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.

Fasciola hepatica juveniles interact with the host fibrinolytic system as a potential early-stage invasion mechanism

“…More specifically, we identify FhCL3 and FhCB3 as potential PLG-binding proteins, and FhCL1, FhCL2 and FhCB2 to a lesser extent. This is consistent with the developmental stage-specific expression of these proteases, being FhCB1-3 and FhCL3 mostly expressed in the juvenile stages of the parasite that are found in the duodenum [ 36 , 48 , 49 ]. However, one of the limitations of our 2D-MS approach is that it is performed under denaturing conditions, which may expose internal PLG-binding epitopes in some proteins that are not employed for PLG-binding in a real physiological context.…”

Fasciola hepaticajuveniles interact with the host fibrinolytic system as a potential early-stage invasion mechanism

“…More specifically, we identify FhCL3 and FhCB3 as potential PLG-binding proteins, and FhCL1, FhCL2 and FhCB3 to a lesser extent. This is consistent with the developmental stage-specific expression of these proteases, being FhCB1-3 and FhCL3 mostly expressed in the juvenile stages of the parasite that are found in the duodenum (46)(47)(48). However, one of the limitations of our 2D-MS approach is that it is performed under denaturing conditions, which may expose internal PLG-binding epitopes in some proteins that are not employed for PLG-binding in a real physiological context.…”

Progresses and challenges in Strongyloides spp. proteomics

“…Such analysis could however be particularly useful in revealing novel facets of the pathological mechanisms associated with human strongyloidiasis, especially when applied in comparative studies. Indeed, this strategy has already been successfully applied to the study of other helminthiases, including schistosomiasis, fasciolosis and echinococcosis, to identify proteins or biological processes altered in the pathological state compared to uninfected controls, during the disease progression or in response to treatment [66][67][68][69][70][71][72][73][74][75][76]. The most commonly investigated samples are plasma/serum and plasma/serumderived EVs (either from animal models or from patients), even though also tissue proteomics has been described [69,70].…”

“…Indeed, this strategy has already been successfully applied to the study of other helminthiases, including schistosomiasis, fasciolosis and echinococcosis, to identify proteins or biological processes altered in the pathological state compared to uninfected controls, during the disease progression or in response to treatment [66][67][68][69][70][71][72][73][74][75][76]. The most commonly investigated samples are plasma/serum and plasma/serumderived EVs (either from animal models or from patients), even though also tissue proteomics has been described [69,70]. Importantly, a number of studies identified parasite proteins within the host's systemic or vesicular proteome, supporting the relevance of host proteomics also in revealing novel diagnostic markers [67,68,71,72,74,75].…”