Fig 1.
Trypanosoma cruzi lipid bodies (LBs) show variation in number, size and electron-density in response to the interaction with host cells.
(A) Trypomastigotes cultured with peritoneal macrophages show increased numbers of cytosolic LBs compared to parasites alone, after 1h of interaction. (B) At this time, several trypomastigotes (arrows) can be seen in contact with the macrophage surface. Macrophage nucleus was outlined in red. (C, D) Transmission electron microscopy (TEM) of T. cruzi amastigotes within a peritoneal (in vitro infection after 24h) (C) and heart inflammatory macrophage (in vivo infection after 12 days) (D). Amastigote LBs are indicated (circles). (E) LB sizes are significantly higher in amastigotes growing in vivo (black bar) compared to amastigotes LBs living in vitro (white bar). (F) LBs from amastigote forms of the parasite within heart macrophages are more electron-dense compared to amastigote LBs from peritoneal macrophages. Cultured peritoneal macrophages and heart macrophages were fixed and processed for TEM[25]. A total of 36 electron micrographs, 50 parasites and 125 LBs were evaluated. LBs were classified as Strongly Electron-Dense (SED) Electron-Dense (ED) or Electron-Lucent (EL) by using the software ImageJ® as in materials and methods. Scale bar, 10 μm (B); 5 μm (C, D). * P<0.05.
Fig 2.
Lipid bodies (LBs) are formed within trypomastigotes in response to arachidonic acid (AA) stimulation.
Metacyclic trypomastigotes were stained with BODIPY (A, Ai, B, Bi), Oil Red O (C, Ci), Nile Red (D, Di) or osmium (E) for LB detection. Panels A and Ai; B and Bi; C and Ci, and D and Di represent identical fields of trypomastigotes seen by contrast phase and fluorescence microscopy after 1 h of incubation with vehicle (A, Ai) or 7.5 μM AA (B-D). Note in (C) that Oil Red O staining enables visualization of LBs at both contrast phase and fluorescence microscopy. Parasite nuclei are visualized after DAPI staining in Ai, Bi, Ci and Di. In (E), osmium staining is observed under bright field microscopy. (F, G) Number of LBs within trypomastigotes after stimulation with exogenous AA for 1 h (F) or 24 h (G). Bars represent the mean ± SEM of LB per parasite from 50 consecutively counted parasites from at least 4 independent pools. * P < 0.05 between groups. Cells were enumerated after osmium staining. In (H, I), trypomastigotes are observed by transmission electron microscopy. Typical non-membrane bound LBs (arrows) are seen in the cytoplasm after stimulation with AA (I). Scale bar, 5 μm (A-E); 1 μm (H, I).
Fig 3.
Raman spectroscopy detects higher content of unsaturated fatty acids and arachidonid acid (AA) in stimulated parasites compared to control unstimulated cells.
Raman spectra of AA (black), AA-stimulated (blue) and unstimulated (red) parasites. Parasites were stimulated or not for 1h with 7.5 μM AA, fixed and analyzed by Raman spectroscopy without labeling.
Fig 4.
Arachidonic acid (AA) is incorporated into parasite lipid bodies (LBs).
Raman spectra of 1mM AA in aqueous solution (black) and from LB fractions isolated by subcellular fractionation from unstimulated (red) and AA-stimulated (blue) parasites show that the chemical composition of LBs is due to AA and not to the presence of other fatty acids. The spectra were normalized using the band at 1659 cm-1. Samples were analyzed without any labeling. Data are representative of 2 independent experiments.
Fig 5.
MALDI-TOF mass spectrum, from 303 to 306 m/z range, acquired from a pure arachidonic acid (AA) solution (A) and from lipid body (LB) fractions isolated from unstimulated and AA-stimulated parasites (B).
In (A), the spectrum shows the molecular weight of the pure AA (m/z 304.24). In (B), a higher content of AA is observed in the LB fraction purified from stimulated parasites (black) compared to the LB fraction from unstimulated cells (red). Samples were analyzed without any labeling. Data are representative of 3 independent experiments.
Fig 6.
Arachidonic Acid (AA) induces prostaglandin E2 (PGE2) production by metacyclic trypomastigotes.
(A) PGE2 levels in supernatants harvested from trypomastigotes stimulated for 24 h with 1.5 or 7.5 μM AA. (B) Oleic Acid (OA) did not induce PGE2 production. Bars represent the mean ± SEM, n = 3, being the graph representative of, at least, 3 independent experiments (*) P < 0.05.
Fig 7.
T. cruzi stimulated by AA expresses PGE synthase, but not COX-2 enzymes.
Expression of COX-2 (A) and PGE synthase (B) in trypomastigote forms of T. cruzi after stimulation or not with AA during 1h. Mice peritoneal macrophages (Mϕ) stimulated by BCG during 24h were used as positive controls [30]. Total T. cruzi (2 x 106 cells/lane) and macrophage cell (1 x 106 cells/lane) lysates were separated by SDS-PAGE (10%) and subjected to Western blotting for COX-2, PGE synthase or β-actin. The images are representative of at least two different blots. The graph represents the densitometric analysis of PGE synthase enzyme (arbitrary units) of the Western blotting bands.
Fig 8.
LBs are sites of PGE2 synthesis in AA-stimulated trypomastigotes.
(A-Aii) Unstimulated trypomastigotes were labeled for PLIN2/ADRP, a marker for LBs, (A) and for PGE2 (Ai). Merged image (Aii) exhibited negative labeling for both ADRP and PGE2. (B-Bii) Arachidonic acid (AA)-stimulated trypomastigotes showed strong labeling for newly-formed PGE2 (Bi) in PLIN2/ADRP-associated LBs (B). The merged image is observed in (Bii). (C-Cii) An IgG1 irrelevant isotype (clone MOPC 21) was used as control for PGE2 labeling in combination with labeling for PLIN2/ADRP. (Cii) is the merged image of (C) and (Ci). The nuclei of parasites are observed after DAPI staining (blue). Cells were immunolabeled by using the EicosaCell technique [31]. Colocalization quantitative analyses showed that PGE2 and PLIN2/ADRP significantly colocalized in cells [Pearson’s correlation coefficient of 0.84 ± 0.02 (mean ± SEM)]. Scale bar, 2 μm (all images). Images are representative of 3 independent experiments.