Macromolecular Synthesis in Leishmania donovani Amastigotes

Abstract

Amastigotes (non-flagellated tissue forms) of Leishmania sp. reside and multiply within the phagolysosomes in tissue macrophages of vertebrate hosts. They are true obligate, intracellular parasites and do not persist as viable entities outside of host cells. This absolute dependence on host cells raises interesting questions about the metabolic capabilities of the amastigote stage. It is assumed that in general, intracellular stages of parasitic protozoa, exhibit reduced metabolic capabilities when compared with insect or culture forms. This hypothesis is largely untested in Leishmania sp. although there is evidence to indicate that several enzymes in amastigotes are less active than their counterparts in promastigotes (Coombs et al., 1982, Molecular and Biochemical Parasitology 5: 199-211; Meade et al., 1984, Journal of Protozoology 31: 156-161). Both forms also differ in their ability to utilize various substrates (Hart et al., 1981, Molecular and Biochemical Parasitology 4: 39-51; Hart and Coombs, 1982, Experimental Parasitology 54: 397-409). The presence of several enzymes of intermediary metabolism in amastigotes of L. donovani (Looker et al., 1983, Molecular and Biochemical Parasitology 9: 15-28; Meade et al., loc. cit.) and L. mexicana (Coombs et al., 1982, loc. cit.) suggests that they are indeed capable of carrying out independent metabolic activities. Since in vitro demonstration of enzyme activity in cell free extracts in and of itself does not ensure functional activity in vivo, the capacity of L. donovani amastigotes to catabolize and incorporate substrates or precursors into macromolecules was tested. Amastigotes of L. donovani (Sudan strain 1S) were isolated and purified from infected hamster spleens as described before (Meade et al., 1984, loc. cit.). Parasites were washed twice and resuspended in basal salts solution (Mukkada et al., 1974, Journal of Protozoology 21: 393-397), pH 5.5 to a density of 0.35 mg protein/ml. Incorporation of glucose and proline into macromolecules was determined by measuring the incorporation of label into cold trichloroacetic acid (TCA) insoluble materials (Mukkada and Simon, 1977, Experimental Parasitology 42: 87-96). Amastigote suspensions (4.9 ml) were incubated on a shaking waterbath at 30 C for 20 min for temperature equilibration. [U-_4C]D-glucose and [U-_4C]-L-proline were added to a final concentration of 0.1 mM and a specific radioactivity of 0.4 A,Ci/umole. Samples of 0.5 ml were withdrawn at various intervals and immediately added to 0.5 ml cold 10% TCA. After 15 min of extraction, 0.5 ml of the suspension was filtered through a millipore filter (0.8 ,tm porosity), washed with 3 ml basal salts and the filter pads with the cells thereon were dropped into scintillation vials containing 10 ml Bray's solution (Bray, 1960, Analytical Biochemistry 1: 279-285); radioactivity was determined in a Packard scintillation spectrometer Model 3003. Radioactivity in amastigotes after extraction with cold TCA represents incorporation of label into macromolecules. Synthesis of nucleic acids was followed by determining the incorporation of [3H] thymidine and [3H] uridine into TCA insoluble materials. Amastigote suspensions were incubated with [3H] thymidine and [3H] uridine at a final concentration of 0.4 mM and specific radioactivity of 0.6 ACi/,umole. Samples of 0.5 ml were withdrawn at intervals, filtered rapidly through glass microfiber filters (1.2 ,um porosity; Whatman GF/C) and immediately washed with 3 ml cold 10% TCA. They were then rinsed with 1 ml ethanol (95%) and transferred to vials containing 10 ml Biofluor scintillation fluid (NEN) and the radioactivity determined. Protein was determined by the method of Oyama and Eagle (1956, Proceedings of the Society of Experimental Biology and Medicine 91: 305-307). Since amastigotes were found to carry out a variety of metabolic activities optimally at pH 5.0-5.5 (data not shown) these studies were conducted at pH 5.5. Label from glucose and proline were readily incorporated into TCA insoluble materials in a time dependent fashion (Fig. 1). Though details are not shown, trapping in methyl benzethonium hydroxide (Weiss et al., 1967, Nature 213: 1020-1022) showed the evolution of significant amounts of labelled CO2 from both substrates which clearly indicate that amasti-