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

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 Tb SMT 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 mechanismbased 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. -Haubrich, B. A., U. K. Singha, M. B. Miller, C. R. Nes, H. Anyatonwu, L. Lecordier, P. Patkar, D. J. Leaver, F. Villalta, B. Vanhollebeke, M. Chaudhuri, and W. D. Nes. Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth. J. Lipid Res. 2015.