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

J Lipid Res. 2015 Feb;56(2):331-41. doi: 10.1194/jlr.M054643. Epub 2014 Nov 25.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cholesterol / metabolism
  • Ergosterol / metabolism*
  • Itraconazole / pharmacology
  • Male
  • Methyltransferases / genetics
  • Methyltransferases / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • RNA / pharmacology
  • Trypanosoma brucei brucei / drug effects
  • Trypanosoma brucei brucei / metabolism*

Substances

  • Protozoan Proteins
  • Itraconazole
  • RNA
  • Cholesterol
  • Methyltransferases
  • Ergosterol