Fig 1.
Overview of experimental design, evaluation of T. cruzi amastigote isolation and LC-MS/MS methods.
(A) Schematic overview of the experimental approach that involved parallel lipidomics analysis of T. cruzi parasites isolated from two different mammalian cell types: C2C12 (mouse skeletal myoblast) and HFF (human foreskin fibroblasts). Host cell infection was established with T. cruzi trypomastigotes (TCT) and intracellular T. cruzi amastigotes (ICA) were isolated from infected host cells 48 hours post infection as detailed in the Methods. T. cruzi amastigote purity was evaluated using (B) transmission electron microscopy, scale bar = 2 μm, and (C) western blot analysis using antibodies to T. cruzi, host mitochondria (ATP5B), endoplasmic reticulum (IRE1) and lipid droplets (TIP47) to probe whole cell lysates of control uninfected HFF (HFFu), T. cruzi-infected HFF (HFFi), and T. cruzi amastigotes purified from HFF (hICA). (D) Positive ion mode base peak chromatogram of lipid extracts derived from C2C12, HFF, and cognate T. cruzi amastigote (cICA and hICA, respectively) analyzed by LC-ESI-MS/MS. The major lipid subclasses eluting at different retention times (min) are indicated above the chromatogram. TG–triacylglycerol, DG–diacylglycerol, Cer–ceramide, CerG–hexosylceramide, SM–sphingomyelin, LPC–lysophosphatidylcholine, PC–phosphatidylcholine, LPE–lysophosphatidylethanolamine, PE–phosphatidylethanolamine, LPS–lysophosphatidylserine, PS–phosphatidylserine, PI–phosphatidylinositol, PG–phosphatidylglycerol.
Fig 2.
Lipid class breakdown in T. cruzi and mammalian cells.
Pie charts display the relative abundance of the major lipid subclasses of mammalian host cells (C2C12 and HFF) and T. cruzi intracellular amastigotes (ICA) and tissue-culture trypomastigotes (TCT) represented as a portion of total lipid content in each sample, averaged for 4 independent experiments. TG–triacylglycerol, DG–diacylglycerol, Cer–ceramide, CerG–hexosylceramide, SM–sphingomyelin, PC–phosphatidylcholine, PE–phosphatidylethanolamine, PS–phosphatidylserine, PI–phosphatidylinositol, PG–phosphatidylglycerol.
Fig 3.
Principle component analysis of host and parasite lipidomes at the lipid species level.
Principle component analysis of lipid species are plotted for the (A) total lipidome, (B) TG subclass, and (C) PI subclass. The first two principle components are plotted (PC1 and PC2) with proportion of variance for each component shown in parenthesis. Each sample is represented and the 95% confidence interval indicated in shaded circle.
Fig 4.
Trends in host and parasite FA composition varies between lipid classes.
The relative proportion of identified FA for (A) PE, (B) PI, (C) PC, and (D) LPC lipid subclasses is plotted (FA area %; calculated as detailed in Methods) for HFF-derived samples (C2C12-derived samples plotted in S4 Fig): uninfected HFF (HFFu), T. cruzi-infected HFF (HFFi) and T. cruzi amastigotes purified from HFF (hICA). The long-chain fatty acid (LCFA) and very long-chain polyunsaturated fatty acid (VLC-PUFA) are plotted separately for clarity. Data are represented as mean ± standard deviation.
Fig 5.
FA composition in TG and DG of T. cruzi intracellular amastigotes mirrors host cells.
FA area % is plotted for (A) TG and (B) DG classes for HFF-derived samples, (C2C12 plotted in S5 Fig): uninfected HFF (HFFu), T. cruzi-infected HFF (HFFi) and T. cruzi amastigotes purified from HFF (hICA). Data are represented as mean ± standard deviation.
Fig 6.
T. cruzi intracellular amastigotes (ICA) scavenge and incorporate exogenous FA, amastigote FA acquisition and proliferation are compromised in DGAT-TG synthesis deficient host cells.
Lipidomic analysis of uninfected HFF (uninfected), T. cruzi-infected HFF (infected) and T. cruzi amastigotes purified from HFF (ICA) show incorporation of exogenous C15:0 FA into (A) TG, (A, inset) total FA, dotted line indicates the average C15:0, C17:0, C17:1 in unlabeled samples, (B) PI, and (C) PE. Representative autoradiographs showing (D) neutral lipid and (E) glycerophospholipid TLC analysis of 14C-palmitate incorporation into uninfected (lanes 1–3), infected (lanes 4–6), and isolated amastigotes (lanes 7–9) from wild type mouse embryonic fibroblasts, diacylglycerol acyl transferase 1/2 knockout MEF DGAT1/2-/-, and DGAT1/2-/- (+DGAT2) cell lines, respectively; (F) Proliferation of T. cruzi amastigotes measured by CFSE intensity at 18 hpi (undivided) and 48 hpi in WT MEF, DGAT1/2-/- and DGAT1/2-/- (+DGAT2) cell lines.