Fig 1.
(A) Transmembrane domain predicted by Phobius [43] in TcGSa: TcCLB.508175.370; TcGSb: TcCLB.503405.10 and TcGS, the sequence used in this work; (B) β-grasp domain predicted by Pfam [44]; (C) Catalytic domain predicted by Pfam. The amino acid sequence of TcGS was aligned to orthologs from Tbb: Trypanosoma brucei brucei; Tbg: Trypanosoma brucei gambiense; Tvivax: Trypanosoma vivax; Lbra: Leishmania brasiliensis; Lmex: Leishmania mexicana; Linf: Leishmania infantum; Lpan: Leishmania panamensis; Sg: Strigomonas galati; Ca: Crithidia acanthocephala; Hm: Herpetomonas muscarum; Ad: Angomonas deanei; At: Arabidopsis thaliana; Oa: Oryza sativa; Hs: Homo sapiens; Mm: Mus musculus; Bt: Bos taurus; Gg: Gallus; St: Salmonella typhimurium; Sc: Saccharomyces cerevisiae; St: Salmonella typhimurium; and Tk: Thermococcus kodakarensis.
Fig 2.
Yeast functional complementation assay.
Saccharomyces cerevisiae SAH35 yeast with an endogenous glutamine synthetase gene controlled by the GAL1 gene promoter was transformed with an empty plasmid, a copy of S. cerevisiae GS gene or the T. cruzi gene (p416, p416-ScGLN1 and p416-TcGS, respectively) and plated at different dilutions (a and e: 104 yeasts; b and f: ≈103 yeasts; c and g: ≈100 yeasts d and h: ≈10 yeasts). The endogenous ScGLN1 gene is not transcribed in a defined medium with glutamate as a ScGLN1 non-repressible nitrogen source and with glucose as a carbon source. Furthermore, when glutamine is supplied, compensation of the glutamine biosynthesis pathway occurs; GS is not required in this case, and it is not essential for yeast clones (line 1—a to d). However, the empty vector transformed yeast do not grow in a medium without glutamine (line 1—e to h). The yeast recover the capacity to proliferate when they are transformed with the same gene (ScGLN1) or with GS from T. cruzi.
Fig 3.
Heterologous expression and purification of recombinant TcGS.
(A) The recombinant protein was analyzed by SDS-PAGE using 10% (v/v) polyacrylamide gels under reducing conditions and visualized by Coomassie Blue staining. M: molecular mass maker; S: Supernatant of lysed bacterial culture overexpressing TcGS; FT: Supernatant after flowing through the column; W1 to W4: Samples of column washes with buffers with crescent concentrations of imidazole (5 to 100 mM); E1 to E5: Elution fractions performed with a buffer containing 500 mM imidazole; PCE: Fraction after passing through Amicon Ultra Centrifugal filters with a 30,000-Da cut-off. (B) Western blot analysis was performed using an anti-His6 antibody raised against the recombinant enzyme elutions E3 to E5, as indicated by the box. (C) Size-exclusion chromatography (SEC) of the elutions. Four methodologies were applied to estimate the presence of TcGS. They are listed as follows in ascending order of specificity: a Bradford assay quantifying the total content of protein in the samples; a dot-blot identifying a TcGS signal in 6 SEC fractions (bottom); an ELISA assay quantifying the total amount of TcGS in the fractions; and a GS activity assay showing the functionality of TcGS in different oligomeric conformations. Error bars represent standard deviation (n = 3). Inset: Calibration of the SEC assay utilized to estimate the number of subunits of the sample.
Fig 4.
Effect of substrate, pH and temperature variations on TcGS activity.
(A) The coupled reactions and malachite green methods were applied to access the kinetic parameters Vmax and KM related to the three substrates of TcGS (Glutamate, ATP and NH3); data were adjusted to a Michaelis-Menten equation. (B) The pH of the media in the reaction catalyzed by TcGS was modified using different buffer systems. Enzymatic activity was determined in the presence of 1 mM glutamate, 1 mM ATP, 2 mM NH4Cl and 100 mM of reaction buffer as follows: MES NaOH (pH 5.0 to 6.5) (filled circles), imidazole HCl (pH 7.0 to 8.0) (open squares), and Tris-HCl (pH 8.5 to 9.0) (open triangles). The reaction was initiated by the addition of the enzyme, and the initial velocities were calculated as linear rates for TcGS-His6. (C) Enzymatic activity was determined by progressively increasing the reaction temperature (from 10 to 60°C). Inset: The activation energy values were estimated by an Arrhenius plot of the specific activity of TcGS. y-axis: log of GS activity according to tested temperature values; x-axis: (molar gas constant x temperature values)-1 x (temperature in Kelvin). (D) TcGS activity was measured in the presence of the three divalent ions (Mg2+, Mn2+ or Co2+), and the effect on activity caused by the replacement of the natural substrate of the enzyme (L-glutamate) by other amino acids was evaluated. Saturating concentrations were used for two GS substrates (NH3 and ATP) and a 1 mM concentration of each glutamate analog. (E) Effect of increasing the EDTA concentration on TcGS activity. (F) Effect of increasing Ca2+ concentration on TcGS activity under standard conditions. Inset: Dose-response curve of Ca2+; IC50 = 205.7 ± 2.8 μM. The values of the enzymatic parameters are available in Table 1. Statistical analysis were made using one-way ANOVA / Dunnet’a Multiple Comparison Test. *p<0.05, **p<0.01, ***p<0.001. Error bars represent standard deviation (n = 3).
