Fig 1.
Ex vivo model for the migration of FhNEJ through mouse intestine.
A) Preparation of the intestines before FhNEJ infection; B) Incubation of ligated intestines in RPMI medium at 39°C 5%CO2 for 2.5 h; C) Image obtained in a stereomicroscope showing the FhNEJ after intestinal passage (white arrow).
Fig 2.
Principal Component Analysis (PCA) of FhNEJ (A) tegument and (B) somatic extracts.
Blue dots represent the control replicates (Control_1 to _3) whereas red dots represent the replicates of FhNEJ after migration across the gut wall (Stimulated_1 to _3). The percentage of variance showed by each principal component (PC1 and 2) is indicated in its corresponding axis. (C) Venn’s diagram showing the distribution of quantified proteins.
Fig 3.
Volcano plots of the significantly differentially expressed proteins in tegument (A) and somatic extract (B) of F. hepatica juveniles after passing through mouse intestinal wall.
The grey dashed line represents the threshold delimiting the differentially expressed proteins. Red dots represent upregulated proteins, while blue dots represent downregulated proteins.
Fig 4.
Bar graphs representing the results of the Gene Ontology analysis performed on the differentially expressed proteins, referred to Biological Process (A) and Molecular Function (B) categories.
Red bars represent the proportion of proteins found in the tegument samples, and grey bars represent the proportion of proteins found in the somatic samples.
Fig 5.
Upregulated (in red) and downregulated (in blue) proteins in tegument (upper panels) and somatic (box lower panel) extracts of F. hepatica juveniles after intestinal passage, compared with control FhNEJ.
Both protein description and the respective Log2FC values are shown. Colour scales represent the relative Log2FC value in each panel.
Fig 6.
SDS-PAGE (A) and immunoblot (B) of tegument extracts from control FhNEJ (1) and FhNEJ after intestinal passage (2).
(A) Sypro staining of total proteins; (B) anti-rFhSrp2 immunoblot. Molecular weights are shown in kDa.
Fig 7.
Histological features of the intestine of mice after hematoxylin-eosin staining.
(A) Fresh intestine from a non-infected mouse showing normal villi (V). (B) Non-infected intestine after incubation for 2.5 hours at 39°C and 5% of CO2 showing detachment of villi (V). (C) Infected intestine after incubation and FhNEJ passage showing detached villi (V) and FhNEJ in the intestinal lumen (arrow) and in the intestinal serosa (arrowhead), both without inflammatory infiltrate. D) Detail of a FhNEJ (arrow) crossing the intestinal crypts. Note that there is no inflammatory infiltrate in the periphery of the FhNEJ.
Fig 8.
Mouse intestine subjected to the ABC method and hematoxylin counterstain.
Cleaved caspase 3 in non-infected fresh intestine (A), non-infected incubated intestine (B) and infected and incubated intestine (C) all of them showing caspase 3+ cells (arrow). Specific areas of the infected and incubated intestine (C) showed a higher number of caspase 3+ cells in the lamina propria (arrows), compared with the fresh and the incubated and non-infected intestine.