Table 1.
Metabolites from cell extracts showing more than 3-fold difference between species.
Table 2.
Metabolites taken up or exported into media during growth of T. vaginalis and T. foetus.
Table 3.
Hydroxy acid levels in cell extracts of T. vaginalis and T. foetus.
Table 4.
Hydroxy acid levels in spent media samples of T. vaginalis and T. foetus cultures.
Fig 1.
13C labelling of intermediates in central carbon metabolism in T. vaginalis and T. foetus.
T. vaginalis and T. foetus were grown at 37°C in MDM containing 100% D-[U-13C6] glucose as the only carbohydrate carbon source. Cell extracts were prepared after 2 h, 6 h and 20 h in culture and analysed by LC-MS. (A) Map of glycolysis and central carbon metabolism in T. vaginalis and T. foetus. Reactions found only in T. foetus are shown by dashed lines. T. vaginalis uses pyrophosphate (ppi) dependent enzymes for the steps indicated [32]. Metabolites detected in the 13C labelling experiment are boxed. (B) Map of pyruvate metabolism in the hydrogenosome in T. vaginalis. Dashed lines indicate the NADH dehydrogenase complex I. Metabolites detected in the 13C labelling experiment are boxed. (C) 13C labelling of metabolites in T. vaginalis and T. foetus. Graphs show the percentage of isotopologues with different numbers of 13C carbon atoms.
Fig 2.
Comparison of total levels of intermediates in central carbon metabolism from 13C labelled extracts.
Total metabolite levels represent the sum of the peak intensities of all isotopologues. Of metabolites detected in 13C-labelled extracts. Graphs show the log2 fold change in total peak intensities after 2 h, 6 h and 20 h growth. Results represent the mean of 3 independent cultures ± SD. Dashed line indicates the threshold for a definite difference between the species (3-fold or log2 1.6-fold change in metabolite level). Positive values represent a relative increase in T. vaginalis, negative values a relative increase. Glucose-6P, glucose-6-phosphate; Triose phosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate; 3-PG, 3-phoglycerate; PEP, phosphoenol pyruvate. * P ≤ 0.001 (Paired T-Test, assuming equal variance).
Fig 3.
Metabolomic analysis of methionine and cysteine metabolism in T. vaginalis and T. foetus.
T. vaginalis and T. foetus were grown for 20 h at 37°C in MDM which contains no added cysteine. Graphs show concentrations of metabolites (nmol/108 cells) calculated from peak intensity values using standard curves. There were no standards available for the mixed disulfide of cysteine and homocysteine (Cys:Hcy disulfide) and its concentration is represented by peak intensity/108 cells. Broken lines indicate the oxidation of sulfur amino acids to form disulfides. Enzymes found in the T. vaginalis genome are: 1, S-adenosylmethionine synthase; 2, S-adenosylmethionine dependent methyltransferase; 3, S-adenosylhomocysteine hydrolase; 4, methionine γ-lyase (MGL); 5, cysteine synthase (CS); 6, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. CS from T. vaginalis is a multi-functional enzyme with broad specificity, as demonstrated in later sections. * More than 3-fold difference between species. P ≤ 0.001 (Paired T-Test, assuming equal variance).
Fig 4.
13C labelling of intermediates in nucleotide and methionine metabolism in T. vaginalis and T. foetus.
T. vaginalis and T. foetus were grown at 37°C in MDM containing 100% D-[U-13C6] glucose as the only carbohydrate carbon source. Cell extracts were prepared after 2, 6 and 20 h in culture and analysed by LC-MS. (A) Map showing pathway from glucose to nucleotide and methionine metabolism in T. vaginalis and T. foetus. Reactions found only in T. foetus are shown by dashed lines. Formation of cystathionine from cysteine and homocysteine has not been confirmed. Metabolites detected in the 13C labelling experiment are boxed. (B) Map showing metabolites labelled with 13C via the glycolytic intermediate 3-phosphoglycerate. Metabolites detected in the 13C labelling experiment are boxed. (C) 13C labelling of metabolites in T. vaginalis and T. foetus. Graphs show the percentage of isotopologues with different numbers of 13C carbon atoms, error bars show standard deviation.
Fig 5.
Comparison of total levels of intermediates in nucleotide and methionine metabolism from extracts of 13C labelled cells.
Total metabolite levels represent the sum of the peak intensities of all isotopologues of metabolites detected in extracts of 13C labelled cells. Graphs show the log2 fold change in total peak intensities after 2, 6 and 20 h growth. Results represent the mean of 3 independent cultures ± SD. Dashed line indicates the threshold for a definite difference between the species (3-fold or log2 1.6-fold change in metabolite level). Positive values represent a relative increase in T. vaginalis, negative values a relative decrease. Ribose-5P, Ribose-5-phosphate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; MTA, 5-methylthioadenosine; OPS, O-phosphoserine; SMC, S-methylcysteine. * P ≤ 0.001 (Paired T-Test, assuming equal variance).
Fig 6.
Effect of growth conditions on methionine metabolism in T. vaginalis.
T. vaginalis and T. foetus were grown for 20 h at 37°C in MDM which contains no added cysteine (MDM), MDM with 10 mM cysteine (CYS) or MDM with 5 μM propargylglycine (PAG). Cell extracts were analysed by LC-MS and the ratios of metabolite peak intensities for different growth conditions were determined. The graph show the log2 of the ratios CYS/MDM and PAG/MDM ± SD. The dotted lines indicate a difference in peak intensity ≥ 3 (or log2 1.6).* P ≤ 0.001 (Paired T-Test, assuming equal variance).
Fig 7.
SMC biosynthesis by T. vaginalis CS.
Production of SMC from OPS and CH3S.Na by recombinant T. vaginalis CS was determined at 37°C. (A) A coupled assay for SMC synthase. The assay couples SMC production by CS to NADH oxidation using MGL and LDH. The rate of NADH oxidation and hence the rate of SMC production can be quantified by measuring the absorbance at 340 nm. (B) The effect of increasing amounts of CS in a coupled assay for SMC biosynthesis. The reaction contained 10 mM OPS, 3 mM CH3SNa and 0, 1 and 5 μg/ml CS. The graph shows the rate of NADH oxidation which is equivalent to the rate of SMC biosynthesis in the coupled assay. (C) Substrate saturation curve for CH3SNa. Reactions contained 10 mM OPS, 85 nM CS and 0.25–12 mM sodium CH3SNa. A plot of enzyme activity against CH3SNa concentration shows Michaelis-Menten kinetics.
Table 5.
Kinetic analysis of SMC synthase and CS synthase activities of T. vaginalis CS.
Fig 8.
Synthesis of OPS and SMC in T. vaginalis and T. foetus.
Cell extracts prepared after 2, 6 and 20 h growth at 37°C in media containing 100% D-[U-13C6] glucose were analysed by LC-MS. Graphs show the mean peak intensities of 3-carbon labelled isotopologues from 3 biological replicates (± SD). (A) 13C labelled SMC in cell extracts; (B) total SMC levels in media samples; (C) 13C labelled OPS in cell extracts; (D), total OPS levels in media samples. Complete, uninfected or complete media control; TvagSPT, spent media sample from T. vaginalis cultures; TfoeSPT, spent media from T. foetus cultures.