Figure 1.
Distribution of total metabolites identified in Trypanosoma cruzi epimastigote samples.
The pie chart depicts the percentages of putatively identified metabolites from each of the metabolite classes. A total of 1,069 metabolites were analysed.
Figure 2.
Putatively identified metabolites mapped onto the KEGG metabolic pathways.
Coloured nodes (putatively identified metabolites) demonstrate coverage of diverse metabolic pathways (grey lines indicate annotated T. cruzi pathways). Node colour represents metabolite abundance in Bzn-treated T. cruzi relative to untreated controls on a continuous colour scale: blue: 0.5, yellow: 1 (no change), red: 2-fold increase. Node size indicates P-value from unpaired Welch's t-test: large: p<0.01, medium: p<0.05, small: P≥0.05. The large blue spot in the lower-right corner represents trypanothione disulphide (relative abundance = 0.7, P-value<0.01). Image generated with iPath2.0 [66].
Figure 3.
Metabolites displaying significant differences between control and 20 µM Bzn treated T. cruzi samples.
Metabolites displaying significant differences between control samples (cBc) and 20 µM Bzn treated samples (cBt) with p<0.05 (unpaired T-test) and fold change >1.4. Bzn derived metabolites were not included.
Table 1.
Bzn in vivo derived metabolites arising after 20 µM Bzn treatment of T. cruzi epimastigotes.
Table 2.
Bzn in vivo derived metabolites arising after 50 µM Bzn treatment of T. cruzi epimastigotes.
Figure 4.
A number of Bzn derived molecules detected after treatment of T. cruzi epimastigotes with 20 µM and/or 50 µM Bzn are represented. A putative pathway for their in vivo formation is shown; double arrows indicate the existence of possible intermediates such as nitroso or nitrenium derivatives. The observed m/z values corrected for proton gain or loss (observed m/z ± 1.007276) are included. Cys, G, Ov, γGC, T and Gsp refer to cysteine, glutathione, ovothiol A, gamma-glutamylcysteine, trypanothione and glutathionylspermidine respectively. All thiols are represented with their functional -S- groups separately. Bold numbers in parenthesis are the metabolite reference numbers (Tables 1 and 2).
Figure 5.
Identification of Bzn-glutathione adducts.
A. Upper plot is a chromatogram obtained from a sample of Bzn treated parasites corresponding to m/z 536.1922 within a 3 ppm mass range (single-charged ion). Middle and bottom plots are magnified mass spectra in the regions of interest within RT 18.5–19. B. Chromatograms and m/z plots corresponding to m/z 268.5997 (di-charged ion) in a sample of Bzn treated parasites. In both A and B each ion is detected in two different but closely eluting chromatographic peaks with retention times centred at 17.8 and 18.6 minutes. Ionization charge states of the molecules are confirmed by the m/z difference for the isotopic 13C peaks, which are expected to be 1.0034 for mono charged ions and 0.5017 for di-charged ions (bottom plots). 34S isotopic peaks are also observed for both ions which are expected at 1.9959 m/z difference for mono-charged ions and 0.9979 for di-charged ions. C. MSMS fragmentation spectrum for precursor ion m/z 536.19. The m/zc = m/z−1.007276 (proton mass). The proposed structures for the precursor ion (Bzn-glutathione adduct) and for some of the most intense fragments are shown. Exact theoretical mass and formula are included together with each fragment structure. A 4-C adduct is depicted though a 5-C adduct could be represented.