Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

Brad A Haubrich,Ujjal K Singha,Matthew B Miller,Craigen R Nes,Hosanna Anyatonwu,Laurence Lecordier,Presheet Patkar,David J Leaver,Fernando Villalta,Benoit Vanhollebeke,Minu Chaudhuri,W David Nes

doi:10.1194/jlr.m054643

Abstract

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

Highlights

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24␤-methyltransferase (TbSMT) and sterol 14␣-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth
In a first attempt to test whether there is an ergosteroldependent disease process linked to T. brucei infections, we examined the response of mice infected with bloodstream form (BSF) cells engineered for RNAi-mediated depletion of TbSMT or infected with wild-type BSF and treated with inhibitors of TbSMT
The sensitivity of trypanosomatid protozoa to regulation of ergosterol biosynthesis by gene silencing and inhibitors offers a unique opportunity for understanding ergosterol biology, drug target identification, and the subsequent development of new anti-parasite agents

Summary

Introduction

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24␤-methyltransferase (TbSMT) and sterol 14␣-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. To further examine the importance of TbSMT and TbSDM enzymes in PCF growth in a manner complementary to RNAi silencing, we exposed wild-type cells to increasing concentrations of AZA, 25-thialanosterol sulfonium salt, or ITC, drugs which should inhibit these enzymes for mechanistic reasons (supplementary Fig. 6) [4, 44].

Results

Conclusion