Introduction
Fasciolosis is a zoonotic parasitic disease caused by liver flukes in the genus Fasciola, of which Fasciola hepatica and Fasciola gigantica are the primary agents for this widely neglected parasitic disease (Mas-Coma et al. Reference Mas-Coma, Bargues and Valero2005). These parasites are widespread and have been identified across over 70 countries from all continents (Fürst et al. Reference Fürst, Keiser and Utzinger2012; Logue et al. Reference Logue, Barbieri, Nielsen and Toldrá2017). Although F. hepatica primarily presents in temperate, tropical, or subtropical regions such as Europe, South America, Middle East, and some part of Asia, F. gigantica is predominantly found in tropical climates including Africa, Asia, and the Middle East (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009). In livestock, infection can lead to substantial economic losses, arising from factors such as morbidity, mortality, decreased productivity, fertility, and increased susceptibility to secondary infections (Beesley et al. Reference Beesley, Caminade, Charlier, Flynn, Hodgkinson, Martinez-Moreno, Martinez-Valladares, Perez, Rinaldi and Williams2018; Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2019).
It is generally recognised that various molecules originating from the host are involved in growth and development of parasites that can influence the progression of parasitic infection (Ednilson Hilário et al. Reference Ednilson Hilário, Rafaella Pontes, Claudio Romero, Katia Cristina, Jorge, R-A and Romel2022). Numerous studies have demonstrated the existence of diverse growth factor receptors and the conservation of signalling pathways in parasitic nematodes and flatworms. Notably, transforming growth factor-beta (TGF-β), epidermal growth factor, and insulin have been identified (Konrad et al. Reference Konrad, Kroner, Spiliotis, Zavala-Góngora and Brehm2003; Spiliotis et al. Reference Spiliotis, Konrad, Gelmedin, Tappe, Brückner, H-U and Brehm2006; Zavala-Góngora et al. Reference Zavala-Góngora, Kroner, Bernthaler, Knaus and Brehm2006). These pathways are believed to potentially mediate communication between the host and the helminth, influencing the dynamic interactions that unfold during infection and direct parasite intrinsic developmental events. Therefore, investigating these signalling components is important for understanding the complexities of parasitism, parasite development, and the identification of new targets for the development of strategies against these diseases (Salzet et al. Reference Salzet, Capron and Stefano2000; You et al. Reference You, Gobert, Jones, Zhang and McManus2011).
The signalling pathway of the TGF-β/bone morphogenetic protein (BMP) family is implicated in numerous biological processes across the animal kingdom (Huminiecki et al. Reference Huminiecki, Goldovsky, Freilich, Moustakas, Ouzounis and Heldin2009; Tzavlaki and Moustakas Reference Tzavlaki and Moustakas2020). Signal transduction of all TGF-β proteins is initiated upon the binding of ligands to their cognate receptors and subsequent phosphorylation of the intracellular mediators known as mothers against decapentaplegic (Smads). These phosphorylated Smads then translocate into the nucleus and function as transcription factor regulating transcription of the downstream target genes (Massagué Reference Massagué2012; Moustakas and Heldin Reference Moustakas and Heldin2009; Tzavlaki and Moustakas Reference Tzavlaki and Moustakas2020).
Within the TGF-β signalling pathway, there are three types of Smad signalling molecules classified based on their functions: receptor-regulated Smads (R-Smads), common-partner Smad (Co-Smad), and inhibitory Smads (Miyazono Reference Miyazono2000; Moustakas et al. Reference Moustakas, Souchelnytskyi and Heldin2001; Samanta and Datta Reference Samanta and Datta2012). Smad4, the Co-Smad present in vertebrates or Medea in Drosophila or SMA in Caenorhabditis elegans (Lagna et al. Reference Lagna, Hata, Hemmati-Brivanlou and Massagué1996; Zhang et al. Reference Zhang, Musci and Derynck1997), acts as a common mediator for both BMP-specific and TGF-β/activins-specific R-Smads. Smad4 possesses a unique characteristic as it is unable to undergo phosphorylation or bind to either TGF-β or BMP receptors. However, it has the capacity to form heteromeric multimeric complexes with almost all activated R-Smads, enabling it to participate in the regulation of TGF-β signalling transduction (Wang et al. Reference Wang, Li, Wang and Chen2013).
