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Proteomics coupled with in vitro model to study the early crosstalk occurring between newly excysted juveniles of Fasciola hepatica and host intestinal cells [1]

['David Becerro-Recio', 'Parasitology Unit', 'Institute Of Natural Resources', 'Agrobiology Of Salamanca', 'Irnasa-Csic', 'Salamanca', 'Judit Serrat', 'Marta López-García', 'Javier Sotillo', 'Parasitology Reference']

Date: 2022-11 

FhNEJ protein expression changes rapidly in response to interaction with the host intestinal epithelial barrier.

In the detergent-soluble extract enriched with tegument of FhNEJ, the top over-represented BPs were proteolysis, microtubule-based process and transport (Fig 6A), while the main MFs identified in this antigenic compartment were transferase activity, metal ion binding and oxidoreductase activity (S1 Fig).

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pntd.0010811.g006

Regarding proteolysis, a considerable number of proteomic studies have demonstrated the high level of protein complexity of the juvenile stages of F. hepatica in terms of proteolysis-related molecules [16,37,38]. This situation is not unexpected considering that this function is pivotal for the parasite invasion process. More specifically, trematode peptidases have been pointed out as key molecules in facilitating parasite migration, nutrition, immune evasion and other important host-parasite interactions [39]. Cathepsin peptidases are among the major proteolytic enzymes involved in FhNEJ virulence and tissue migration and they represent the most abundant proteases in the secretions of F. hepatica and a paradigmatic example of evolutionary divergence in response to selection pressure [40]. In fact, 23 and 11 different sequences of cathepsins belonging to the L and B classes have been identified in the F. hepatica genome [41], and their expression shows a stage-specific temporal shift that correlates with juvenile development and the organ invasion process [42,43].

The high abundance of proteases among the up-regulated proteins in the detergent-soluble extract enriched with tegument of FhNEJ upon incubation with MPSIEC is remarkable (S1 Table). These included cathepsin L (identified as cathepsin L4 (CL4) after comparison of its prosegment with those corresponding to F. hepatica consensus sequences of CL1 and CL5 prosegments [44]), cathepsin L3 peptidase (CL3) and peptidase D. The first and third upregulated proteins in FhNEJ tegument upon contact with host cells (CL4 and CL3, respectively) have been found in the transcriptome of FhNEJ axenically maintained in vitro for 1, 3 and 24 h [37], but only CL3, and not CL4, has been found in protein extracts of FhNEJ tegument [17]. Our results suggest that CL4 would be overexpressed in the detergent-soluble extract enriched with tegument of FhNEJ only when subjected to interact with host cells. On the contrary, CL3 would be expressed in FhNEJ after in vitro excystment without requiring host stimulus. Thus, CL4 may play a role at the host-parasite interface upon contact of FhNEJ with the host. Importantly, CL3 is known for its collagenolytic activity, which is especially efficient in digesting type I and II collagens [45]. Provided that FhNEJ must overcome four layers in the small intestine wall (mucosa, submucosa, muscularis externa and serosa) that are highly rich in collagen [46] in order to reach the peritoneum, the over-expression of proteases with collagenolytic activity in the host-parasite interface could be crucial for parasite invasion. Moreover, this important function might be followed by the activity of other up-regulated proteases such as peptidase D (also known as prolidase), whose most studied substrates are dipeptides that are generated during collagen breakdown [47]. Together with these proteases, the protease inhibitor serpin 2 is also upregulated in this compartment of FhNEJ after incubation with MPSIEC. Serpin 2, characterized as a surface molecule in this parasite, can both protect the FhNEJ from host proteolysis and play a role in immune evasion [48].

