Cholesterol crystals in reservosomes of Trypanosoma cruzi

The LDL endocytosis provides cholesterol supply to Trypanosoma cruzi epimastigotes. Cholesterol reaches reservosomes (lysosome like organelles) being used according to cell demand or is storage in lipid droplets. But a remnant fraction remains in reservosome lumen where solidifies. In this work we investigated the crystalline properties of these cholesterol solids. First, ultrathin sections, freeze fracture and deep etching replicas suggested collectively different spatial configurations such as needles, plaques or rounded structures. Cryo-EM images showed hemi- and membrane profiles in close association with sterol solids, possibly flanking the growth of these structures. Second, the analysis in situ of parasites by polarized light microscopy pointed to the birefringence of cholesterol. In this way, we used fractions of reservosome lipid inclusions to determine the spectral signature by FTIR, and X-ray diffraction defined the crystallinity of the lipid inclusions. Additionally, our analyses showed that cholesterol was arranged in two polymorphs of anhydrous crystal. Cholesterol crystals had triclinic configuration. Polymorph 1 presented the following unit cell parameters: a = 14.21Å, b = 33.86Å, c = 10.56Å, V = 5028.8Å while the polymorph 2: a = 27.32 Å, b = 38.24 Å, c = 10.66 Å, V = 9776.98 Å. Differences in crystalline densities were also found by our group. The polymorph 1 was more packed and denser than the second crystal analyzed. The densities were estimated in 5.11 g/cm³ and 2.63 g/cm³, respectively. Third, cholesterol crystals did not impair metacyclogenesis being rapidly dismantled if parasites were kept under nutritional starvation.