TUNEL assays in resveratrol-treated trophozoites.

<p><b>A)</b> TUNEL assays of untreated or IC₅₀ resveratrol-treated trophozoites using dUTP-FITC and visualized under the laser confocal microscope. Nuclei were stained with DAPI. Squares in merge images were magnified in the zoom panels. <b>B)</b> Flow cytometry analysis of dUTP-FITC-treated trophozoites. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls.</p

Effect of resveratrol in <i>in vitro</i> virulence of <i>E</i>. <i>histolytica</i> trophozoites and in <i>E</i>. <i>invadens</i> encystment.

<p><b>A)</b> Ingested erythrocytes after different incubation times with untreated or resveratrol -treated (110 μM_, 12 h and washing out) trophozoites. Values represent the mean ± standard error of three independent experiments. **p<0.01, ***p<0.001. <b>B)</b> Representative images of trophozoites with ingested erythrocytes at different incubation times. <b>C)</b> Destruction of MDCK cell monolayers incubated with untreated or resveratrol-treated (110 μM_, 12 h and washing out) trophozoites. Bottom: representative images of MDCK monolayers incubated with trophozoites and stained with methylene blue. C: MDCK cells that were not in contact with trophozoites. 0.4% ethanol-treated trophozoites were used as positive controls. Values represent the mean ± standard error of three independent experiments. *p<0.05,** p<0.01,*** p<0.001. <b>D)</b> Cysts formed were counted under the epifluorescence microscope and number of cysts in untreated cells was taken as 100%. Values represent the mean ± standard error of three independent experiments., ** p<0.01,***p<0.001.</p

Oxidative stress produced by resveratrol in trophozoites.

<p><b>A)</b> ROS production in trophozoites untreated or treated with IC₅₀ resveratrol. Quantification was performed by flow cytometry using the DCFDA reagent. <b>B)</b> Lipid peroxides in total lipids of trophozoites. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Values represent the mean ± standard error of three independent experiments. ***p<0.001.</p

Heterodimerization of the <i>Entamoeba histolytica</i> EhCPADH virulence complex through molecular dynamics and protein–protein docking

<p>EhCPADH is a protein complex involved in the virulence of <i>Entamoeba histolytica</i>, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with <i>in silico</i> results.</p

Immunochemistry of livers from hamsters inoculated with virulent trophozoites.

<p>Paraffin sections of livers from hamsters treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146287#pone.0146287.g010" target="_blank">Fig 10</a>. <b>A)</b> Non-challenged hamsters (healthy liver). <b>B)</b> Challenged hamsters treated with ethanol. <b>C)</b> Challenged hamsters treated with resveratrol that did no develop hepatic abscesses. <b>D)</b> Challenged hamsters treated with resveratrol that develop small abscesses. <b>E)</b> Challenged hamsters treated with metronidazole. <b>F)</b> Challenged hamsters treated with ethanol and developed only with the pre-immune serum. <b>G)</b> Parasitic burden quantified in 15 sections of livers from hamsters treated as above.</p

PS externalization produced by resveratrol in trophozoites.

<p><b>A)</b> Confocal microscopy of untreated or IC₅₀ resveratrol-treated trophozoites incubated with Annexin V-FITC and PI. Merge: fluorescence channels and phase contrast. <b>B)</b> Flow cytometry analysis of trophozoites incubated with Annexin V-FITC and PI. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Q1: Trophozoite scheme with nucleus stained (red) representing entrance of PI. Q2: Trophozoite scheme representing PI stained (nucleus) and Annexin V plasma membrane stained. Q3: Trophozoite scheme representing plasma membrane integrity. Q4: Trophozoite scheme representing Annexin V (loss of plasma membrane asymmetry) stained without nucleus stained.</p

Ultrastructure of resveratrol-treated trophozoites.

<p>TEM of untreated, 0.4% ethanol-, IC₅₀ resveratrol- and 0.5 mM H₂O₂-treated trophozoites.</p

Hepatic abscess formation by resveratrol-treated and untreated trophozoites.

<p><b>A)</b> Trophozoites were incubated with 110 μM resveratrol for 12 h, then they were changed to fresh TYI-S-33 medium without resveratrol and cell proliferation was measured at different times by spectrophotometry using the WST-1 reagent. -•- Untreated trophozoites. -○- Trophozoites pretreated with 110 μM resveratrol for 12 h and then incubated in fresh TYI-S-33 medium. -▲- Trophozoites cultured in the presence of 110 μM resveratrol during 72 h. <b>B)</b> Hamsters were intraportally inoculated with 3 x 10⁶ untreated or resveratrol-treated (110 μM, 12 h, drug removal) trophozoites. After seven days, livers were examined. <b>C)</b> Damage was evaluated as the weight of the abscesses formed divided by the weight of the whole liver, before the injured areas were removed. Values represent the mean ± standard error of liver damage in inoculated animals. n = 8. ***p<0.001.</p

Effect of resveratrol in <i>E</i>. <i>histolytica</i> trophozoites.

<p><b>A)</b> Trophozoites growth was measured at different times by spectrophotometry using the WST-1 reagent. <b>B)</b> Viability of trophozoites after 48 h incubation with different concentrations of resveratrol was evaluated under light microscope by Trypan blue exclusion. IC₅₀ was calculated as described in Materials and Methods. Values represent the mean ± standard error of three independent experiments.</p

Biochemical changes in resveratrol-treated trophozoites.

<p><b>A)</b> Cytosolic Ca²⁺ was measured in extracts of untreated or IC₅₀ resveratrol-treated trophozoites, using Fluo-4 AM. <b>B)</b> Calpain activity of trophozoites extracts incubated with Suc-Leu-Leu-Val-Tyr-AMC was measured by fluorescence spectroscopy. <b>C)</b> SOD activity of trophozoite extracts was monitored by spectrophotometry using the SOD kit. 0.4% ethanol- and 0.5 mM H₂O₂-treated trophozoites were used as controls. Values represent the mean ± standard error of three independent experiments. ***p<0.001.</p