A genome-scale metabolic model of parasitic whipworm

Abstract

Genome-scale metabolic models are widely used to enhance our understanding of metabolic features of organisms, host-pathogen interactions and to identify therapeutics for diseases. Here we present iTMU798, the genome-scale metabolic model of the mouse whipworm Trichuris muris. The model demonstrates the metabolic features of T. muris and allows the prediction of metabolic steps essential for its survival. Specifically, that Thioredoxin Reductase (TrxR) enzyme is essential, a prediction we validate in vitro with the drug auranofin. Furthermore, our observation that the T. muris genome lacks gsr-1 encoding Glutathione Reductase (GR) but has GR activity that can be inhibited by auranofin indicates a mechanism for the reduction of glutathione by the TrxR enzyme in T. muris. In addition, iTMU798 predicts seven essential amino acids that cannot be synthesised by T. muris, a prediction we validate for the amino acid tryptophan. Overall, iTMU798 is as a powerful tool to study not only the T. muris metabolism but also other Trichuris spp. in understanding host parasite interactions and the rationale design of new intervention strategies.

Fig. 1. GSMM reconstruction for T. muris.

a Reconstruction of a draft metabolic network for T. muris and addition of extra reactions in KEGG and Rhea based on T. muris orthologs with Trichuris spp. b Improving the draft model by organising its biomass reactions, subsystems, metabolite information and annotations. c Identification of blocked reactions by FVA and filling identified gaps computationally and manually. d Allocation of reactions in iTMU798. (Created with BioRender.com).

Fig. 2. iTMU798 comparison with C. elegans, B. malayi GSMMs and the experimental data.

The number of (a) unique reactions and (b) unique metabolites in iTMU798, iCEL1314, Worm1 & iDC625 and their overlaps. c The number of essential genes in iTMU798 and their comparison using their orthologs in C. elegans to iCEL1314, Worm1 and the OGEE database 28. d The comparison of unique metabolites in iTMU798 with the detected metabolites in two individual studies^,.

Fig. 3. The prediction of essential enzymes and allocation of their associated reactions in subsystems in iTMU798.

a The number of reactions associated with essential genes (black dots) and the number of reactions of subsystems found for associated reactions (red bars). b Enzymes that are responsible for the biosynthesis of selenocysteine and its incorporation to selenoprotein structure. c The role of thioredoxin reductase (TrxR) (b, c created with BioRender.com).

Fig. 4. Inhibition of TrxR and GR activities of T. muris.

a T. muris TrxR activity is not affected by DMSO (1%) but its activity is significantly reduced by auranofin at both 10 µM and 1 µM (n = 3 biologically independent samples). Measured using a commercial TrxR activity kit. b Even though T. muris does not have glutathione reductase, it has a GR activity, and it is significantly reduced by auranofin at both 10 µM and 1 µM (n = 3 biologically independent samples). Statistical analysis was carried out using a two-sided, unpaired one-way ANOVA with Bonferroni’s multiple comparison tests. Data are presented as mean ± S.D. and P < 0.05 is considered significant. Source Data and statistical details are provided as a Source Data file.

Fig. 5. Validation of iTMU798: Auranofin treatment of T. muris in vitro affects worm survival and absence of tryptophan reduces worm fitness.

a T. muris larvae were treated with auranofin at 1 µM, 0.1 µM and 0.01 µM and 1% DMSO for 30 h in vitro (three biological replicates with a total of ~150 L1 worms). Data are presented as mean ± S.D. in percentile. Additional intermediate doses of auranofin on L1 worms with additional time points are provided with the Source Data file. b Adult worms were treated with auranofin at 10 µM, 1 µM, and 0.1 µM and 1% DMSO for 46 h in vitro (three biological replicates with a total of ~24 adult male and female worms). Data are presented as mean ± S.D. in percentile. c Eggs that are released from adult worms to the petri dish were counted following the auranofin treatment for 46 h (at least two biological replicates with a total of 8–12 female worms). d Adult worms following auranofin treatment were also counted based on their motility: moving, limited moving, not moving. e The number of eggs released from adult females following the incubation for 48 h in media with all amino acids (Control), media without amino acids (No AA) or media lacking amino acids supplemented with 0.2% (w/v) casamino acids (CA) (four biological replicates with a total of 7–13 female worms). Statistical analysis was carried out using a two-sided, unpaired Welch ANOVA with Dunnett’s T3 multiple comparison (c) and one-way ANOVA with Bonferroni’s multiple comparison tests (e). Data are presented as mean ± S.D. (c, e) and P < 0.05 is considered significant. Source Data and statistical details are provided as a Source Data file.