Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi

Abstract

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)₂) that is the principal intracellular thiol-redox metabolite in trypanosomatids.

Methods and results: We utilized genetic overexpression (TryS^hi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryS^hi (vs. control) parasites. In comparison to controls, the TryS^hi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryS^hi exhibited 36-71% higher resistance to H₂O₂ (50-1000 μM) and heavy metal (1-500 μM) toxicity. Treatment with TryS inhibitors (5-30 μM) abolished the proliferation and survival advantages against H₂O₂ pressure in a dose-dependent manner in both TryS^hi and control parasites. Further, epimastigote and trypomastigote forms of TryS^hi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment.

Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.

Keywords: Anti-parasite drugs; Chagas disease; Paullones; Small molecule inhibitors; Trypanosoma cruzi; Trypanothione synthetase.

Figure 1:. Stable integration of pTREX.TryS expression vector in T. cruzi.

(A) Scheme of pTREX.TryS and pTREX expression vectors integration into the ribosomal locus of T. cruzi genome. Full-length TryS coding sequence (1960 bp) was amplified by using the Tc SylvioX10/c4 DNA as template in a PCR reaction, and cloned downstream of HX1 intergenic region (HX1) at EcoRI/HindIII sites in pTREX plasmid. (B&C) Sylvio X10/c4 epimastigotes were electroporated with pTREX or pTREX.TryS plasmids and transfectants were selected in presence of G418. Representative PCR amplification of neomycin resistance gene (Neo, B) and T7 – ribosomal locus (C) in pTREX and pTREX.TryS (TryS^hi) transfectants are shown. Note that no amplification of pTREX-derived sequences was observed in wild-type (WT) parasites.

Figure 2:. TryS enzyme is over-expressed in pTREX.TryS-transfected T. cruzi.

The pTREX and pTREX.TryS (TryS^hi) transfectants were cloned as described in Materials and Methods. (A&B) Representative Western blot images are shown for the levels of TcTryS and TcGAPDH in clonal populations of epimastigote (A) and trypomastigotes (B) forms. Detection of GAPDH (housekeeping protein) was used as loading control. Ponceau staining of the gel is also shown as an evidence for equal loading of epimastigote lysates. (C) Shown are representative bright field (a&c) and fluorescence (b&d) images of pTREX-transfected (a&b) and pTREX.TryS-transfected (c&d) epimastigotes. For immunofluorescence, parasites were stained with anti-TryS antisera and Alexa488-conjugated secondary antibody (green) together with DAPI (blue, DNA marker).

Figure 3:. Recombinant TryS is produced as an active enzyme in epimastigote and trypomastigote forms of T. cruzi.

Clonal epimastigote (Epi) and trypomastigote (Tryp) forms of pTREX- and pTREX.TryS-transfectants were expanded as described in Materials and Methods. Bar graphs present the (A) specific enzymatic activity (SEA) of TryS, (B) total trypanothione (T(SH)₂) content by an enzymatic recycling method, and (C) content of total GSH. The data in bar graphs are representative of ≥ 2 independent experiments (three determinations per sample per experiment) and plotted as mean value ± SEM (* and *** correspond to p < 0.05 and p < 0.001, respectively, ns stands for non significant).

Figure 4:. Effect of TryS overexpression on parasite growth.

(A) The growth of synchronized epimastigote cultures of pTREX and pTREX.TryS (TryS^hi) transfectants was monitored by cell counting under a light microscope. Data are representative of three independent experiments (three determinations per sample per experiment), and plotted as mean value ± SEM. (B) The doubling time of TryS^hi and control epimastigotes was calculated in GraphPad Prims 5. Rate constant k fitted for an exponential growth curve differed significantly between the two datasets (p < 0.05). (C) pTREX and TryS^hi clonal epimastigote populations were pulse labeled with CFSE and examined 3 days later by flow cytometry. The decline in CFSE fluorescence intensity corresponds to cell division. (D) A fixable viability dye was used to gate CFSE⁺ viable cells. For both pTREX and TryS^hi populations, 99% of the analyzed parasites were viable.

Figure 5:. Effect of TryS inhibitors on T. cruzi replication and viability.

Clonal cultures of TryS^hi and control epimastigote and trypomastigote forms of T. cruzi were incubated in presence of varying concentrations of TryS inhibitors (0, 5, 10, and 30 µM of KuOrb39 or KuOrb54). (A) The percentage (%) of growth inhibition of epimastigotes at 24 h post-incubation was determined by counting live, motile parasites by light microscopy. (B) Live motile trypomastigotes were quantitated by light microscopy, and percentage (%) of cell death calculated at 6 h post-incubation. Data are representative of two independent experiments (three determinations per sample per experiment), and plotted as mean value ± SEM (* p < 0.05).

Figure 6:. Role of TryS in T. cruzi resistance to oxidative stress.

T. cruzi transfectants, pTREX and pTREX.TryS (TryS^hi), were cloned and expanded as described in Materials and Methods. Cloned epimastigote (A-C) and trypomastigote (D-F) populations were incubated for 48 h and 6 h respectively, in presence of 0–1000 µM H₂O₂ and 0–30 µM of TryS inhibitors KuOrb39 or KuOrb54. (A&D) IC₅₀ concentration of H₂O₂ that resulted in 50% inhibition of TryS^hi (vs. control) parasites. (B&E) Parasite growth or survival inhibition curves fitted after incubation with increasing H₂O₂ concentrations. (C&F) Percentage (%) inhibition of growth or survival of TryS^hi (vs. control parasites) in presence of H₂O₂ at its ~IC₅₀ value and varying concentrations of TryS antagonists. For all experiments, viable, motile parasites were visually quantitated by light microscopy. All experiments were conducted at least twice (three determinations per sample per experiment), and data are plotted as mean value ± SEM (* p < 0.05).

Figure 7:. TryS inhibition is detrimental to parasite resistance to nifurtimox.

Clonal cultures of TryS^hi and control epimastigotes (A&B) and trypomastigotes (C-D) of T. cruzi were incubated for 48 h or 6 h respectively, with 0–400 µM nifurtimox (NFX) in presence or absence of KuOrb54 (10 µM or 30 µM). (A&C) IC₅₀ concentration of NFX that resulted in 50% inhibition of TryS^hi (vs. control) parasite forms. (B&D) Parasite growth or survival inhibition curves fitted after incubation with increasing concentrations of NFX. (E) Percentage (%) inhibition of trypomastigotes’ survival in presence of 2.5 µM NFX (IC₅₀ value) and KuOrb 54 (10 and 30 µM). All experiments were conducted twice (three determinations per sample per experiment), and data are plotted as mean value ± SEM (* p < 0.05).

Figure 8:. TryS inhibitors increase T. cruzi susceptibility to benznidazole.

Clonal cultures of pTREX and TryS^hi epimastigote (A-B) and trypomastigote (C-D) forms of T. cruzi were incubated for 48 h and 6 h respectively, with benznidazole (BZ, 0–500 µM) in presence or absence of KuOrb54 (10 µM and 30 µM). (A&C) IC₅₀ concentration of BZ that resulted in 50% inhibition of TryS^hi (vs. control) epimastigote and trypomastigote forms of T. cruzi. (B&D) Parasite growth or survival inhibition curves fitted after incubation with increasing concentrations of benznidazole. (E) Percentage (%) inhibition of TryS^hi (vs. control) trypomastigotes’ survival in presence of 60 µM BZ (IC₅₀ value) and TryS antagonist KuOrb 54 (10 and 30 µM). All experiments were conducted twice (three determinations per sample per experiment), and data are plotted as mean value ± SEM (* p < 0.05).