Figure 1.
Human colon explants incubated with E. histolytica HM1:IMSS or Rahman trophozoites.
Longitudinal tissue sections of colon explants incubated during 1 or 7E. histolytica HM1:IMSS or Rahman trophozoites. The upper panel (A) corresponds to the alcian blue staining of the mucus in the top followed by staining of the tissue where the epithelial cells and the crypts of Liberkün (counterstain in red with Safranin) are visible. The lower panel (B) corresponds to immunohistochemistry revealing the presence of trophozoites in the top by immunostaining for the Gal/GalNAc lectin and the tissue by counterstaining with Hematoxylin/Eosin (bleu). Note the presence of Rahman trophozoites on top of the mucus layer even after 7 h of incubation and the massive destruction of the mucosa in the presence of HM1:IMSS. Scale bar = 50 µm.
Figure 2.
Experimental design and strategy for microarray data analysis.
A. Scheme representing the four experimental conditions and transcriptome comparisons. B. Cross-comparative analysis accomplished to identify strain-specific and common responses under the two conditions tested. The transcriptome data were processed as indicated in order to identify gene expression profiles associated with the strain and the colon explant contact. A/B designates the pool of genes overexpressed in condition A compared to condition B. + means that the common overexpressed genes of the comparisons are kept, whereas - indicates genes that were discarded. Note that the profiles obtained by this strategy are composed of genes with increase transcript levels in a particular strain versus the other.
Figure 3.
Results of the Principal Component Analysis.
The figure represents the projection of the 48 arrays on the first three Principal Components. The sum of these principal three axes corresponds to 64.5% of the total variance. The representation corresponds to: The Rahman strain in contact with mucus (Red), Rahman strain in axenic culture (Blue), HMI:IMSS strain in contact with mucus (Green), and HMI:IMSS strain in axenic culture (Purple).
Figure 4.
Summary of the microarray data.
The transcriptomic modulations obtained in the 4 comparisons. In total, 614 transcripts with a fold change greater than two were significantly modulated (Bonferroni adjusted p-value<0.05).
Table 1.
Classification of modulated transcripts into functional categories.
Table 2.
The E. histolytica Rahman ubiquitous gene expression profile.
Table 3.
The E. histolytica Rahman specific gene expression profile in contact with the mucus.
Table 4.
Common gene expression profile in response to the mucus layer contact.
Table 5.
The E. histolytica HM1: IMSS ubiquitous gene expression profile.
Table 6.
The E. histolytica HM1:IMSS specific gene expression profile in contact with the mucus.
Table 7.
The E. histolytica HM1:IMSS specific gene expression profile in contact with the mucus.
Figure 5.
Transcriptomic landscape describing the virulent and non-virulent signatures of E. histolytica.
The scheme summarizes the transcriptomic map of E. histolytica highlighting two categories of genes: those permanently expressed in each strain and those overexpressed when amoebas are in contact with human colon explants. The signature of HM1:IMSS is on the left and the Rahman non-virulent signature is on the right. Central in the scheme are the genes common to the two strains and only upregulated when the parasites are in contact with the mucus. The adhesion-cell surface molecules are depicted in green; proteases in black; carbohydrates metabolism in red; lipid metabolism in purple. Only the most striking differences were highlight in this figure, others categories are described in tables 2, 3, 4, 5, and 6.
Figure 6.
Enzymes overexpressed and involved in carbohydrate metabolism specific to the virulent HM1:IMSS strain.
Genes involved in glycan metabolism with significant and specific overexpression in HM1:IMSS are shown in green. The genes were chosen according to a Bonferroni adjusted p value≤0.05 and without fold change cutoff (Table S9).
Figure 7.
Functional characterization of E. histolytica β-amylase.
A. Amino acid sequence alignment of full-length β-amylase homologues in Entamoeba histolytica (EHI_192590) and Glycine max (BMY1: 547931). Identical residues are highlighted. The red boxes indicate the two conserved catalytic domains containing two glutamic acids (E185 and E378) involved in the catalytic reaction. B. Bioinformatic structural model of β-amylase in E. histolytica predicted using LOMETS software. A 3D structural search of E. histolytica β-amylase match β-amylase from G. max as the best-hit template. The upper panel shows the predicted structural model of E. histolytica β-amylase. The middle panel represents the crystal structure of G. max β-amylase [26] and the lower panel shows the merge between the two structures. Note (i) the strong structural homology between the two enzymes and (ii) the N-terminal tail of E. histolytica β-amylase, which was predicted as a transmembrane domain (white arrow). C. Cellular localization of E. histolytica β-amylase. Trophozoites were fixed and labelled by immunofluorescence for β-amylase. Confocal microscopy image analysis revealed a cell surface localization in non-permeabilized trophozoites, and in cytoplasmic dots in permeabilized parasites. Scale bar = 5 µm. D. Immunodetection of β-amylase (48 kDa) in crude extracts of HM1:IMSS or Rahman strains. 30 µg of proteins were loaded and resolved on a 12% SDS-PAGE gel. Proteins were transferred onto PVDF membranes and probed with an anti β-amylase specific antibody. Actin was used as a loading control. Rahman strain synthetizes roughly 13% of β-amylase compared to HM1:IMSS taken as 100%.
Figure 8.
Depletion of ß-amylase in HM1:IMSS trophozoites prevent mucus layer degradation.
A. Immuno-detection of β-amylase in dsRNA treated parasites. Trophozoites were treated for 24 or 48 h with control dsRNA or β-amylase specific dsRNA. Crude extracts were analysed by western blotting. Protein loading was normalized with respect to actin. After 48 h, β-amylase amounts were reduced by 75.5% (SEM ± 4.6%; n = 3) in comparison to the control. B. Quantification of the mucus layer degradation. After 7 h of incubation with control dsRNA treated trophozoites or β-amylase specific dsRNA treated trophozoites colonic explant were fixed and stained with Alcian blue to visualize the mucus layer. In the presence of β-amylase specific dsRNA treated trophozoites the thickness of the mucus layer is not altered as compared to the control tissue (132.7 µm vs 133.7 µm) while in the presence of GFP dsRNA treated trophozoites the mucus layer thickness decrease to 13.58 µm. C. Histological study of mucosal invasion. Trophozoites treated for 48 h with control dsRNA or β-amylase specific dsRNA were incubated with the human colon explant and tissue section were analysed as described in Figure 1. Decrease in β-amylase abundance inhibits mucosa invasion after 7 h of incubation. β-amylase deficient trophozoites were still associated with the mucus layer while control-treated trophozoites have depleted the mucus layer and invaded the lamina propria. Scale bar = 50 µm.
Figure 9.
Schematic of Entamoeba histolytica activities leading to mucus layer depletion and invasion of the human colon.
During its vegetative life style (A), E. histolytica exploits lipids (from lipid-rich food particles, bacteria, and shed epithelial cells) and carbohydrates (undigested glycan-rich food particles provided by the bolus or shed mucus fragment) present in the colonic environment. (B) When dietary polysaccharides are scarce, we hypothesize that E. histolytica turns to host mucus by first deploying a set of polysaccharide hydrolases that depletes the protective oligosaccharide side chains of mucin which can be then targeted by cysteine proteases leading to the depletion of the protective mucus barrier and allowing subsequent invasion of the mucosa. This adaptive foraging could reflect the coevolved functional versatility of E. histolytica glycobiome and the structural diversity of host mucus glycans involved in the interaction.