Fig 1.
Characterization of parasite-encoded COP9 signalosome.
(A) Multiple sequence alignment of the COP9 signalosome subunit 5 (CSN5) metalloprotease site from medically important protozoan parasites. Identical (green), conserved (blue), semi-conserved (pink) and non-conserved residues (red). (B) Experimental approach for identifying CSN2 interacting proteins from E. histolytica cells by co-immunoprecipitation (Co-IP) followed by mass spectrometry. (C) Scatter plot and Pearson’s correlation analysis of log2 ratios label-free quantification intensities for proteins identified by mass spectrometry in anti-Flag co-immunoprecipitations from E. histolytica cells expressing Flag-CSN2 compared to the vector control. CSN2 and CSN5 subunits (red), other COP9 subunits 1, 3 and 6 (blue), and non-COP9 subunits (grey). (D) Diagram of experimental procedure for validating CSN5 and CSN2 interaction. (E and F) Reciprocal co-immunoprecipitation with anti-Flag (E) and anti-E. histolytica CSN5 (F) antibodies show interaction between CSN5 and CSN2.
Fig 2.
COP9 signalosome activity is essential for E. histolytica protein degradation.
(A) Cell proliferation assay showing the effect of CSN5 knockdown on E. histolytica viability. (B) Immunoblot with anti-E. histolytica Nedd8 antibody demonstrating accumulation of neddylated proteins in CSN5 knockdown cells compared to the vector control. Actin was used as a loading control. (C) Schematic of cullin1 deneddylation by the COP9 signalosome. Accumulation of neddylated cullin1 in CSN5 knockdown cells compared to vector control (24 h). Immunoblot of amebic lysate with anti-E histolyica cullin1 antibody. Immunoprecipitated cullin1 was analyzed by immunoblotting using anti-E. histolytica Nedd8 antibody. (D) Dominant negative effect of the catalytically inactive CSN5 mutant. Cell proliferation assay evaluating the effect of a single amino acid mutation (D147N) within the CSN5 metalloprotease site on parasite viability. (E) Immunoblot analysis with anti-E. histolytica cullin1 and Nedd8 antibodies on CSN5 WT and mutant (D147N) overexpressing cells showing levels of neddylated cullin1. (F and G) CSN5 dominant negative mutant expression impairs protein degradation. Confocal images and quantification of WT and CSN5 mutant expressing live cells accumulating GFP. Mean fluorescence intensity, MFI. (H) Fluorometric assay of GFP accumulation. CSN5 mutant results in GFP accumulation. Data represent mean ± SD of quintuples from one experiment and are representative of three independent experiments. *P < .05, ** P < 0.01, *** P < 0.001, two-tailed t test.
Fig 3.
Pharmacological inhibition of COP9 by ZnDTC phenocopy CSN5 genetic disruption.
(A) Illustration of zinc-ditiocarb (ZnDTC) formation from disulfiram and zinc. Dose response curve showing amebicidal effect of ZnDTC and metronidazole after 48 hours of incubation at indicated doses. EC50 for ZnDTC (11.21 ± 6.39 nM) and metronidazole (10.26 ± 6.06 μM) was calculated from nonlinear robust regression fit of the dose response curves. (B) Target validation using viability assay of vector control, CSN5 knockdown and CSN5 overexpression parasites incubated with increasing doses of ZnDTC. CSN5 overexpression reduces sensitivity to ZnDTC while CSN5 knockdown increases sensitivity to ZnDTC. (C) Immunoblot analysis for neddylated cullin1 with anti-E. histolytica cullin1 and Nedd8 antibodies on ZnDTC treated cells. (D) Quantification of live cells accumulating GFP, in ZnDTC treated cells with representative confocal micrographs. (E) Fluorometric assay of GFP degradation. ZnDTC inhibits GFP degradation in a dose dependent manner. Data represent mean ± SD of quintuples from one experiment and are representative of three independent experiments. ***P < .001, two-tailed t test.
Fig 4.
ZnDTC has potent activity against E. histolytica in a preclinical animal model of amebic colitis.
(A) Representative live bioluminescent images of mice infected with luciferase-expressing E. histolytica during the treatment period. (B) Infection rate measured by ameba culture of cecal content. (C) Representative H&E staining and immunohistochemical analysis of the cecum of infected mice after 5 days of treatment. Specific anti–E. histolytica macrophage migration inhibitory factor antibody was used for immunohistochemical staining of trophozoites (brown). Numerous parasites in the infected control, absent in the ZnDTC treated mice. Scale bar, 50μm. (D) Histology score, combined epithelial damage and infiltration scores. (E) Reduced levels of the MPO marker of intestinal inflammation in mice treated with ZnDTC. Data represent mean ± SD (n = 6 mice per group). **P < .01, ***P < .001, Fisher’s exact test and Mann-Whitney U test were performed for statistical analysis.