Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts

Abstract

Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-β-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.

Figure 1

Analysis of purified flagella. Flagella were purified from T. brucei procyclic cells as described in Methods. (A and B) Immunofluorescence microscopy of purified flagella and whole cells using antibodies specific for tubulin (cytoskeleton) and procyclin (surface), and whole cell body (WCB) protein (cell bodies). Scale bar = 5 μm. (C) Western blot analysis of a whole cell lysate (WCL), a crude extract (CE) and purified flagella (Flag) using antisera specific for WCB (cell bodies), procyclin (surface), calflagin (flagellum) and PFR2 (flagellum). Relative protein levels normalized to the flagellar signal by densitometry are shown beneath each band. Numbers on the right represent molecular weight markers (in kDa).

Figure 2

Sterol analysis by GC-MS of whole cell and flagellum extracts of procyclic T. brucei cells. Base-peak chromatograms of whole cell (top) and flagellar (bottom) extracts are shown. Peaks: 1, cholesterol; 2, zymosterol; 3, cholesta-5,7,24-trienol; 4, ergosta-5,7,25(27)-trienol; 5, lanosterol; 6, stigmastanol (internal standard). Base-peak chromatograms were plotted based on the quantitative ion specific for each sterol ion species in positive-ion mode, as follows: mass to charge ratio (m/z) 368 (cholesterol), 351 (zymosterol), 349 (cholesta-5,7,24-trienol), 363 (ergosta-5,7,25(27)-trienol), 393 (lanosterol), and 383 (stigmastanol). 10X indicates magnification of the chromatogram. See Supplementary Fig. 1 for total ion spectra of each of these species.

Figure 3

Sterol chelating agents disrupt membrane liquid order of bloodstream and procyclic cells differently. T. brucei bloodstream (A) and procyclic (B) cells were incubated with or without methyl-β-cyclodextrin (MBCD) or Amphotericin B (Amp B), stained with C-laurdan, and analyzed by fluorescence microscopy. Generalized polarization (GP) was calculated at each pixel in the two-dimensional image to determine the ratio of emission spectra obtained at 435 nm and 500 nm (see Methods). Higher liquid order (raft-enriched) is reflected by a higher GP (yellow-red), while lower liquid order is reflected by a lower GP (blue-green). The fluorescence images at the two wavelengths are shown in the middle panels and line drawings showing the cell body and flagellar membranes in black and red, respectively, are shown below these. Amp B treatment disrupts cell morphology but C-laurdan staining remains. MBCD has a milder effect on the cell morphology. The GP scale is provided at the bottom. Scale bar = 5 μm. (C) Higher power view of the anterior tips of untreated bloodstream and procyclic cells containing a thin cell body extension and mostly flagellum and having high GP. (D) Quantitative image analysis of individual pixels from cell images like those in (A and B) showing a reduction in liquid order in bloodstream cells by MBCD and in procyclic cells by Amp B from two biological replicates of procyclic cells and three biological replicates of bloodstream cells.

Figure 4

Sterol chelating agents disrupt localization of a dually-acylated flagellar membrane protein in bloodstream and procyclic cells differently. T. brucei (A) bloodstream cells and (B) procyclic cells were incubated with MBCD or Amp B, extracted with ice-cold 1% Triton X-100, fixed, and examined by immunofluorescence microscopy using antibodies specific for PFR2 (paraflagellar rod) or calflagin (dually-acylated flagellar membrane protein). Control WT cells were visualized only after fixation. Scale bar = 2 μm.

Figure 5

Sterol chelating agents remove sterols in bloodstream and procyclic cells differently. (A) T. brucei bloodstream cells and procyclic cells were stained with sterol-specific Filipin after pretreatment with or without MBCD or Amp B and visualized by fluorescence microscopy. (B) Cells were incubated with MBCD or Amp B, extracted with ice-cold 1% Triton X-100, fixed, and examined by immunofluorescence microscopy using antibodies specific for galactosylceramide (GC) and calflagin (dually-acylated flagellar membrane protein). Scale bar = 2 μm.

Figure 6

The lipid raft association of calflagin is affected differently by methyl-β-cyclodextrin (MBCD) and amphotericin B (Amp B) treatment of bloodstream and procyclic cells. Lipid raft extracts were prepared from T. brucei bloodstream and procyclic cells with or without pretreatment with MBCD or Amp B. Extracts were run on Optiprep gradients and gradient fractions were analyzed by western blotting using antibodies specific for calflagin (lipid raft) and PFR2 and mt-hsp70 (non-raft controls). I = input material loaded on the gradient.

Figure 7

Bloodstream and procyclic cells are differentially susceptible to MBCD- and Amp B-mediated killing. T. brucei bloodstream and procyclic cells were cultured in the presence of different concentrations of methyl-β-cyclodextrin (MBCD) (20 μM to 20 mM) or Amphotericin B (Amp B) (1.32 nM to 43.2 μM) for 48 hrs. Metabolic activity was determined using a standard Alamar blue assay as described in Methods to generate IC₅₀ curves. The IC₅₀ values are given in Table 2. Error bars indicate SEM.