Interestingly, some proteins traditionally associated with the nucleus such as histones H4 and H2B and histone deacetylase were also up-regulated in the FhNEJ detergent-soluble extract enriched with tegument after co-incubation with host intestinal epithelial cells. These proteins have important functions in chromosomic DNA organization [49] and they can also be released to the cytosol and the extracellular space to perform other functions, such as antimicrobial responses [50]. In fact, it has been demonstrated that histones (e.g. H4 or H2B) and their derivatives, which are cleaved from histones by cathepsins [51], directly eliminate a wide range of pathogens in a similar way to other antimicrobial peptides [50]. This mechanism, by which proteins exhibit ‘unexpected’ functions separate from their canonical activities, is referred to as ‘moonlighting’. Moonlighting is directly linked to unconventional protein secretion routes that allow for the translocation of cytosolic antigens to the host-parasite interface [52]. This is also the case of surface-located glycolytic enzymes, such as phosphoglycerate mutase, which is up-regulated in the tegument and the soma of FhNEJ in this study. This enzyme normally participates in energy metabolism, but when located extracellularly it also plays a role in parasite invasion and establishment within the host [53].

Potentially linked to immune regulation, we found glutathione-S-transferase (GST) and the calcium-binding EF-hand protein 4 (CABP4) to be overexpressed in this protein fraction of FhNEJ upon incubation with MPSIEC. F. hepatica GST (also found here overexpressed in the somatic extract of FhNEJ) has shown anti-inflammatory properties [54], and F. gigantica CABP4 stimulated IL-2 and IFN-γ Th1-type cytokines and reduced IL-10 (Th2) expression levels by activated goat monocytes [55]. As observed in other trematode species (e.g., schistosomes [56]), these proteins could play a role in the first steps of infection by protecting FhNEJ from inflammatory responses as well as eliciting a transient Th1-type immune response during early infection.

Down-regulated proteins in the detergent-soluble extract enriched with tegument of the parasite include components of the muscle contraction machinery (troponin, actin, dynein, titin, etc.) and proteins involved in metabolite transport (fatty acid binding protein, vesicle-associated membrane protein-associated protein A) along with other multi-purpose proteins (annexin, peptidyl-prolyl cis-trans isomerase). Noteworthy, some biological processes with up-regulated proteins (i.e. proteolysis or antioxidant response) also exhibit down-regulated representatives, and might therefore be finely regulated during the early phases of F. hepatica infection. This could be the case of the above-mentioned cathepsins. In fact, the results obtained in this study showed a down-regulation in the expression of two isoforms of cathepsin B after contact of FhNEJ with MPSIEC. FhCB peptidases appear to be highly expressed by FhNEJ and down-regulated in adult flukes [40, 57], although defined isoforms of CBs could have important roles in the excystment process but not in the subsequent migration of FhNEJ. Overall, these results suggest an important role of the interaction between FhNEJ and the host intestinal wall in the cathepsin expression shift observed in F. hepatica in correlation to parasite development and migration through host tissues.

Interestingly, the downregulated protein with the highest Log2FC in this FhNEJ extract, also corresponding to the fifth most downregulated protein in somatic extracts of FhNEJ upon MPSIEC incubation, is CD59, a complement regulatory protein that protects cells from the destructive action of the complement system. Homologue CD59 proteins have been previously described to be expressed and secreted by FhNEJ in vitro [37, 58], together with other parasite proteins that could impair the host’s complement response, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [37, 38, 58], which is similarly downregulated in the tegument extract of FhNEJ upon incubation with MPSIEC. Thus, it is possible that the down-regulation of CD59-like molecule and GAPDH herein identified could be an indication of depletion of these molecules in the tegument compartment as a result of increased secretion upon interaction with the host to counteract complement attack. However, it is also known that FhNEJ employ multiple mechanisms to inhibit complement attack during invasion [59], so alternative strategies to avoid this host defence mechanism could also be taking place.

In the somatic FhNEJ extract, the main GO terms in the BP category were proteolysis, carbohydrate metabolic process, protein folding and microtubule-based process (Fig 6B), and transferase activity, peptidase activity and metal ion binding in the MF category (S1 Fig). Up-regulated proteins in this compartment exhibited a profile similar to that described in the tegument, including proteins involved in peptide degradation (CL3, dipeptidyl peptidase), antioxidant response (GST) or metabolic enzymes (phosphoglycerate mutase), as well as proteins involved in signalling cascades (Mitogen-activated protein kinase) or cell-cell adhesion (tetraspanin). Tetraspanins are transmembrane proteins involved in important functions linked with the maintenance of the integrity of the adult fluke tegument [60]. Noteworthy, tetraspanins from other helminth trematodes such as Schistosoma mansoni or Opistorchis viverrini have elicited promising results as vaccine candidates against schistosomiasis and opisthorchiasis, respectively [61, 62]. However, the biological role of the identified up-regulation of this protein in the internal tissues of the FhNEJ should be further investigated.

