Metabolomic profiling and stable isotope labelling of Trichomonas vaginalis and Tritrichomonas foetus reveal major differences in amino acid metabolism including the production of 2-hydroxyisocaproic acid, cystathionine and S-methylcysteine

Abstract

Trichomonas vaginalis and Tritrichomonas foetus are pathogens that parasitise, respectively, human and bovine urogenital tracts causing disease. Using LC-MS, reference metabolomic profiles were obtained for both species and stable isotope labelling with D-[U-13C6] glucose was used to analyse central carbon metabolism. This facilitated a comparison of the metabolic pathways of T. vaginalis and T. foetus, extending earlier targeted biochemical studies. 43 metabolites, whose identities were confirmed by comparison of their retention times with authentic standards, occurred at more than 3-fold difference in peak intensity between T. vaginalis and T. foetus. 18 metabolites that were removed from or released into the medium during growth also showed more than 3-fold difference between the species. Major differences were observed in cysteine and methionine metabolism in which homocysteine, produced as a bi-product of trans-methylation, is catabolised by methionine γ-lyase in T. vaginalis but converted to cystathionine in T. foetus. Both species synthesise methylthioadenosine by an unusual mechanism, but it is not used as a substrate for methionine recycling. T. vaginalis also produces and exports high levels of S-methylcysteine, whereas only negligible levels were found in T. foetus which maintains significantly higher intracellular levels of cysteine. 13C-labeling confirmed that both cysteine and S-methylcysteine are synthesised by T. vaginalis; S-methylcysteine can be generated by recombinant T. vaginalis cysteine synthase using phosphoserine and methanethiol. T. foetus contained higher levels of ornithine and citrulline than T. vaginalis and exported increased levels of putrescine, suggesting greater flux through the arginine dihydrolase pathway. T. vaginalis produced and exported hydroxy acid derivatives of certain amino acids, particularly 2-hydroxyisocaproic acid derived from leucine, whereas negligible levels of these metabolites occurred in T. foetus.

Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

“…The 2-hydroxy-acid dehydrogenase(s) responsible for the reduction of the 2-keto acids to form lactates has not been identified. In lactic acid bacteria (28) and protozoa, such as Trichomonas vaginalis (29), these reactions are catalyzed by the canonical NAD-dependent hydroxy acid dehydrogenase lactate dehydrogenase (LDH), an enzyme with broad substrate specificity (30). Leishmania species do not encode LDH, but genes for malate dehydrogenases (MDH) and a putative D-lactate dehydrogenase (D-LDH) are present.…”

Metabolomics in infectious diseases and drug discovery

“…107,108 A comparison between metabolomic networks of closely related pathogens can also be achieved by stable isotope labelling to understand the functions of enzymes. 109,110 The technique is also applied to investigate the effect of drug candidates and drug combinations for clinical treatment. Cobbold and McConville summarized a mass spectrometrybased protocol for detecting the effects of antimalarial drugs on Plasmodium falciparum, and through its coupling with stable isotope tracing, the method can provide a broader understanding of overall metabolomic perturbations.…”

Anti-trichomonad activities of different compounds from foods, marine products, and medicinal plants: a review

“…Nozaki, Ali [130] reported that the metabolism of sulfur amino acids in T. vaginalis probably influences their virulence and defense against stress. Based on an extensive bioanalytical study, Westrop, Wang [131] showed that there are major differences in cysteine and methionine synthesis and metabolism in T. vaginalis and T. foetus, that both trichomonads synthesize methylthioadenosine by an unusual mechanism, that T. foetus has higher levels of ornithine and citrulline than T. vaginalis and released high levels of the polyamine putrescine, and that T. vaginalis but not T. foetus exported 2hydroxyisocaproic acid derived from leucine. Because these biochemical events and associated enzymes probably contribute to the viability of the protozoa, inhibition of the active sites of the enzymes might transform pathogenic to nonpathogenic trichomonads.…”

“…vaginalis probably influences their virulence and defense against stress. Based on an extensive bioanalytical study, Westrop, Wang [ 131 ] showed that there are major differences in cysteine and methionine synthesis and metabolism in T . vaginalis and T .…”