Arginine Decarboxylase Inhibitors Reduce the Capacity of Trypanosoma Cruzi to Infect and Multiply in Mammalian Host Cells FELIPE KIERSZENBAUM*T, JULIA J
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Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4278-4282, June 1987 Medical Sciences Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells FELIPE KIERSZENBAUM*t, JULIA J. WIRTH*, PETER P. MCCANNt, AND ALBERT SJOERDSMAt *Department of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824; and tMerrell Dow Research Institute, Cincinnati, OH 45215 Communicated by William Trager, March 3, 1987 (received for review November 6, 1986) ABSTRACT The capacity of blood (trypomastigote) forms for and reactions influencing the invasion of host cells by T. of Trypanosoma cruzi to infect mouse peritoneal macrophages cruzi (4-12), our current understanding of this process- or rat heart myoblasts in vitro was inhibited by treatment of the which could underlie the development of effective chemo- trypomastigotes with DL-a-difluoromethylarginine (F2Me prophylaxis and chemotherapy-is limited. Particularly de- Arg), monofluoromethylagmatine, or (E)-a-monofluorometh- ficient is knowledge about the role of polyamines and yl-3-4-dehydroarginine-all irreversible inhibitors of arginine polyamine biosynthesis in T. cruzi infectivity. Polyamines are decarboxylase. Similar results were obtained when F2MeArg- present in a number of trypanosomatids and ornithine decar- treated parasites were incubated with rat heart myoblasts. The boxylase (OrnDCase), which generates putrescine from or- inhibitory effects were characterized by marked reductions in nithine, has been shown to play an important role in the both the proportion of infected cells and the number of proliferation of African trypanosomes (reviewed in refs. 13, parasites per 100 host cells. The concentrations of the arginine 14, and 15). Although polyamines (e.g., putrescine, spermi- decarboxylase inhibitors that affected infectivity had no de- dine, and spermine) are present in T. cruzi trypomastigotes tectable effect on either the concentration or motility of the (P.P.M., A. J. Bitonti, F.K., and J.J.W., unpublished re- parasite and, therefore, could not have affected the collision sults), treatment with DL-a-difluoromethylornithine or other frequency. F2MeArg appeared to inhibit the ability of T. cruzi catalytic inhibitors of OrnDCase does not alter the capacity to penetrate the host cells since the drug had no significant of these organisms to infect phagocytic or nonphagocytic effect on the extent of parasite binding to the surface of the host host cells in vitro (unpublished data). It is conceivable, cells. The inhibitory effect of F2MeArg was markedly reduced however, that T. cruzi relies on an alternative pathway for or abrogated in the presence of either agmatine or putrescine, polyamine biosynthesis. This possibility has been explored in as would have been expected if F2MeArg acted by inhibiting the present work in which we studied the effects of irrevers- arginine decarboxylase. Addition of F2MeArg to macrophage ible inhibitors of arginine decarboxylase (ArgDCase)-an or myoblast cultures immediately after infection or at a time enzyme heretofore identified in bacteria (16-19), plants (20- when virtually all of the intracellular parasites had trans- 24), and, apparently, in the parasitic worm Ascaris lumbri- formed into the multiplicative amastigote form, resulted in a coides (25)-on T. cruzi infection. markedly reduced parasite growth rate. This effect was also prevented by exogenous agmatine. These results indicate the MATERIALS AND METHODS importance of polyamines and polyamine biosynthesis in the following two important functions of T. cruzi: invasion of host Animals. The 6- to 9-week-old inbred CBA/J mice used to cells and intracellular multiplication. Furthermore, concentra- obtain resident peritoneal macrophages were purchased from tions of the inhibitors tested that affected the parasite did not The Jackson Laboratory. Four-week old Crl-CD-1(ICR)BR alter the viability of the host cells, the cellular density of the Swiss mice from Charles River Breeding Laboratories were cultures, or the ability of uninfected myoblasts to grow. Thus, used to maintain and produce blood forms of T. cruzi. arginine decarboxylase inhibitors may have a potential appli- Mouse Peritoneal Macrophages. The methods used to cation in chemotherapy against T. cruzi infection. purify unelicited mouse peritoneal macrophages and for setting up cultures ofthese cells on 3-mm diameter glass wells Trypanosoma cruzi is the protozoan causing Chagas disease cut in sterile, Teflon-coated microscope slides (Cel-Line, or American trypanosomiasis-a major health problem in Newfield, NJ) have been described in detail (26). The South and