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

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

ACAT-related activity assay.

<p>The ability of <i>T</i>. <i>cruzi</i> epimastigotes to esterify cholesterol was tested using Sandoz 58-035-58-035 for 16–18 h (A) or Avasimibe for 24 h (B) in different concentrations, followed by addition of BSA-³H-palmitate (800,000 DPM; 1 mg/mL) for 24 h. Lipid analysis was performed by TLC; CHOE spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of two independent experiments in duplicate, and analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p

<i>Trypanosoma cruzi</i> Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions

<div><p><i>Trypanosoma cruzi</i> epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.</p></div

Free and total (after saponification) cholesterol and ergosterol contents in lipid starved <i>T</i>. <i>cruzi</i> epimastigotes, kept in LIT medium supplemented with 10% dFCS for 0, 24, 48 and 72h.

<p>All samples correspond to 5 μg/mL of protein.</p><p>Free and total (after saponification) cholesterol and ergosterol contents in lipid starved <i>T</i>. <i>cruzi</i> epimastigotes, kept in LIT medium supplemented with 10% dFCS for 0, 24, 48 and 72h.</p

Incorporation of ³H-cholesterol into cholesteryl esters in epimastigotes.

<p>Parasites were incubated in LIT medium with 10% FCS (control) or 10% dFCS (starved) in the presence of LDL-³H-cholesterol (1,000,000 DPM; 2 mg/mL) for 3 days at 28°C. Parasites were washed and their lipids were extracted and analysed by TLC. The cholesterol (CHO) and cholesteryl-ester (CHOE) spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of three independent experiments analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p

Morphometric analysis of the number and the area occupied by lipid bodies in the cytoplasm of epimastigotes cultivated in LIT medium supplemented with 10% dFCS.

<p>¹ n = 30 parasite profiles</p><p>*<i>P</i> > 0.05. The results are expressed as the mean ± S.E.M using one-way ANOVA.</p><p>Morphometric analysis of the number and the area occupied by lipid bodies in the cytoplasm of epimastigotes cultivated in LIT medium supplemented with 10% dFCS.</p

ACAT-related activity assay.

<p>The ability of <i>T</i>. <i>cruzi</i> epimastigotes to esterify cholesterol was tested using Sandoz 58-035-58-035 for 16–18 h (A) or Avasimibe for 24 h (B) in different concentrations, followed by addition of BSA-³H-palmitate (800,000 DPM; 1 mg/mL) for 24 h. Lipid analysis was performed by TLC; CHOE spots were scraped and the lipid eluted from silica. The lipid-associated radioactivity was measured by liquid scintillation counting. The results are expressed as the mean (±SD) per 1 mg of protein of two independent experiments in duplicate, and analysed by one-way ANOVA followed by the Bonferroni test (*<i>P</i><0.05).</p

Quantification of NBD-cholesterol distribution in epimastigotes according to time.

<p>Starved parasites (A) uptake more LDL-NBD-cholesterol than controls (B), comparing fluorescence intensity in reservosomes/vesicles at time zero (immediately after washing off the tracer). The fluorescence intensity in reservosomes varies with time in starved parasites, but not significantly in control reservosomes, except after 2h. Starved parasites quickly redistribute the exogenous NBD-chol to their membranes (C) after 30 min, while in control cells (D) the NBD-chol rate increases only after 2 h of traffic. The results are expressed as the mean (±SD) and analysed with the one-way ANOVA test in comparison to time zero (*/**/*** significantly different, <i>P</i> < 0.05).</p

Transmission electron microscopy of epimastigotes cultivated in LIT medium supplemented with 10% dFCS.

<p>(A) 0 h—Reservosomes show many lipid inclusions in their lumen (asterisk). (B) After 6 h of lipid starvation, reservosomes filled with lipid inclusions could still be observed, but after 24 h (C), 48 h (D) and 72 h (E), an expressive decrease in reservosome lipids was registered. The insets show that there were reservosomes with typical lipid inclusions even after 24 to 72 h of starvation. The arrows point to lipid bodies throughout the cytoplasm. K- Kinetoplast, M—Mitochondrion, N—Nucleus, R- reservosomes. Bars correspond to 0.25 μm.</p