemia patterns and survival of mice inoculated with Trypanosoma cruzi obtained from insects fed with trypomastigotes resuspended in normal mouse blood with ADP-ribosyltransferase inhibitors and of the control group. Parasitemias (0-O) and survival (O---O) of the control group without inhibitors. Parasitemia (O-O) and survival (O---O) of the group of mice inoculated with parasites obtained from insects fed with: A-5 mM theophylline, B-2.5 mM benzamide, C-2.5 kI4 3-aminobenzamide, D- 10 mM nicotinamide

which metacyclic differentiation was induced by “metabolic stress,” the parasites being in a depleted culture medium containing a single amino acid. In our system, differentiation is reached during the exponetial growth period and needs specific stimulating factors present in a predominantly 17K protein fraction common to Triatoma infestans intestinal homogenate (Isola et al. 1981) and hemolymph (Isola et al. 1986a). Results presented here indicate that all four ADP-ribosyltransferase inhibitors employed, interacting in vitro during morphogenesis interfered with the process triggered by the stimulating factors. Moreover, both benzamide and nicotinamide almost entirely abolished morphogenesis when added during the two steps of the experimental assays, suggesting that these enzyme antagonists were also interfering with mechanisms other than those triggered by specific stimulating factors. The lack of total inhibition with 3-aminobenzamide when added to both steps suggests that this compound might act only during the interaction of the parasite and stimulating factors or that it might become inactivated during culture. In support of the former

possibility is the fact that the addition of the enzyme inhibitor to the culture every 48 hr failed to decrease morphogenesis (data not shown). Given the equally low metacyclic numbers recorded at Day 15, the delay observed in morphogenesis has little, if any, effect on final count. The failure of nicotinic acid to interfere in the differentiation process strengthens the hypothesis that ADP-ribosyltransferase is a key participant. Differences in infectivity among the samples obtained from those insects fed with infected blood with or without the ADP-ribosyltransferase inhibitors indicate that these compounds interfered with morphogenesis in vivo. Whether the optimal in vivo morphogenic inhibition effect of theophylline is due to a lesser degradation of this compound in the insect digestive tract remains to be demonstrated. Since theophylline (Beavo et al. 1970) possesses pharmacologic effects other than the inhibitions of ADP-ribosyltransferase, alternative mechanisms cannot be ruled out for this antagonist. Williams (1983) reported morphogenic inhibition from the amastigote to trypomas-

ISOLA,

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AND GONZiLEZ

tigote in a mammalian cell line, and from amastigote to flagellated forms in an axenic medium, using ADP-ribosyltransferase inhibitqrs throughout the experimental period. Even when blocking at levels other than the epimastigote to trypomastigote, differentiation cannot be ruled out in our in vivo experiments; the fact that a similar number of parasites obtained from insects fed with infected blood in the presence of the inhibitors induced milder infection in mice than in controls is indicative that the epimastigote to trypomastigote differentiation was ineffectively triggered. Williams also reported that epimastigote proliferation in a suitable medium for duplication of this stage (Warren) was practically unaffected by 3-aminobenzamide, benzamide, and nicotinamide. In the present report, 3-aminobenzamide and benzamide as well as nicotinamide were found to inhibit epimastigote proliferation between 30 and 50% as compared to control values. Whether this discrepancy is due to the use of different culture media remains to be determined. Results presented here and those reported by Williams suggest strongly that the morphogenic process shared common mechanisms involving ADP-ribosyltransferase, even in quite different systems. This enzyme is therefore a promising target for therapeutic and prophylactic trials.

CAPPA

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lationship between poly (ADP-Rib) synthesis, intracellular NAD levels and muscle or cartilage differentiation from mesodermal cells of embryonic chick of the National Academy of limb. Proceedings Sciences USA 72, 1852-1857. CONTRERAS, V. T., MOREL, C. M., AND GOLDENBERG, S. 1985. Stage specific gene expression

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The authors thank Dr. A. 0. Stoppani for reviewing the manuscript prior to submission for publication. Stella Maris Gonztiez Cappa is a Career Research Member of the Consejo National de Investigaciones Cientifcas y Tkcnicas from Argentina. This work was supported by a grant from Secretaria de Estado de Ciencia y Tecnologia (SECYT) and from Consejo Nacional de Investigaciones Cientificas y Tkcnicas (CONICET) from Argentina. REFERENCES ALVARENGA, N. J. 1977. “Evolu~lo de Trypanosoma cruzi no trato digestivo de Triatoma infestans,”

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