Phenotypic analysis of trypanothione synthetase knockdown in the African trypanosome

Abstract

Trypanothione plays a pivotal role in defence against chemical and oxidant stress, thiol redox homoeostasis, ribonucleotide metabolism and drug resistance in parasitic kinetoplastids. In Trypanosoma brucei, trypanothione is synthesized from glutathione and spermidine by a single enzyme, TryS (trypanothione synthetase), with glutathionylspermidine as an intermediate. To examine the physiological roles of trypanothione, tetracycline-inducible RNA interference was used to reduce expression of TRYS. Following induction, TryS protein was reduced >10-fold and growth rate was reduced 2-fold, with concurrent 5-10-fold decreases in glutathionylspermidine and trypanothione and an up to 14-fold increase in free glutathione content. Polyamine levels were not significantly different from non-induced controls, and neither was the intracellular thiol redox potential, indicating that these factors are not responsible for the growth defect. Compensatory changes in other pathway enzymes were associated with prolonged suppression of TryS: an increase in trypanothione reductase and gamma-glutamylcysteine synthetase, and a transient decrease in ornithine decarboxylase. Depleted trypanothione levels were associated with increases in sensitivity to arsenical, antimonial and nitro drugs, implicating trypanothione metabolism in their mode of action. Escape mutants arose after 2 weeks of induction, with all parameters, including growth, returning to normal. Selective inhibitors of TryS are required to fully validate this novel drug target.

Figure 1. Effect of TRYS RNAi on trypanosome growth and enzymatic phenotype

(A) Growth of cell line A: RNAi-induced cells (●), non-induced cells (○); ‘g’ indicates the generation time. The results are means±S.D. for three separate cultures. (B) Western blot analysis of RNAi-induced and non-induced trypanosomes. The blot is representative of two separate experiments. (C) Ratio of induced:non-induced band densitometry measurements: TryS (●), TryR (■), γ-GCS (△), ODC (▽). Densitometry measurements were obtained from (B).

Figure 2. Growth of procyclic TRYS RNAi revertant trypanosomes and changes in thiol content

(A) Growth of cell line A: RNAi-induced cells (●), non-induced cells (○); ‘g’ indicates the generation time. (B) Free glutathione (■) and trypanothione (●) content of RNAi-induced cells; free glutathione (□) and trypanothione (○) content of non-induced cells are also shown. (C) Total glutathione content of RNAi-induced cells (▲) and non-induced cells (△). Total glutathione was calculated as [glutathione+glutathionylspermidine+2×trypanothione]. All results are the means±S.D. for separate cultures.

Scheme 1. Biosynthetic pathway of trypanothione and chemotherapeutic intervention points

SOD, superoxide dismutase (all other abbreviations for enzymes are explained in the main text); metabolites: AdoSMe, methylthioadenosine; dAdoMet, S-adenosylmethionine; γ-GluCys, γ-glutamylcysteine; GspdSH, glutathionylspermidine; Put, putrescine; Spd, spermidine; T(SH)₂, dihydrotrypanothione; T(S)₂, trypanothione disulphide; drugs: BSO, buthionine sulphoximine; DFMO, difluoromethylornithine; RAs=O, arsenicals; RNO₂, nitro compounds (nifurtimox, megazol); Sb³⁺, trivalent antimony.