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

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.

Keywords: anti-parasite drugs; cholesterol; ergosterol biosynthesis; inhibitor; knockdown; ribonucleic acid interference; sparking function.

Fig. 1.

Comparative sterol biosynthesis pathways across kingdoms showing representative routes to fungal ergosterol (Cryptococcus neoformans associated with AIDS) and protozoan (T. brucei associated with sleeping sickness) and animal cholesterol (Homo sapiens as the human host). Insects do not synthesize sterols as typified by the vector of T. brucei Glossina spp. [adapted from (–, . Boxed structures represent final products of functional significance.

Fig. 2.

Sterol analysis of BSFs of different origins. A: Representative total ion current chromatogram of neutral lipids from T. brucei BSFs [peak 1, cholesterol; peak 2, campesterol; peak 3, sitosterol cf. (4)]. B: UV spectra of HPLC fraction which had the α_c of ergosterol derived by semi-preparative HPLC and analytical HPLC of the neutral lipid fraction of T. brucei brucei (b-1), T. brucei gambiense (b-2), and T. brucei rhodesiense (b-3), as described in text.

Fig. 3.

Effect of RNAi knockdown and inhibitor treatment of TbSMT and TbSDM in PCF. Growth of control engineered cells from the TbSMT RNAi line (A) and the TbSDM RNAi line (B) in FGM and LDM in the absence and presence of DOX. Growth curves were performed in triplicate conducting three independent experiments described in the Experimental Procedures; error bars are not shown because, in most cases, they approximate the data symbols. mRNA steady state levels and dsRNA induction of TbSMT (C) and TbSDM (D) analyzed by Northern blot as described in the Experimental Procedures. Partial total ion current chromatogram of DOX-induced TbSMT cells harvested from 3 to 5 days; inset above GC peak of cholesta-5,7,24-trienol overlapping ergosterol corresponds to the enhanced high end mass spectrum (E). Structures of compounds that accumulate in TbSMT and TbSDM RNAi cells lines and in treated cells, as reported in supplementary Table 1 (F). Growth of PCF in LDM without inhibitor (diamond symbol), or with ED₅₀ concentrations of either AZA or ITC (square and triangle symbols, respectively), or a combination of AZA and itraconzaole at ED₅₀ concentrations (× symbol) (G).

Fig. 4.

Growth of BSFs cultured in vitro with and without inhibitor treatment in FGM or LDM and Kaplan-Meier survival analysis of mice infected with and without DOX-induced TbSMT RNAi cells and with inhibitors of TbSMT. A: TbSMT RNAi strain cultured with and without DOX in FGM or LDM. B: Northern blot of TbSMT RNAi cell line. Pro, PCF; Bs, BSF. C: Survival data for T. brucei-infected mice. Solid blue line, control group (infected, wild-type BSF); dashed green line (infected, TbSMT RNAi BSF cell line). D: Survival data for T. brucei-infected mice. Solid blue line, control group (infected, wild-type BSF); red dashed line, AZA; and green-dotted line, 25-thialanosterol sulfonium salt (TL+), treated mice at 5 mg/kg. E: Rescue experiment of BSF-TbSMT RNAi cells in FGM supplemented with AZA at 1 μM (ED₅₀ concentration) with increasing concentrations of ergosterol (Erg) as shown. For all panels, the average of three replicates is shown. Error bars are not shown because, in most cases, they approximate the data symbols.