In our previous works, we described the existence of the TGF-β protein family in liver flukes, specifically FhTLM in F. hepatica (Japa et al. Reference Japa, Hodgkinson, Emes and Flynn2015) and FgTLM in F. gigantica (Japa et al. Reference Japa, Prakhammin and Flynn2022). These findings strongly suggest the presence of the signal mediator homologue components within the TGF-β pathway of these liver flukes, emphasising the importance of further exploring into this component of signalling molecules. In this study, we present our findings on the identification and characterisation of FgSmad4, the Co-Smad homologue in F. gigantica. Additionally, we investigated the gene expression patterns of the FgSmad4 throughout the various developmental stages of F. gigantica.
Material and Methods
Ethics approval
The use of animals in this study was reviewed and approved by the Animal Ethics Committee of the University of Phayao, Thailand, approval number 1-023-65.
Preparation of parasites
Adult
Adult F. gigantica was obtained from naturally infected buffaloes in Phayao, Thailand, found in local abattoirs (19°11’18.00" N 99°52’27.59" E). The liver flukes were removed from bile ducts and gall bladder stored in RPMI-1640 supplemented with gentamycin (10 μg/mL). After collection, the flukes underwent multiple washes with sterile phosphate-buffered saline and were then preserved in Trizol for subsequent RNA extraction and preparation of liver fluke eggs.
Unembryonated egg
Unembryonated eggs from the uterus were collected from mature flukes. The eggs were thoroughly rinsed with sterile distilled water until the fluid was transparent. The fluke eggs were collected and preserved in Trizol for RNA extraction. Additionally, aliquots of the fluke eggs were reserved and stored in the fridge for subsequent use in inducing the formation of embryonated eggs and miracidia.
Embryonated egg
The liver fluke eggs were induced to undergo embryonation through laboratory incubation under dark conditions at 30 °C for 14 days. Following this incubation period, the development of the eggs was observed using a stereo microscope; only those that had undergone embryonation were selected for RNA preparation.
Miracidium
The F. gigantica miracidia were obtained through egg hatching protocol as outlined by Moxon et al. (Reference Moxon, LaCourse, Wright, Perally, Prescott, Gillard, Barrett, Hamilton and Brophy2010). In brief, after inducing embryonation in the eggs for 14 days, they were exposed to direct light at room temperature to stimulate miracidia hatching. Following this, the miracidia were examined under a stereo microscope and collected in Trizol for further experimentation.
Intra-molluscan larval stages (redia, cercaria) and metacercaria
The larval stages of F. gigantica, including rediae, cercariae, and metacercariae, were generated from experimentally infected snails. Specifically, snails of the Radix (Lymnaeae) rubiginosa species were maintained in the laboratory and used to establish F. gigantica infection. Laboratory infection of the F. gigantica was conducted according to Japa et al. (Reference Japa, Prakhammin and Flynn2022). After approximately 45 days of infection, the infected snails were monitored for cercarial emission under a stereo microscope. The released cercariae were immediately collected and stored in Trizol for RNA extraction.
To prepare metacercaria, a cellophane sheet was placed over the water surface to facilitate cercarial attachment and their subsequent transformation into metacercariae. Following this, the F. gigantica infected snails were dissected to retrieve the redial stages. The larval stages of F. gigantica acquired from the snails were preserved in Trizol and stored at –20 °C until they were ready for RNA extraction and subsequent analysis.