Table 1.
Enzymatic characterization overview.
Fig 5.
Subcellular localization of GS in T. cruzi.
(A) All stages of T. cruzi life cycle (E–Epimastigote; MT–Metacyclic trypomastigote; A–Amastigote; IE–Intracellular epimastigote; and CDT–Trypomastigote) were harvested by centrifugation, immobilized on glass slides, incubated with anti-GS (green) and labeled with DAPI for DNA staining (blue) and MitoTracker Red Mito Sox for mitochondrial staining (red). Bars are 1 μm. N—nucleus, k–kinetoplast. (B) E were selectively permeabilized with increased digitonin concentrations (0–5 mg/ml), and the resulting supernatants (S) and pellets (P) were used for enzymatic assays. The enzymatic activities of pyruvate kinase (filled circles—cytosol marker), hexokinase (filled squares—glycosomal marker), citrate synthase (right-side up filled triangles—mitochondrial marker), and GS (upside-down filled triangles) were determined for all resulting fractions (S and P). The data correspond to the ratio between activities on S and P and were expressed as a percentage of the enzyme.
Fig 6.
Expression profile of GS in T. cruzi.
(A) Quantification of transcripts for the TcGS gene in the different stages of T. cruzi. The mRNA was quantified by qRT-PCR using primers for a fragment of the gene. The TcGAPDH gene was used as a normalizer (kept in-house). The expression ratio was calculated by the 2-ΔΔCt method, in which E stage expression was defined as 1. The cDNA of CHO-K1 cells was used as a negative control since this strain is used to obtain the intracellular forms of T. cruzi. The differences between the expression profiles were evaluated by a one-way ANOVA test (p <0.05). In (B), the specific activity of GS was evaluated in the different stages of T. cruzi. The measurement of enzymatic activity was performed as described previously for the parasite extracts. (C) shows a representative Western blot performed with protein extracts of the T. cruzi stages. The extracts were resolved on denaturing polyacrylamide gel (10% acrylamide), transferred to a nitrocellulose membrane and incubated with anti-GS antibody (Sigma- Aldrich, St. Louis, Missouri, USA). The membranes were incubated with a secondary anti-rabbit IgG (Sigma- Aldrich, St. Louis, Missouri, USA) antibody for chemiluminescent detection. In this case, the stripping process was performed by adding 0.2 M NaOH. After two washes with PBS, incubation was performed using the same process described above, but the primary antibody was changed to anti-GAPDH (Sigma- Aldrich, St. Louis, Missouri, USA). Lanes: E: Epimastigote; A: Amastigote; CDT: Trypomastigotes; IE: Intracellular epimastigotes; MT: Metacyclic trypomastigotes; CHO: non-infected CHO-K1 cells. (D) Densitometry analysis of Western blot bands (three independent experiments including that of panel C). Asterisks indicate statistical analysis by 1way ANOVA / Tukey’s Multiple Comparision Test for (A) and Dunnet’s Multiple Comparision Test for (B) and (D); *p<0.05, **p<0.01, ***p<0.001, error bars represent standard deviation (n = 3).
Fig 7.
(A) The extract of epimastigotes (filled circles) and the recombinant enzyme TcGS (filled squares) were both incubated with different MS concentrations for 30 min. Then, the specific activity was measured and the inhibition percentage was calculated. The values were adjusted to a dose-response curve (IC50 for cell-free extracts: 19.66 ± 0.37 μM, IC50 for TcGS: 14.70 ± 0.54 μM). The data refer to three independent experiments. (B) GS activity was measured with different MS concentrations for 30 min; at the same time, it was measured in different glutamate concentrations. The data were adjusted to a Michaelis-Menten equation. (C) The KM values for each MS concentration were compared. (D) The relationship between KM/Vmax per MS concentration revealed the inhibitory constant value (Ki = 4.12 ± 0.21). Statistical analysis were made using one-way ANOVA / Dunnet’a Multiple Comparision Test. *p<0.05, **p<0.01, ***p<0.001. Error bars represent standard deviation (n = 3).
