Mathematical modelling of polyamine metabolism in bloodstream-form trypanosoma brucei: An application to drug target identification

© 2013 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis article has been made available through the Brunel Open Access Publishing Fund.We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.The work was carried out under a PhD programme partly funded by Prof. Ray Welland, School of Computing Science, University of Glasgo

Pyrimidine biosynthesis is not an essential function for trypanosoma brucei bloodstream forms

<p>Background: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.</p> <p>Methodology/Principal Findings: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.</p> <p>Conclusions/Significance: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.</p&gt

Ethanolamine phosphoglycerol attachment to eEF1A is not essential for normal growth of Trypanosoma brucei

Eukaryotic elongation factor 1A (eEF1A) is the only protein modified by ethanolamine phosphoglycerol (EPG). In mammals and plants, EPG is attached to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote, Trypanosoma brucei, a single EPG moiety is attached to domain III. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but the role of this unique protein modification in cellular growth and eEF1A function has remained elusive. Here we report, for the first time in a eukaryotic cell, a model system to study potential roles of EPG. By down-regulation of EF1A expression and subsequent complementation of eEF1A function using conditionally expressed exogenous eEF1A (mutant) proteins, we show that eEF1A lacking EPG complements trypanosomes deficient in endogenous eEF1A, demonstrating that EPG attachment is not essential for normal growth of T. brucei in culture

A molecular mechanism for eflornithine resistance in African trypanosomes

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered

Characterization of RNA transcripts from the alpha tubulin gene cluster of Leptomonas seymouri.

A tandem cluster of alpha tubulin genes was identified in the trypanosomatid protozoan Leptomonas seymouri. One repeat unit and the first gene of the cluster, with its upstream flanking region, were cloned and analyzed for their transcriptional and coding capacities. The 12-15 copies per cell of the 4 kb repeat encode a stable 2 kb transcript, which contains a mini-exon at its 5' end and two closely spaced polyadenylation sites. Transcription of the alpha tubulin gene cluster in isolated nuclei was unidirectional. Intergenic and coding regions were transcribed at the same rate, and nascent intergenic and coding region transcripts were quantitatively linked. These results are consistent with the possibility that the primary transcripts are polycistronic, or that there is a single small intercistronic gap