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

Aabha I Sharma,João I Mamede,David M Engman,Cheryl L Olson,Felipe Gazos-Lopes,Igor C Almeida,Conrad L Epting

doi:10.1038/s41598-017-08770-9

Aabha I Sharma, João I Mamede + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-08770-9

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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.

Highlights

All eukaryotic cilia, whether motile, sensory or primary, share a highly conserved structure and serve as environmental sensors in a wide range of organisms and cell types, from protists to humans[1,2,3,4]
What was not clear from previous studies is whether the flagellar enrichment of sterols, the key component of lipid rafts, is due to selective concentration of one or more individual sterol species, or general enrichment of all sterols found in the cell body membrane
We used MBCD treatment of T. brucei bloodstream cells to determine the requirement of sterols for calflagin targeting to the flagellar membrane, lipid raft association, and formation of detergent-resistant axoneme-associated particles[9]

Summary

Introduction

Whether motile (flagella), sensory or primary, share a highly conserved structure and serve as environmental sensors in a wide range of organisms and cell types, from protists to humans[1,2,3,4]. Such as Gp63, variant surface glycoprotein in bloodstream cells and procyclin in procyclic cells, that account for majority of the surface proteins in T. brucei, are not lipid raft associated[9, 33] due to the unusual acyl and alkyl chain composition of the GPI anchor[33]. Procyclic cells synthesize sterols de novo and produce 24 β-alkyl sterols typical of fungi and other protozoans[38, 39] They scavenge cholesterol from the bloodmeal the tsetse consumes from its mammalian hosts. The sterol in bloodstream cells is ~96% cholesterol, whereas the sterol in procyclic cells cultured in vitro contain approximately 60% scavenged cholesterol and the rest derivatives of ergosterol and cholesterol[40,41,42]

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