Search

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.

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10(Δpepc) ), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10(Δpepc) had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using (13)C-U-D-glucose and (13)C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10(Δpepc) and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of (13)C,(15)N-U-glutamine was similar in both parasite lines, although the flux was lower in D10(Δpepc); it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.

Abstract Globally increasing levels of artificial light at night (ALAN) are associated with shifting rhythms of behaviour in many wild species. However, it is unclear whether changes in behavioural timing are paralleled by consistent shifts in the molecular clock and its associated physiological pathways. Inconsistent shifts between behavioural and molecular rhythms, and between different tissues and physiological systems, disrupt the circadian system, which coordinates all major body functions. We therefore compared behavioural, transcriptional and metabolomic responses of captive great tits (Parus major) to three ALAN intensities or to dark nights, recording activity and sampling brain, liver, spleen and blood at mid-day and midnight. ALAN advanced wake-up time, and this shift was paralleled by advanced expression of the clock gene BMAL1 in all tissues, suggesting close links between behaviour and clock gene expression across tissues. However, further analysis of gene expression and metabolites revealed that clock shifts were inconsistent across physiological systems. Untargeted metabolomic profiling showed that only 9.7% of the 755 analysed metabolites followed the behavioural shift. This high level of desynchronization indicates that ALAN disrupted the circadian system on a deep, easily overlooked level. Thus, circadian disruption could be a key mediator of health impacts of ALAN on wild animals.

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides.