RESUMEN

The relative amounts of ovothiol A (N(1)-methyl-4-mercaptohistidine) and trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] have been determined in all life cycle stages of representative trypanosomatids (Leishmania spp, Crithidia fasciculata, Trypanosoma cruzi and T. brucei). Ovothiol A is present in all insect stages with intracellular concentrations of >1 mM for five species of Leishmania promastigotes and <0.25 mM for other trypanosomatids. In Leishmania promastigotes, ovothiol A can exceed trypanothione content particularly in late logarithmic and stationary phases of growth. In the other trypanosomatids, it represents less than 10% of the total thiol pool. Although amastigotes of L. major and L. donovani contain equivalent amounts of glutathione and trypanothione, ovothiol A is present in the former but absent in the latter. Ovothiol A is present in all developmental stages of T. cruzi but absent in bloodstream trypomastigotes of T. brucei. No ovothiol reductase activity could be detected in dialysed parasite extracts. Ovothiol disulphide is not a substrate for trypanothione reductase, although it can be reduced by the concerted action of trypanothione and trypanothione reductase. No ovothiol-dependent peroxidase activity was present in Leishmania extracts. Although ovothiol A can act as a non-enzymatic scavenger of hydrogen peroxide, it is less efficient than trypanothione. Second order rate constants were determined with trypanothione>glutathionylspermidine>ovothiol>glutathione. Given the presence of an active trypanothione peroxidase system in all these trypanosomatids, it is concluded that under physiological conditions, ovothiol is unlikely to play a major role in the metabolism of hydrogen peroxide in intact cells. Nonetheless, since ovothiol is absent in host macrophage, kidney and CHO cells, this metabolite may have other important functional roles in trypanosomatids that could be exploited as a chemotherapeutic target.

Asunto(s)

RESUMEN

Concentrations of free polyamines were investigated in Trypanosoma granulosum cultured in a semidefined medium containing traces of polyamines. Spermidine content peaked in early logarithmic growth while putrescine was not detectable. Unlike African trypanosomes and Leishmania, spermine was measured at equivalent amounts to spermidine in mid to late logarithmic stage cells. Addition of d,l-alpha-difluoromethylornithine to cultures did not decrease polyamine content nor was ornithine decarboxylase activity detected. In contrast, incubation of parasites with tritiated putrescine showed rapid uptake and subsequent conversion to spermidine and spermine. At late logarithmic growth, parasites contained glutathione (77% of total sulphydryl groups) and ovothiol A as major low molecular mass thiols with glutathionylpolyamine conjugates undetectable. However, the addition of exogenous putrescine elevated trypanothione and glutathionylspermidine content to 48% of total sulphydryl groups. Correspondingly, the addition of exogenous cadaverine increased homotrypanothione content. This first report of polyamines and low molecular mass thiols in Trypanosoma granulosum indicates intriguing similarities with the metabolism of the human pathogen Trypanosoma cruzi.

Asunto(s)

RESUMEN

We report the cloning, expression and functional characterisation of a peroxidase belonging to the peroxiredoxin family from the potato cyst nematode Globodera rostochiensis, the first molecule of this type from any nematode parasitic on plants. The G. rostochiensis peroxiredoxin catalyses the breakdown of hydrogen peroxide, but not cumene or t-butyl hydroperoxide, in a trypanosomatid reducing system comprising trypanothione reductase, trypanothione and tryparedoxin. In common with its homologues from Onchocerca volvulus and Brugia malayi, the G. rostochiensis enzyme is present on the surface of invasive and post-infective juveniles despite the apparent lack of a cleavable N-terminal signal peptide. The possibility that the G. rostochiensis peroxiredoxin plays a role in protection of the parasite from plant defence responses is discussed.

Asunto(s)

RESUMEN

In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.

Asunto(s)

RESUMEN

Entamoeba histolytica lacks glutathione reductase activity and the ability to synthesise glutathione de novo. However, a recent report suggested that exogenous glutathione can be taken up and conjugated to spermidine to form trypanothione, a metabolite found so far only in trypanosomatids. Given the therapeutic implications of this observation, we have carefully analysed E. histolytica for evidence of trypanothione metabolism. Using a sensitive fluorescence-based HPLC detection system we could confirm previous reports that cysteine and hydrogen sulphide are the principal low molecular mass thiols. However, we were unable to detect trypanothione or its precursor N1-glutathionylspermidine [ < 0.01 nmol (10(6) cells)(-1) or < 1.7 microM]. In contrast, Trypanosoma cruzi epimastigotes (grown in a polyamine-supplemented medium) and Leishmania donovani promastigotes contained intracellular concentrations of trypanothione two to three orders of magnitude greater than the limits of detection. Likewise, trypanothione reductase activity was not detectable in E. histolytica [ < 0.003 U (mg protein)(-1)] and therefore at least 100-fold less than trypanosomatids. Moreover, although E. histolytica were found to contain trace amounts of glutathione (approximately 20 microM), glutathione reductase activity was below the limits of detection [ < 0.005 U (mg protein)(-1)]. These findings argue against the existence of trypanothione metabolism in E. histolytica.

Asunto(s)

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Intracellular levels of glutathione and glutathionylspermidine conjugates have been measured throughout the growth phases of Escherichia coli. Glutathionylspermidine was present in mid-log-phase cells, and under stationary and anaerobic growth conditions accounted for 80% of the total glutathione content. N1,N8-bis(glutathionyl)spermidine (trypanothione) was undetectable under all growth conditions. The catalytic constant kcat/Km of recombinant E. coli glutathione reductase for glutathionylspermidine disulphide was approx. 11,000-fold lower than that for glutathione disulphide. The much higher catalytic constant for the mixed disulphide of glutathione and glutathionylspermidine (11% that of GSSG), suggests a possible explanation for the low turnover of trypanothione disulphide by E. coli glutathione reductase, given the apparent lack of a specific glutathionylspermidine disulphide reductase in E. coli.

Asunto(s)