Newly excysted juvenile (NEJ)
The newly excysted juveniles of F. gigantica were obtained through an in vitro excystation process of the F. gigantica metacercariae as outlined by McVeigh et al. (Reference McVeigh, McCammick, McCusker, Morphew, Mousley, Abidi, Saifullah, Muthusamy, Gopalakrishnan, Spithill, Dalton, Brophy, Marks and Maule2014) and Japa et al. (Reference Japa, Prakhammin and Flynn2022). Initially, the outer cysts were manually removed from the metacercariae. Afterward, the metacercariae were incubated in a 0.5% bleach solution for 4 min, followed by extensive washing with sterile water (five washes) and transferring to a new Petri dish. They were thoroughly suspended in an excystation solution and incubated for 1 hour at 37 °C. The NEJs were observed under stereo microscope and then collected in RPMI-1640 containing gentamycin (10 μg/mL). The NEJs underwent multiple washes with 1X D-phosphate-buffered saline and were finally preserved in Trizol for RNA extraction.
RNA isolation
Total RNA from each life stage of F. gigantica including adult, unembryonated egg, embryonated egg, miracidium, redia, cercaria, metacercaria, and NEJ was isolated using Trizol reagent and the RNeasy Micro Kit (Qiagen, Germany) with some modifications. The parasite was homogenised in 1 mL of Trizol reagent. The homogenate was then centrifuged at 12,000g for 5 min. The resulting supernatant was combined with 200 μL of chloroform and incubated at room temperature for 15 min, followed by another centrifugation for 5 min. The clear upper phase containing RNA was thoroughly mixed with an equal volume of 70% ethanol before being transferred to an RNeasy MinElute spin column and centrifuged for 1 min. Then, 350 μL of Buffer RW1 was added to the column before centrifugation as previously described. The flowthrough was discarded, and the column was loaded with PRE buffer and centrifuged. The column was then washed twice with 80% ethanol. The RNA was eluted from the column by 100 μL of sterile diethylpyrocarbonate-treated water. The purified RNA samples from each life stage were stored at –80 °C until further use.
Identification of the Co-Smad in the F. gigantica genome
To identify the Co-Smad sequence within the F. gigantica genome, we conducted a tblastn search using mammalian Smad4 protein sequences as the query sequence against the F. gigantica genome database available at https://parasite.wormbase.org/. Afterwards, gene-specific primers (GSPs) for the isolation of FgSmad4 via 5′ and 3′ rapid amplification of cDNA ends (RACE) were designed based on partial sequences acquired from tblastn searches against the F. gigantica genome/cDNA database.
Isolation of FgSmad4 cDNA
Total RNA extracted from adult F. gigantica specimens was used for the synthesis of first-strand cDNA library using the SMARTer RACE 5′/3′ Kit (Takara, Japan) following the manufacturer’s protocol.
The 5′ and 3′ ends of the FgSmad4 cDNA were amplified through two rounds of nested polymerase chain reaction (PCR) using GSP primers (10 μM) and the corresponding primers supplied by the kit. The GSP primer sequences are provided in Table 1. The PCR cycle for the first-round RACE amplification were as follows: pre-denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 3 min, and a final extension at 72 °C for 10 min. The nested PCR was carried out using 1 μL of the initial PCR product along with nested GSP primers targeting both ends. The nested PCR conditions consisted of 25 cycles following the same program used in the first PCR.
Table 1. Primer sequences used in 5′/3′ RACE experiments and life stage expression of FgSmad4
Cloning and Sequencing
The 5′ and 3′ RACE PCR products were excised and purified from the agarose gel using a gel extraction kit (Qiagen, Germany). The purified PCR products were then cloned into the pRACE vector and transformed into Stellar competent cells. Transformed colonies were selected, and plasmids containing the inserts were isolated using the Wizard Plus SV minipreps DNA purification system (Promega, UK). Subsequent sequencing was carried out bi-directionally using promoter primers, conducted by U2Bio (Korea).