Down-regulated proteins in the soma of FhNEJ included proteins also down-regulated in their tegument (fatty acid binding protein, GAPDH, peptidyl-prolyl cis-trans isomerase). Additionally, legumain like and leucyl aminopeptidase, superoxide dismutase, transketolase and pyruvate dehydrogenase, representing functions similar to those down-regulated in the tegument of FhNEJ, were also found as down-regulated in the somatic extracts of FhNEJ.

Regarding antioxidant enzymes, our data revealed that the expression of four isoforms of one of their main representatives, GST, is up-regulated within the FhNEJ somatic and tegument extracts after co-culture with MPSIEC. It has been described that GSTs account for 4% of F. hepatica excretory/secretory products and that these enzymes have a key role in detoxifying both endogenous and exogenous toxins arising from the oxidative host defence response [63]. For that reason, this parasitic antigen has been proposed as a vaccine candidate against fasciolosis [64]. However, our data also revealed the presence of a GST isoform that is down-regulated within the FhNEJ somatic extract. This particular shift in the expression pattern of parasitic isoforms of the same protein upon interaction with host cells could be explained by finely regulated biochemical redundancy mechanisms, which would hamper the deleterious effects of the immune response against these specific antigens. This bidirectional regulation of molecules that are important for parasite survival and development could also explain the reported high variability in protection rates and the partial success of vaccination studies against fasciolosis that included these diversely-regulated antigens in their formulations [10,65].

The comparison of the results of the present analysis with that performed before by our group by using the iTRAQ methodology after 24 h co-incubation of FhNEJ and MPSIEC [14], shows that proteolysis seems to play a key role in FhNEJ after co-culture with MPSIEC, at both 3h and 24h post-incubation (pi), as this functional category is up-regulated at both times. However, the protease profile shows some differences between the two incubation times, as cathepsin L3 is the major isoform together with peptidase D after 3h pi, whereas at 24h pi cathepsin L1 and peptidase C1 are up-regulated and cathepsins L3, L4 and B, along with papain and legumain, are down-regulated. This suggests that at 24h pi the parasite is already expressing adult stage specific isoforms, while juvenile isoforms are already decaying. Regarding regulation of proteolysis, this seems to play a more important role in the earliest stages (3h pi), as serpin 2 is up-regulated at this time, while cystatin is found as down-regulated at 24h pi. Similarly, response to oxidative stress is represented by several GST isoforms that are up-regulated at 3h pi but not at 24h pi.

On the other hand, muscle contraction and cytoskeleton reorganization processes seem to be more represented at 24h pi, as at this time several related proteins are found to be up-regulated while they are not found at 3h pi, including dynein, cadherin, myophilin and catenin alpha. Several metabolic enzymes could be also differentially expressed at both times pi. The landscape at 3h pi is mainly aerobic (including several up-regulated respiratory enzymes such as cytochrome c oxidase and quinone oxidoreductase), while at 24h pi it seems to evolve to an anaerobic profile with a greater abundance of glycolytic enzymes (e.g., aldolase, GAPDH). Interestingly, histone isoforms are found as up-regulated proteins at both times pi, with histone H4 present at both times, while histone H2B is found at 3h and histone H2A at 24h. In summary, comparison of the results obtained here at 3h pi obtained by SWATH and the previous results at 24 h pi obtained by iTRAQ, show that proteolysis and its regulation, muscle contraction and metabolism could be differentially regulated in FhNEJ upon MPSIEC co-incubation at different times pi. Nevertheless, this should be further explored in more comparable studies, since the methods used to check up- and down-regulated proteins in FhNEJ after co-culture with host MPSIEC between the two above-mentioned studies are different regarding the proteomic and statistical approaches.

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0010811

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