Fig 8.
Effect of extracellular NH4+, MS and their combination on E proliferation.
(A) The effect of different concentrations of NH4+ on E replication was evaluated by culturing them in liver infusion tryptose (LIT) medium at 28°C supplemented with different concentrations of NH4OH (in all cases, the pH was adjusted to 7.4). Parasites were quantified daily as previously described [46]. Symbols: filled circles: 1 μM; filled squares: 5 μM; right-side up filled triangles: 10 μM; upside-down filled triangles: 50 μM; right-side up filled triangles: 100 μM; left filled triangles: 500 μM; filled diamonds: 1 mM; open diamonds: 5 mM; open circles: 10 mM; open squares: 50 mM; right-side up open triangles: 75 mM; right open triangles: 100 mM; asterisks: untreated (control); x symbol: 0.5 μM antimycin and 60 μM rotenone (100% growth inhibition control). Inset: dose-response curve (EC50 = 17.77 ± 0.99 mM). (B) The effect of different concentrations of MS and 5 mM NH4OH on E replication was evaluated by culturing them in LIT medium at 28°C. Parasites were quantified daily. Symbols: filled circles: 0.05 mM; filled squares: 0.1 mM; right-side up filled triangles: 0.5 mM; upside-down filled triangles: 1 mM; right filled triangles: 2.5 mM; asterisks: 5 mM; filled diamond: 10 mM; open diamonds: 5 mM MS without NH4OH; open circles: 10 mM MS without NH4OH; open squares: control (no treatment); right-side up open triangles: 5 mM NH4OH without MS; upside-down open triangles: 0.5 μM antimycin and 60 μM rotenone (100% growth inhibition control). Inset: dose-response curve for MS in the presence of NH4OH (EC50 = 438.4 ± 47.4 μM). (C) The combined effect of 1 mM MS and 5 mM NH4OH on epimastigote proliferation measured at the mid-exponential phase of untreated cultures (4th day of proliferation). Statistical analysis were made using one-way ANOVA / Dunnet’a Multiple Comparision Test. *p<0.05, **p<0.01, ***p<0.001. Error bars represent standard deviation (n = 3).
Fig 9.
Effect of MS on the intracellular cycle of T. cruzi.
(A) The effect of MS on CDT bursting was evaluated by treating the cells throughout the entire infection cycle (concentrations varied between 5 and 500 μM). CDT forms were counted in the culture media at day 5 post-infection. Inset: dose-response curve for the effect of MS treatment on CDT bursting (EC50 = 20.02 ± 0.91 μM). (B) Effect of MS on the different moments of intracellular infection. The mammalian host cells were infected under synchrony conditions in such a way that all infected cells were displaying the same intracellular stages at any moment, as previously described [15]. The infected cells were treated with 20 μM MS (dose corresponding to the EC50) for 3 h during the invasion, for 24 h immediately after invasion (in our system, this time period is when parasites remain inside the parasitophorous vacuole—PV), between 24 and 48 h (intracellular parasites are already in A form in the host-cell cytoplasm) or between 48 and 120 h post-infection (intracellular parasites are in IE form). The measured effect was CDT bursting, which was quantified by counting parasites in a Neubauer chamber. (C) Representative images of the PV of cells treated (or not, as a control) with MS. The assays were performed by labeling CHO-K1 infected cells with LysoNIR. After 3 h of infection the cultures were submitted to a 24-h treatment (or not) with 83 μM MS (corresponding to EC80) and washed. Nuclear DNA (N) was stained with Hoechst 33342. Yellow arrows: example of unlabeled parasites; green arrows: example of labeled parasites. Bottom: the total number of parasites and number of parasites labeled with LysoNIR 200 cells were counted and used to calculate the percentage of parasites within the PV at different times post-infection. Statistical analysis were made using one-way ANOVA / Dunnet’a Multiple Comparision Test. *p<0.05, **p<0.01, ***p<0.001, Error bars represent standard deviation (n = 3).
Fig 10.
Schematic proposal for the role of TcGS in the progression of intracellular infection by T. cruzi.
CDT forms invade the mammalian host cells by forming a parasitophorous vacuole, which involves the recruitment of lysosomes to fuse the vacuole containing the parasite. The lysosome fusion event triggers the acidification of the vacuole and promotes amastigogenesis [7,9,74]. It is well established that A are amino acid consumers; thus, ammonium production (which should be managed by the metabolic network involving glutamate dehydrogenases, aspartate aminotransferases and TcGS) is expected (Panel A). We propose that if TcGS is inhibited, then the production of ammonium overloads the transamination system, and an impairment of PV evasion occurs. Furthermore, if GS is inhibited after PV evasion and during A proliferation, infection is also impaired (Panel B).