Bioinformatics analyses
The nucleotide sequence was manually edited and annotated; overlapping sequence of 5′ and 3′ RACE was removed. The complete sequence of FgSmad4 cDNA was deposited in the GenBank database under accession no PP856693. The complete cDNA sequence of FgSmad4 was subsequently translated into the predicted amino acid sequence using the Expasy translation tools (https://web.expasy.org/translate/). Both nucleotide and protein sequences of FgSmad4, were performed homology searches using the blast software available at the National Centre for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST/). The nucleotide sequences were assessed using blastn against entries in the GenBank and WormBase ParaSite databases (https://parasite.wormbase.org/Multi/Tools/Blast). Similarity search of the protein sequence was conducted using PSI-blast and blastp at NCBI.
To determine exon–intron organisation, the cDNA sequences were used for blastn searches against the F. gigantica genome. This was carried out through online searches on the WormBaseParaSite database and local blastn analysis within BioEdit (Hall Reference Hall1999; Hall et al. Reference Hall, Biosciences and Carlsbad2011). A graphical representation of the gene organisation was generated using GeneMaper 2.5.
In silico analyses of Fgsmad4 protein properties
The FgSmad4 protein sequence was analysed for its properties using available online tools. Conserved domain prediction was conducted using CD-Search at the NCBI website (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The molecular weight and theoretical isoelectric point were predicted using the tool available at ExPASy (https://web.expasy.org/compute_pi/). Other predictions for general physiological and chemical properties, including amino acid composition, estimated instability index, and grand average of hydropathicity, were performed using the ProtParam tool on the ExPASy website (https://web.expasy.org/protparam/).
Prediction of two-dimensional and three-dimensional structures of FgSmad4
The FgSmad4 model structure was predicted in silico using the Phyre2 online tool (intensive method) available at http://www.sbg.bio.ic.ac.uk/~phyre2 (Kelley et al. Reference Kelley, Mezulis, Yates, Wass and Sternberg2015). Visualisation of the modeled protein was carried out using UCSF Chimera version 1.16 (Pettersen et al. Reference Pettersen, Goddard, Huang, Couch, Greenblatt, Meng and Ferrin2004).
Phylogenetic analysis
Sequences of Co-Smad protein from various parasitic helminths and their mammalian hosts were included into the phylogenetic tree analyses. Multiple alignment of these sequences was conducted using the Muscle method embedded in Seaview (Gouy et al. Reference Gouy, Guindon and Gascuel2009). All positions with gaps or incomplete data were excluded. Following the removal of informative sites, a total of 65 and 106 amino acids corresponding to conserved sequences in the MH1 and MH2 domains were analysed. The Drosophila melanogaster DAD sequence [BAA22841.1] was incorporated as the outgroup for the phylogenetic construction of both MH1 and MH2. The PhyML algorithm implemented in Seaview was utilised to generate the maximum likelihood phylogenetic tree, using the WAG model (Whelan and Goldman Reference Whelan and Goldman2001). The reliability of branching order was assessed by bootstrap analysis consisting of 1,000 replicates. The final tree visualisation was generated using FigTree software version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).
Reverse transcription PCR
First-strand cDNA was synthesised from RNA extracted at each life cycle stage of F. gigantica, including unembryonated egg, embryonated egg, miracidium, redia, cercaria, metacercaria, NEJ, and adult. The synthesis reaction was performed in a 20-μL volume using the GoScript Reverse Transcription System (Promega, UK), following the manufacturer’s instructions.
Quantification of FgSmad4 gene expression levels with real-time PCR
Life stage expression primer for FgSmad4 was designed from the conserved sequence flanking intron specific for FgSmad4 cDNA to yield PCR products of 275 bp. Standard PCR using Taq DNA polymerase (Qiagen, Germany) and subsequent sequencing were performed to confirm the correct PCR product and to ensure the absence of non-specific PCR product or primer dimers.
