Proteomics coupled with in vitro model to study the early crosstalk occurring between newly excysted juveniles of Fasciola hepatica and host intestinal cells

Abstract

Fasciolosis caused by the trematode Fasciola hepatica is a zoonotic neglected disease affecting animals and humans worldwide. Infection occurs upon ingestion of aquatic plants or water contaminated with metacercariae. These release the newly excysted juveniles (FhNEJ) in the host duodenum, where they establish contact with the epithelium and cross the intestinal barrier to reach the peritoneum within 2-3 h after infection. Juveniles crawl up the peritoneum towards the liver, and migrate through the hepatic tissue before reaching their definitive location inside the major biliary ducts, where they mature into adult worms. Fasciolosis is treated with triclabendazole, although resistant isolates of the parasite are increasingly being reported. This, together with the limited efficacy of the assayed vaccines against this infection, poses fasciolosis as a veterinary and human health problem of growing concern. In this context, the study of early host-parasite interactions is of paramount importance for the definition of new targets for the treatment and prevention of fasciolosis. Here, we develop a new in vitro model that replicates the first interaction between FhNEJ and mouse primary small intestinal epithelial cells (MPSIEC). FhNEJ and MPSIEC were co-incubated for 3 h and protein extracts (tegument and soma of FhNEJ and membrane and cytosol of MPSIEC) were subjected to quantitative SWATH-MS proteomics and compared to respective controls (MPSIEC and FhNEJ left alone for 3h in culture medium) to evaluate protein expression changes in both the parasite and the host. Results show that the interaction between FhNEJ and MPSIEC triggers a rapid protein expression change of FhNEJ in response to the host epithelial barrier, including cathepsins L3 and L4 and several immunoregulatory proteins. Regarding MPSIEC, stimulation with FhNEJ results in alterations in the protein profile related to immunomodulation and cell-cell interactions, together with a drastic reduction in the expression of proteins linked with ribosome function. The molecules identified in this model of early host-parasite interactions could help define new tools against fasciolosis.

Fig 1. Experimental design of the in vitro interaction model between F. hepatica juveniles (FhNEJ) and mouse intestinal epithelial cells (MPSIEC).

(A) Parasitic extracts and host cell lysates were obtained before (FhNEJ 1, MPSIEC 1) and after co-culturing for 3 h (FhNEJ 2, MPSIEC 2) and their proteins analyzed by SWATH-MS. (B, C, D) Representative images of MPSIEC (at the background of each image) stimulated with the FhNEJ (arrows) are shown (optical microscope, 10x). Panel A created with BioRender.com.

Fig 2. Principal Component Analysis (PCA) of each group of samples analysed in the study.

PCA analyses of FhNEJ detergent-soluble extract enriched with tegument (FhNEJ tegument; A), FhNEJ somatic (B), MPSIEC cytosol (C) and MPSIEC membrane (D) protein extracts. Blue dots represent the three control replicates (incubated alone for 3h; control) whereas red dots represent the three replicates co-incubated for 3h (stimulated). The percentage of variance explained by each principal component (PC) is indicated on its corresponding axis.

Fig 3. Volcano plots representing the changes in the proteomic profile of each group of samples analysed in the study.

Volcano plots corresponding to FhNEJ detergent-soluble extract enriched with tegument (FhNEJ tegument; A), FhNEJ somatic (B), MPSIEC cytosol (C) and MPSIEC membrane (D) protein extracts. Red dots represent up-regulated proteins after stimulation challenge, and blue dots represent down-regulated proteins. Only DEPs with q-value <0.05 were considered as differentially expressed, and no fold change threshold was considered.

Fig 4. Differentially expressed proteins in FhNEJ after incubation with MPSIEC.

The first 15 proteins with the highest (represented in red) or lowest (represented in blue) fold change within the FhNEJ detergent-soluble extract enriched with tegument (FhNEJ Tegument extract) and somatic extract (FhNEJ Somatic extract) are shown. Icons created with BioRender.com.

Fig 5. Differentially expressed proteins in MPSIEC after incubation with FhNEJ.

The top 15 proteins with highest (represented in red) or lowest (represented in blue) fold change within the MPSIEC membrane and cytosol extracts are shown. Icons created with BioRender.com.

Fig 6. ReViGO plot showing the main GO terms within the Biological Process (BP) category in FhNEJ.

The BP GO terms corresponding to up- and down-regulated proteins in the detergent-soluble extract enriched with tegument (A) and soma (B) extracts of FhNEJ after incubation with MPSIEC are shown. The size and colour of each circle represents the Nodescore of each GO term, and the spacing between circles refers to the similarity between the represented terms.

Fig 7. ReViGO plot showing the main GO terms within the Biological Process (BP) category in MPSIEC.

The BP GO terms corresponding to up- and down-regulated proteins in the cytosol (A) and membrane (B) extracts of MPSIEC after incubation with FhNEJ are shown. The size and colour of each circle represents the Nodescore of each GO term, and the spacing between circles refers to the similarity between the represented terms.

Fig 8. Interaction plot (STRING) showing protein-protein relations within the set of up-regulated MPSIEC proteins.

Edges between network nodes represent protein-protein associations, which are classified as “known interaction” (light blue: from curated databases; purple: experimentally determined), “predicted interactions” (green: gene neighbourhood; red: gene fusions; blue: gene co-occurrence) or “others” (yellow: textmining; black: co-expression; pale blue: protein homology) (https://version-11-5.string-db.org/cgi/network?networkId=blUqyaKyKA9R).

Fig 9. Interaction plot (STRING) showing protein-protein relationships within the set of down-regulated MPSIEC proteins.

Edges between network nodes represent protein-protein associations, which are classified as “known interaction” (light blue: from curated databases; purple: experimentally determined), “predicted interactions” (green: gene neighbourhood; red: gene fusions; blue: gene co-occurrence) or “others” (yellow: textmining; black: co-expression; pale blue: protein homology) (https://version-11-5.string-db.org/cgi/network?networkId=bEnbwSoK41Pk).