Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods

Abstract

The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.

Fig. 1

Genotypic analysis of WT, SKO and cDKO cell lines. A. Schematic representation of the stepwise generation of the TRYS cDKO cell line in T. brucei. (i) One allele of TRYS was replaced with the puromycin-resistance gene (PAC) by homologous recombination, generating ΔTRYS::PAC cell line (SKO); (ii) a tetracycline-inducible ectopic copy of TRYS was introduced into the rDNA, generating TRYS^TiΔTRYS::PAC cell line; (iii) while tetracycline induces the expression of the ectopic copy, the remaining allele was replaced with a hygromycin resistance gene (HYG) by homologous recombination, resulting in conditional double knockout cell line TRYS^TiΔTRYS::PAC/ΔTRYS::HYG (cDKO). B. Confirmation of genotype of T. brucei TRYS conditional double knockout cell line. Southern blot analysis of PstI-digested genomic DNA (∼5 μg) from wild-type T. brucei cells (lane 1), ΔTRYS::PAC (lane 2), TRYS^TiΔTRYS::PAC (lane 3) and TRYS^TiΔTRYS::PAC/ΔTRYS::HYG (lane 4) cells; the TRYS ORF probe shows allelic TbTRYS at 3 kb and the ectopic copy TbTRYS^Ti at ∼10 kb.

Fig. 2

Growth characteristics and biochemical analysis of cDKO cells in vitro. A. The growth of the cDKO cell line in HMI9-T media was monitored in the presence (closed circles) and absence of tetracycline (open circles, dashed line). The arrow indicates the point where the viable cells are no longer recoverable when subcultured for up to 10 days in medium containing tetracycline. B. Immunoblots of cell extracts of WT, SKO and cDKO (plus and minus tetracycline) cells were probed with antiserum to T. brucei TRYS and to T. brucei PTR1 as a control (1 × 10⁷ parasites in each lane). C. Intracellular T[SH]₂ (closed circles) and GSH (open circles) levels in cDKO cells following the removal of tetracycline from cultures. Initial levels of T[SH]₂ and GSH in untreated cells were 0.42 and 0.54 nmol(10⁸ cells)⁻¹ respectively. Each data point represents the means ± standard deviations from triplicate determinations.

Fig. 3

Virulence of cDKO parasites in mice. Groups of five mice were infected with either WT or cDKO cells (1 × 10⁴ parasites). Mice infected with tetracycline-treated parasites were dosed with doxycycline in their drinking water 3 days prior to, and throughout, the infection. Data are presented in the form of a Kaplan–Meier survival plot. Symbols: WT, blue; cDKO in the presence of doxycycline, red; cDKO in absence of doxycycline, green.

Fig. 4

Chemical targeting of TRYS in T. brucei. A. Chemical structure of TRYS inhibitor DDU 86439 (N-(3-(dimethylamino)propyl)-2-(3-(3-fluorophenyl)-1H-indazol-1-yl)acetamide). B. EC₅₀ values were determined for DDU 86439 against WT (closed circles), SKO (open squares), TRYS-overexpressing (open circles) and cDKO cells grown in the absence of tetracycline for 2 days prior to analysis (closed squares, dotted line). The curves are the non-linear fits of data using a two-parameter EC₅₀ equation provided by GraFit (see Experimental procedures). EC₅₀ values of 7.1 ± 0.2, 1.2 ± 0.05, 23.3 ± 0.3 and 0.46 ± 0.01 μM were determined for WT, SKO, TRYS-overexpressing and cDKO cell lines respectively. Data are the mean of duplicate measurements. C. Analysis of intracellular T[SH]₂ (closed circles) and GSH (open circles) levels in T. brucei bloodstream parasites (WT) following incubation with compound DDU 86439 (72 h). Data are represented as a percentage of the thiol levels of untreated cells (0.51 and 0.42 nmol(10⁸ cells)⁻¹ for GSH and T[SH]₂ respectively; Table S1) and each measurement is the mean of three individual measurements.

Fig. 5

In vitro and in vivo characterization of a TRYS-amidase ‘dead’ cell line. A. The growth of the cDKO(C57A) cell line in HMI9-T medium was monitored in the presence (closed circles) and absence of tetracycline (open circles, dashed line).The arrow indicates the point where the viable cells are no longer recoverable when subcultured for up to 10 days in medium containing tetracycline. B. Intracellular T[SH]₂ (closed circles) and GSH (open circles) levels in cDKO(C57A) cells following the removal of tetracycline from cultures. Each data point represents the means ± standard deviations from triplicate determinations. C. Groups of five mice were infected with either WT or cDKO(C57A) cells (1 × 10⁴ parasites). Mice infected with tetracycline-treated parasites were dosed with doxycycline in their drinking water 3 days prior to, and throughout, the infection. Data are presented in the form of a Kaplan–Meier survival plot. Symbols: WT, blue; cDKO in the presence of doxycycline, red; cDKO in absence of doxycycline, green.

Fig. 6

The effect of polyamines on the growth and virulence of cDKO(C57A) parasites. A. EC₅₀ values were determined for DFMO against cDKO (closed circles) and cDKO(C57A) cells (closed circles). The curves are the non-linear fits of data using a two-parameter EC₅₀ equation provided by GraFit (see Experimental procedures). EC₅₀ values of 17.5 ± 0.5 and 16.6 ± 0.8 μM were determined for cDKO and cDKO(C57A) cell lines respectively. Data are the mean of duplicate measurements. B. Groups of five mice were infected with either cDKO or cDKO(C57A) cells (1 × 10⁴ parasites). All mice were infected with tetracycline-treated parasites and dosed with doxycycline in their drinking water. One set of the cDKO and cDKO(C57A)-infected mice received daily intraperitoneal injections of spermidine (100 mg kg⁻¹). Data are presented in the form of a Kaplan–Meier survival plot. Symbols: cDKO, blue; cDKO plus spermidine, red; cDKO(C57A), green; cDKO(C57A) plus spermidine, yellow.