The real-time PCR was carried out in a volume of 20 μL using QuantiNova SYBR Green PCR Kit (Qiagen, Germany) consisting of 10 μL of 2x SYBR Green PCR Master mix. The amplification reaction was performed in a real-time PCR (Bio-Rad, CFX96). The PCR condition was used with the following conditions: 2 min at 94 °C for denaturation, 40 cycles of 94 °C for 5 s, 57 °C for 10 s, and 72 °C for 30 s, and 10 min of a final extension step at 72 °C.
To quantify the relative expression level of FgSmad4 transcript in the life stage of F. gigantica, we used 2−ΔCT method as described by Silver et al. (Reference Silver, Best, Jiang and Thein2006). The expression of FgSmad4 was normalised by F. gigantica glyceraldehyde-3-phosphate dehydrogenase (FgGAPDH). Each reaction was performed in triplicate (technical replicates), and for each developmental stage, three independent biological replicates were conducted.
Life stage expression data of FgSmad4 were presented as mean ± standard error of the mean derived from three independent experiments. A bar chart was created using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA, USA). Statistical analysis was conducted using one-way ANOVA, with p-values calculated using Tukey’s post hoc test, and significance was considered for p-values < .05 in all statistical comparisons.
Results
Database search for F. gigantica Co-Smad sequences (FgSmad4)
Nucleotide sequences of Smad4 from mammalian hosts of F. gigantica, including human (Homo sapiens Smad4 [NP_001393970.1]), cattle (Bos taurus Smad4 [NP_001069677.1]), and buffalo (Bubalus bubalis Smad4 [NP_001277793.1]), retrieved from the GenBank database, were employed in a tblastn search. The search identified corresponding nucleotides in the genome of F. gigantica, PRJNA230515 - Uganda_cow_1. tblastn analysis revealed three matching regions within the FGIG_03163 transcript, located at positions 124-498 nt, 1474-1872 nt, and 2038-2262 nt, with identity scores of 77.6%, 72.2%, and 70.7%, respectively. Diagrams detailing the matching positions are presented in Figure 1.
Figure 1. The tblastn results illustrate the corresponding positions identified in the F. gigantica genome. The red highlighted positions represent matches found through a tblastn search using mammalian Smad4 as the query sequence.
Identification and Isolation of FgSmad4 cDNA
The isolated FgSmad4 cDNA consisted of 4,014 bp in length encoding for a protein comprising of 771 amino acids. Within the FgSmad4 cDNA, there was a 5′-untranslated region (UTR) of 51 nt in length, an open reading frame spanning 2,316 nt, and a 3’ UTR of 1,647 nt, followed by a poly A tail. The start codon (ATG) and stop codon (TGA) were predicted to be located at nucleotides 52–54 and 2,365–2,367, respectively.
In silico characterisation of the predicted FgSmad4 protein
The FgSmad4 cDNA encoded a 771 amino acid polypeptide, predominantly composed of glycine (11.3%), serine (11.3%), proline (8.8%), leucine (8.0%), and alanine (6.4%). The protein sequence analyses revealed the conservation of key Smad protein domain features, including the Mad Homology 1 (MH1) domain at the N-terminal and the Mad Homology 2 (MH2) domain at the C-terminal, with FgSmad4 consisting of 125 amino acids in the MH1 domain (33–157 amino acids) and 246 amino acids in the MH2 domain (491–736 amino acids). Additionally, essential structural elements of the Co-Smad protein subfamily were identified, including the Nuclear Localisation Signal (NLS; FARRAIESLVKKLKEKRED) and a DNA-Binding Motif (DBM; RTLDGRMQIAG) in the MH1 domain, along with the Nuclear Export Signal (NES; VDLAALSL) situated at the initiation of the linker region (Figure 2).
