Diamine auxotrophy may be a universal feature of Trypanosoma cruzi epimastigotes

Abstract

Polyamines play an important and central role in normal cell growth and differentiation in many cells. In trypanosomatids, spermidine is also an essential precursor in the biosynthesis of the unique glutathione-spermidine conjugate, trypanothione. Our previous study has shown that the epimastigote stage of Trypanosoma cruzi (Silvio strain) is incapable of significant de novo synthesis of putrescine or cadaverine from their amino acid precursors [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027]. In this study we show that when grown to late log phase in medium containing trace amounts of putrescine (0.22 microM) and spermidine (0.63 microM), Y-strain epimastigotes contain low levels of polyamines with free glutathione as their principal low molecular mass thiol (> 97% of total glutathione). Following passage into fresh medium, trypanothione and glutathionylspermidine content increase to 46% of total glutathione by mid log phase but returns to less than 3% by late log phase. In contrast, when supplemented at inoculation with exogenous putrescine, glutathione-spermidine conjugates reach 80% of total glutathione by early log phase and remain elevated throughout growth. Supplementation with exogenous putrescine or spermidine during polyamine starvation (late log phase) results in increased conjugate levels (> 74% of total glutathione) and is associated with large increases in total putrescine and spermidine. Likewise, supplementation with exogenous cadaverine and aminopropylcadaverine results in similar increases in trypanothione analogues and total cadaverine and aminopropylcadaverine. In contrast, ornithine, arginine, lysine, agmatine and other amino acid precursors have no effect on polyamine or conjugate levels. No significant ornithine or arginine decarboxylase activities could be detected (< 0.8 pmol min-1 [mg protein]-1). Similar results were obtained for epimastigotes representing all the major zymodeme classes, providing evidence that diamine auxotrophy may be a universal feature of this stage of the life-cycle.