Evolutionary Adaptations of Parasitic Flatworms to Different Oxygen Tensions

During the evolution of the Earth, the increase in the atmospheric concentration of oxygen gave rise to the development of organisms with aerobic metabolism, which utilized this molecule as the ultimate electron acceptor, whereas other organisms maintained an anaerobic metabolism. Platyhelminthes exhibit both aerobic and anaerobic metabolism depending on the availability of oxygen in their environment and/or due to differential oxygen tensions during certain stages of their life cycle. As these organisms do not have a circulatory system, gas exchange occurs by the passive diffusion through their body wall. Consequently, the flatworms developed several adaptations related to the oxygen gradient that is established between the aerobic tegument and the cellular parenchyma that is mostly anaerobic. Because of the aerobic metabolism, hydrogen peroxide (H2O2) is produced in abundance. Catalase usually scavenges H2O2 in mammals; however, this enzyme is absent in parasitic platyhelminths. Thus, the architecture of the antioxidant systems is different, depending primarily on the superoxide dismutase, glutathione peroxidase, and peroxiredoxin enzymes represented mainly in the tegument. Here, we discuss the adaptations that parasitic flatworms have developed to be able to transit from the different metabolic conditions to those they are exposed to during their life cycle

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (<i>Dugesia dorotocephala</i>)

<div><p>A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth <i>Dugesia dorotocephala</i> was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the <i>D</i>. <i>dorotocephala</i> extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a <i>D</i>. <i>dorotocephala</i> extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.</p></div

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (<i>Dugesia dorotocephala</i>)

<div><p>A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth <i>Dugesia dorotocephala</i> was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the <i>D</i>. <i>dorotocephala</i> extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a <i>D</i>. <i>dorotocephala</i> extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.</p></div

Effect of GSSG and protein concentration on the full progress curves of NADPH consumption with GSSG as substrate.

<p>The GSSG reductase activity was measured at 25°C and pH 7.8 as described in Materials and Methods. A) Effect of GSSG. The enzyme assays were carried out at the following micromolar concentrations of the disulfide: (○) 67; (□) 150; (●) 350; (■) 500; (▲) 700; (▼) 1000. An enzyme concentration of 15 nM was used. B) Effect of protein concentration. The following nanomolar concentrations of protein were used: (●) 1.9; (○) 2.9; (▼) 3.8; (Δ) 7.7; (■) 15.3. Inset at panels A and B shows the dependence of the lag time on either GSSG or protein, respectively.</p

Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

Curcuma is a traditional ingredient of some Eastern cuisines, and the spice is heralded for its antitumoral and antiparasitic properties. In this report, we examine the effect of the curcuminoides which include curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), as well as curcumin degradation products on thioredoxin glutathione reductase from Taenia crassiceps cysticerci Results revealed that both DMC and BDMC were inhibitors of TGR activity in the micromolar concentration range. By contrast, the inhibitory ability of curcumin was a time-dependent process. Kinetic and spectroscopical evidence suggests that an intermediary compound of curcumin oxidation, probably spiroepoxide, is responsible. Preincubation of curcumin in the presence of NADPH, but not glutathione disulfide (GSSG), resulted in the loss of its inhibitory ability, suggesting a reductive stabilizing effect. Similarly, preincubation of curcumin with sulfhydryl compounds fully protected the enzyme from inhibition. Degradation products were tested for their inhibitory potential, and 4-vinylguaiacol was the best inhibitor (IC50 = 12.9 μM), followed by feruloylmethane (IC50 = 122 μM), vanillin (IC50 = 127 μM), and ferulic aldehyde (IC50 = 180 μM). The acid derivatives ferulic acid (IC50 = 465 μM) and vanillic acid (IC50 = 657 μM) were poor inhibitors. On the other hand, results from docking analysis revealed a common binding site on the enzyme for all the compounds, albeit interacting with different amino acid residues. Dissociation constants obtained from the docking were in accord with the inhibitory efficiency of the curcumin degradation products

Correction: Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

[This corrects the article DOI: 10.1371/journal.pone.0220098.]

Effect of GSSG and protein concentration on the full progress curves of NADPH consumption with GSSG as substrate.

<p>The GSSG reductase activity was measured at 25°C and pH 7.8 as described in Materials and Methods. A) Effect of GSSG. The enzyme assays were carried out at the following micromolar concentrations of the disulfide: (○) 67; (□) 150; (●) 350; (■) 500; (▲) 700; (▼) 1000. An enzyme concentration of 15 nM was used. B) Effect of protein concentration. The following nanomolar concentrations of protein were used: (●) 1.9; (○) 2.9; (▼) 3.8; (Δ) 7.7; (■) 15.3. Inset at panels A and B shows the dependence of the lag time on either GSSG or protein, respectively.</p

Electrophoretic analysis of the disulfide reductase activities purified from <i>D</i>. <i>dorotocephala</i>.

<p>A) Protein band pattern of the purified sample. Lane 1: An aliquot from the last purification step was incubated for 10 min at 80°C in the presence of 1% SDS and 5 mM β-mercaptoethanol; then, it was loaded on a top of a 12% polyacrylamide gel and ran during 3 h. Lane 2: molecular weight markers. B) Densitometric analysis of the 65 kDa and 55 kDa protein bands. The relative intensity of the latter was estimated with the software IMAGEJ.</p

Summary of the purification procedure of the GSSG reductase activity from <i>Dugesia dorotocephala</i>.

<p>Summary of the purification procedure of the GSSG reductase activity from <i>Dugesia dorotocephala</i>.</p