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. 2015 Feb;110(1):48-55.
doi: 10.1590/0074-02760140097. Epub 2015 Feb 13.

Mechanisms of growth inhibition of Phytomonas serpens by the alkaloids tomatine and tomatidine

Jorge Mansur Medina  1 , Juliany Cola Fernandes Rodrigues  2 , Otacilio C Moreira  3 , Geórgia Atella  1 , Wanderley de Souza  2 , Hector Barrabin  1
Affiliations

Mechanisms of growth inhibition of Phytomonas serpens by the alkaloids tomatine and tomatidine

Jorge Mansur Medina et al. Mem Inst Oswaldo Cruz. 2015 Feb.

Abstract

Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.

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Figures

Fig. 1:
Fig. 1:. structure of tomatine (A) and tomatidine (B).
Fig. 2A, B:
Fig. 2A, B:. growth curve of Phytomonas serpens in the presence of tomatine and tomatidine. Promastigotes (1 × 106/mL) were grown in medium supplemented with 10% foetal bovine serum at 27ºC. After 24 h of growth, tomatine or tomatidine were added at the indicated concentrations. Each point represents the mean ± standard error of the means of three independent experiments. *: p < 0.05; **: p < 0.001 (2-way ANOVA with Bonferroni's post test). The half maximum inhibitory concentration values obtained after 48 h of treatment were 9.9 μM (for tomatine) and 14.2 μM (for tomatidine). Dimethyl sulfoxide alone had no effect on cell proliferation (data not shown).
Fig. 3
Fig. 3. tomatine induced pyruvate kinase (PK) leakage from Phytomonas serpens. Cells were harvested after two days of growth and resuspended in phosphate buffered saline at the concentrations indicated below. The tomatine (A) or tomatidine (B) was added and after 10 min, the cells were spun down and PK was measured in 0.5 mL of the supernatant (see Materials and Methods). : control without alkaloids; : 1 x 106 cell/mL treated with 10 μM tomatine; □: 3 x 106 cell/mL treated with 10 μM tomatine; : 3 x 106 cell/mL treated with 200 μM digitonin; ▲: 30 mU of purified PK; : 3 x 106 cell/mL treated with 10 μM tomatine and measured in the absence of lactate dehydrogenase; º : 1 x 106 cell/mL treated with 10 μM tomatidine; : 3 x 106 cell/mL treated with 100 μM tomatidine. Each set of curves was repeated three times and the Figure is representative of these experiments.
Fig. 4:
Fig. 4:. ultrastructural alterations of Phytomonas serpens induced by tomatidine. A: ultrathin section of P. serpens without treatment which presents a normal ultrastructure of organelles such as kinetoplast (k) and nucleus (N); B-D: electron micrograph of P. serpens treated with 30 μM tomatidine for 48 h presenting many vacuoles (arrows), in some cases localised to the posterior region of the parasite; E: after treatment with 50 μM tomatidine for 48 h, it is possible to observe the presence lipid bodies (arrowheads). Bars = 1 μM (A-C, E) and 0.25 μM (D).
Fig. 5:
Fig. 5:. fatty acid incorporation in Phytomonas serpens vacuoles. P. serpens was grown for two days in the presence of 5 μM labelled BODIPY-palmitic acid (green) and in the absence (A) or presence (B) of 50 μM tomatidine as described in Materials and Methods, then harvested and resuspended in phosphate buffered saline for fluorescence microscopy. Right panels are images obtained by interferential contrast microscopy. The inserts represents typical cells in detail. Bars = 10 μM; inserts = 0.25 μM.
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