Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation

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

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

Morphometric analysis of the area of reservosomes and the area occupied by lipid inclusions inside them in epimastigotes cultivated in LIT medium supplemented with 10% dFCS.

<p>¹ n = 30 parasite profiles;</p><p>² n = 50 reservosomes</p><p>*<i>P</i> < 0.05 in relation to time zero. The results are expressed as the mean ± S.E.M using one-way ANOVA</p><p>Morphometric analysis of the area of reservosomes and the area occupied by lipid inclusions inside them in epimastigotes cultivated in LIT medium supplemented with 10% dFCS.</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

<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