Mapping the metabolism of five amino acids in bloodstream form Trypanosoma brucei using U-13C-labelled substrates and LC-MS

Abstract

The metabolism of the parasite Trypanosoma brucei has been the focus of numerous studies since the 1940s. Recently it was shown, using metabolomics coupled with heavy-atom isotope labelled glucose, that the metabolism of the bloodstream form parasite is more complex than previously thought. The present study also raised a number of questions regarding the origin of several metabolites, for example succinate, only a proportion of which derives from glucose. In order to answer some of these questions and explore the metabolism of bloodstream form T. brucei in more depth we followed the fate of five heavy labelled amino acids - glutamine, proline, methionine, cysteine and arginine - using an LC-MS based metabolomics approach. We found that some of these amino acids have roles beyond those previously thought and we have tentatively identified some unexpected metabolites which need to be confirmed and their function determined.

Keywords: amino acid metabolism; mass spectrometry; parasitic protozoa; trypanosomes.

Figure 1. Glutamine labelled experiments results

The bar charts show the measured proportions of the different number of ¹³C: grey represents fully unlabelled metabolite, blue 1 carbon ¹³C, brown 4 carbons ¹³C and orange 5 carbons ¹³C. The green dots on the molecules represent the theoretical individual ¹³C starting with a fully labelled precursor following this pathway.

Figure 2. Trypanothione biosynthesis pathway as seen using 50% U-¹³C glutamine, cysteine or methionine

The bar charts show the measured proportions of the different number of ¹³C (colour code is shown at the top of the figure). The green dots on the molecules represent the theoretical individual ¹³C coming from fully labelled glutamine, blue carbons come from fully labelled cysteine and red from fully labelled methionine.

Figure 3. Putative methylthio-d-ribose in the U-¹³C methionine experiment (A and B) and in Kim et al. [18] spend medium experiment (C and D)

Figure 4. Pyruvate (A and B) and alanine (C and D) as seen in the 50% U-¹³C glutamine experiment

Figure 5. S-Adenosylhomocysteine recycling pathway

The bar charts show the measured proportions of the different number of ¹³C (colour code is shown at the top of the figure). The blue dots on the molecules represent the theoretical individual ¹³C coming from cysteine and red from methionine.

Figure 6. Coenzyme A biosynthesis pathway

The bar charts show the measured proportions of the different number of ¹³C (colour code is shown at the top of the figure). The blue dots on the molecules represent the theoretical individual ¹³C coming from cysteine.

Figure 7. Peak of ornithine as seen in the medium (CMM) when arginine is fully labelled, with or without FBS but no cells

(Intensity of the peak shown as a function of retention time).

Figure 8. Growth of wild type and Δak cells

Figure 9. OPLS-DA scores plot of the metabolomics pH experiment

(A) OPLS-DA including all four groups: WT and Δak cells at pH 7.4 and 8.7. (B) OPLS-DA including only Δak cells at both pH. (C) OPLS-DA including only WT cells at both pH.