Fig 1.
Typical 600 MHz 1H-NMR spectrum of Schistosoma extract obtained in H2O.
1: Betaine; 2: Alanine; 3: Threonine; 4: myo-Inositol; 5: sn-Glycero-3-phosphocholine (3GPC); 6: Glutamine; 7: Proline; 8: Valine; 9: Isoleucine; 10: AMP; 11: UDP-N_Acetylglucosamine; 12: NAD+; 13: Tyrosine; 14: Histidine; 15: Tryptophan; 16: Adenosine; 17: Niacinamide; 18: Choline; 19: Methanol; 20: Lactate; 21: Leucine; 22: Gluthatione; 23: Glutamate; 24: Glucose; 25: Glucose-1-phosphate; 26: Glucose-6-phosphate; 27: Glycine; 28: Aspartate; 29: Asparagine; 30: O-Phosphocholine; 31: Succinate; 32: Acetate; 33: ADP+ATP; 34: Lysine; 35: Methionine; 36: Serine; 37: Phenylalanine.
Table 1.
Metabolites identified in S. mansoni worm extracts and classified according to their biological role.
Fig 2.
OPLSY score and loading plots of PHX and GA treatments.
(a,b) PHX treatment (N: 68; A: 2+5+0; R2X: 0.717; R2Y: 0.733; Q2: 0.439; CV Anova: 1.3E-05); (c,d) GA treatment (N:57; A: 2+3+0; R2X: 0.757; R2Y: 0.728; Q2: 0.619; CV Anova: 3.5E-03). Boxes and circles represent samples collected after 6 h and 24 h respectively. Experiments were repeated 2–5 times, with three technical replicates each. GA3-GPC: 3-glycerophosphocholine; Glucose-6: glucose-6-phosphate; Glucose-1: glucose-1-phosphate; GSH: glutathione; PC: phosphocholine; Phenilalan: Phenylalanine; UDP-N-Ac-g: UDP-N-acetylglucosamine.
Fig 3.
OPLS-DA score plots of PHX (10 μM) and GA (1μM) treated-samples.
(a) 6 h treatment; N: 33; A: 2+4+0; R2X: 0.773; R2Y: 0.913; Q2: 0.857; CV Anova: 4.9E-08 and (b) 24 h treatment; N: 27; A: 2+3+0; R2X: 0.725; R2Y: 0.934; Q2: 0.857; CV Anova: 6.3E-10. Red and blue circles are samples treated with PHX and GA respectively and green circles are vehicle-treated samples (controls).
Fig 4.
SUS-plots of PHX and GA treatments.
(a) Samples were treated with the maximum concentration of PHX and GA, respectively 10 μM and 1 μM, for (a) 6h and (b) 24h. Metabolite concentrations showing VIP>1 altered by GA, PHX, or both compounds are showed in blue, red, and grey. Concentrations of choline and 3-glycerophosphocholine (marked in yellow) show opposite behavior in GA and PHX treatment. Abbreviations are the same of Fig 2; Glu tot: concentration sum of glucose, glucose-1-phosphate and glucose-6-phosphate.
Fig 5.
Summary of metabolites altered in all treatment conditions.
a) Metabolites modulated by both time of exposure and drug concentration upon treatment perturbed (time x concentration); b) metabolites whose concentration show opposite behavior in PHX- and GA-treated samples. Abbreviations are the same of Fig 2.
Fig 6.
Heatmap and biclustering of treatment conditions and modulated metabolites.
Data are represented as log2 fold-changes using time-specific vehicle as reference.
Fig 7.
Neighbor-extended networks of metabolites impacted by PHX and GA treatments.
Green and red dots represent respectively an increase or a decrease of the metabolite level in compound-treated samples. Gray nodes represent “neighbor” metabolites.
Table 2.
Summary of metabolic pathways enriched in neighbor-extended networks.
Fig 8.
A scheme of putative connections of some metabolites impacted by PHX treatment and enriched PHX-associated metabolic pathways.
Metabolites modulated by PHX are in bold and metabolites in close proximity, as inferred by extended pathway analysis, are in gray. This scheme is based on the nowadays knowledge of metabolic pathways of animals obtained from KEGG pathway (https://www.genome.jp/kegg/pathway.html), and/or MetaCyc (https://metacyc.org); accordingly to UniProt database). All S. mansoni enzymes are inferred by homology, with the exception of cholinesterase (f.) and choline oxidase/aldehyde dehydrogenase (g. and/or h.) activity that were experimentally demonstrated to be present in the parasite [35,36]. In addition, a glycerol/glutamine metabolic axis (n.) was experimentally demonstrated for S. mansoni [37]. a. Smp_054800, Smp_162910; b. Smp_025160, Smp_031190, Smp_166500, Smp_166530, Smp_171820; c. Smp_132570; d. Smp_124730; e. Smp_146910; g. Smp_094500, Smp_212180; h. methyltransferases; i. betaine-homocysteine S-methyltransferase; j. dimethyl-glycine dehydrogenase; k. sarcosine dehydrogenase; l. Smp_144570, Smp_179710; m. alanine-glyoxylate aminotransferase; n. glycerol kinase. 2-oxoacid refers to pyruvate or 2-oxoglutarate which react with glycine to give alanine or glutammate, respectively. * Highlights metabolites of the Kennedy pathway. Single-headed or double-headed arrows stand for alleged one-way or both-way reactions. Dotted lines indicate reactions inferred by analogy but not annotated in KEGG, while continuous lines indicate reactions with enzymes annotated in KEGG.