Central America (1). The infection is produced in monolayers consisted of >99% cells with typical macrophage most cases by trypomastigote forms borne by reduviid morphology and positive staining for nonspecific esterase insects and, less frequently, by trypomastigotes contaminat- activity. ing blood used in transfusions (2, 3). Mammalian host cell Rat Heart Myoblasts. Confluent monolayers of rat heart invasion by T. cruzi is essential for the production of disease myoblasts (American Type Culture Collection, CRL 1446) and the continuation of the life cycle of the parasite. Estab- were prepared on gelatin-coated microscope slide wells (see lishment ofthis parasite in mammalian hosts requires that the above) using Dulbecco's modified Eagle's medium (DMEM) invasive trypomastigotes attain a cytoplasmic localization containing penicillin at 100 units/ml and streptomycin at 100 because it is in the host cell cytoplasm that they can transform ,ug/ml and 10% (vol/vol) heat-inactivated fetal bovine serum into the multiplicative amastigote form. Amastigotes divide (GIBCO). Cell division was reduced to an insignificant level and many eventually transform into new trypomastigotes that after the myoblast cultures attained confluence. are released from bursting infected cells and disseminate the Parasites. Blood (trypomastigote) forms of Tulahuen strain infection to vital tissues, including those of the heart and T. cruzi were obtained from mice that had been infected 2 nervous system. Although several laboratories, including our weeks previously with 2 x 105 parasites intraperitoneally. own, have examined some of the biochemical requirements Abbreviations: ArgDCase, arginine decarboxylase; F2MeArg, DL- a-difluoromethylarginine; FMeAgm, monofluoromethylagmatine; The publication costs of this article were defrayed in part by page charge FMedHArg, (E)-a-monofluoromethyl-3,4-dehydroarginine; Orn- payment. This article must therefore be hereby marked "advertisement" DCase, ornithine decarboxylase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4278 Downloaded by guest on October 1, 2021 Medical Sciences: Kierszenbaurn et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4279 The trypomastigotes were purified by centrifugation over terminated by washing with DMEM, fixed with absolute Isolymph (Gallard Schlesinger, Carle Place, NY) (27) fol- methanol, and examined microscopically as described above. lowed by chromatography through a DEAE-cellulose column All assays were set up in triplicate. (28). The eluted parasites were concentrated by centrifuga- Assays for Arginine Decarboxylase and OrnDCase. Purified tion (800 x g, 15 min, 200C) and resuspended at 3-5 x 106 trypomastigotes and amastigotes suspended at concentra- organisms per ml in the desired medium. The viability of tions ranging from 107 to 6 x 109 organisms per ml in 50 mM blood-derived T. cruzi used in this work, determined by the Tris HCl (pH 7.5) containing 5 mM dithiothreitol and 1 mM criterion of motility, was >99.9%. The method for preparing EDTA were disrupted by sonication (three 1-min pulses at T. cruzi amastigotes has been described in detail (29). In all maximum setting; Biosonik III, Bronwill, NY). The presence cases, parasite concentrations were determined microscop- of arginine decarboxylase or OrnDCase activity in these ically using a hemacytometer. materials was tested by a modification (19) of the method of Reagents. The following inhibitors of arginine decarbox- Wu and Morris (32) that measures [14C]02 released from ylase: DL-a-difluoromethylarginine (F2MeArg) (19), mono- [1-'4C]arginine or [1-14C]ornithine, respectively. fluoromethylagmatine(FMeAgm)and(E)-a-monofluorometh- Presentation of Results and Statistics. Each set of results yl-3,4-dehydroarginine (FMedHArg) (30) were synthesized presented in the tables of this paper is typically repre- at the Merrell Dow Research Institute. Putrescine was pur- sentative of two to four repeated experiments. Differences chased from Sigma. Agmatine (Aldrich) was purified by between means are expressed in terms of the percentage ion-exchange chromatography on Dowex-50 using a gradient change, calculated by the equation: % change = [(experi- from 0 to 1.5 M HCl and determined to be free of other mental activity - control activity)/control activity] x 100, polyamines by HPLC and thin-layer electrophoresis. Solu- and were considered to be significant if P < 0.05 as deter- tions of these reagents were prepared in DMEM. mined by the Mann-Whitney "U" test. Treatment of Parasites with Inhibitors of Arginine Decar- boxylase. Trypomastigotes were incubated at 370C for 3-4 hr RESULTS in DMEM alone or containing various concentrations of the tested inhibitors. These treatments were not extended be- Effects of Arginine Decarboxylase Inhibitors on T. cruzi yond