Sterol composition and biosynthesis in Tryanosoma cruzi amastigotes

A detailed analysis of the endogenous sterols present in the clinically rel evant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14 alpha demet hylase inhibitor ketoconazole and two inhibitors of Delta(24(25))-sterol me thyl transferase, 20 piperidin-2-yl-5 alpha-pregnan-3 beta-20-R-diol (22,26 -azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated an d purified from their host cells and neutral lipids were extracted, separat ed and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-chol esta-7-en-3 beta-ol (ergosta-7-en-3 beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3 beta-ol; these c ells also contained cholesterol (up to 80% by weight of total sterols), pro bably derived from host cells. Amastigotes that proliferated in the presenc e of 10 nM ketoconazole (minimal inhibitory concentration, MIG) for 24 h ha d a sharply reduced content of endogenous 4-desmethyl sterols with a concom itant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrol anosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100 -300 nM) accumulated large amounts of C-27 sterols whose structure suggeste d, in the case of 22,26-azasterol, that Delta(14) sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorpo ration of [2-C-14]-acetate into the parasite's endogenous C4-desmethyl ster ols with an IC50 of 50 nM, indistinguishable from the Value reported previo usly for the extracellular epimastigote form. Taken together, the results s howed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both Delta(5) and Delta(22 ) reductases. They also suggest that the 100-fold higher potency of antifun gal azoles as antiproliferative agents against amastigotes, when compared w ith epimastigotes, is most probably due to a smaller pool of endogenous ste rols in the intracellular parasites. (C) 1999 Elsevier Science B.V. All rig hts reserved.