Figure 2. Translated amino acid sequences of FgSmad4 derived from a 2,316-bp open reading frame (ORF). The start codon (ATG) and stop codon (TGA) are indicated in underlined bold text. The conserved MH1 domain is shaded with a grey background, whereas the MH2 domain is highlighted with black letters on a pink background. Nuclear localization signals (NLS) are represented by red letters within a red box, and nuclear export signals (NES) are boxed in green. The DNA binding domain is highlighted within light blue block.
Further computational analyses predicted a molecular weight of 82.06 kDa and a theoretical isoelectric point of 6.43 for the translated FgSmad4 protein. Additionally, the deduced FgSmad4 protein was characterised as hydrophilic, with a grand average of hydropathicity value of –0.411. The calculated instability index was 55.14, predicting overall protein instability.
Sequence comparison of FgSmad4 with other Co-Smad proteins
The FgSmad4 identified in this study was identical to mothers against decapentaplegic of F. gigantica in Genbank database [TPP57878.1]. Similarity search by PSI-blast indicated that the entire sequence of FgSmad4 was similar to mothers against decapentaplegic of Fasciolopsis buski [KAA0186896.1] and Schistosoma haematobium [XP_051073969.1] with similarity of 79% and 53.37%, respectively.
Comparison of the MH1 domain, FgSmad4 exhibited the highest similarity with other Smad4 proteins from trematodes, particularly CsSmad4 and OvSmad4 from Clonorchis sinensis [KAG5446872.1] and Opisthorchis viverrini [KER20450.1], showing similarities of 84.80%, and 84.00%, respectively. The FgSmad4 protein displayed an average of 80.00% homology in sequence to the MH1 region of Co-Smads from mammals (HsSmad4, BtSmad4, and BbSmad4) but revealed lower identity to cestodes (66.67%) and nematodes (50.00%–62.40%).
In the MH2 domain of FgSmad4, the greatest similarities were observed within platyhelminthes (74.80%–96.77%), with similarities of 85.55%, 86.21%, and 96.77% to C. sinensis, O. viverrini, and F. buskii, respectively. The similarity for cestodes was found to be 74.80%. The MH2 domain showed closer homology to mammals (65.45%) than to nematodes (34.96%–43.90%) (Table 2).
Table 2. Comparison of FgSmad4 and other Co-Smad homologues from selected organisms
Genomic structure of FgSmad4
The FgSmad4 gene organisation was determined by blastn search of FgSmad4 cDNA sequences against the F. gigantica genome database via online and local analyses. Within the FgSmad4 gene, there were nine exons, covering a total length of approximately 56.64 kb. The sizes of these nine exons were: 63, 294, 215, 194, 723, 193, 189, 357, and 1,786 bp, respectively (Figure 3).
Figure 3. Genomic organization of FgSmad4. Exons, introns, and UTRs are depicted in shaded boxes, lines, and striped boxes, respectively. Numbers denote the size of exons and introns in base pairs.
Multiple sequence alignment
To identify conserved domain sequences between FgSmad4 and related species, multiple sequence alignments were performed on Co-Smad sequences from various organisms. The alignment revealed that FgSmad4 shares 50%–85% amino acid identity in the MH1 domain and 36%–97% in the MH2 domain. Additionally, conserved amino acid domains were also found to be present in other Co-Smad homologues across different organisms (Figure 4).
Figure 4. Multiple sequence alignment of FgSmad4 protein and other Co-Smad homologues. Conserved amino acid sequences and specific residues are highlighted in vertical columns. Asterisks (*) denote exact matches across all aligned sequences, colons (:) indicate strong similarity, dots (.) signify weak similarity, and the absence of a symbol indicates no similarity among amino acids. The boxed sequences indicate conserved motifs, including the nuclear localisation signal (NLS), the DNA binding motif (DBM), and the nuclear export signal (NES). The sequences included in the alignment are as follows: FgSmad4 (Fasciola gigantica), SmSmad4 (Schistosoma mansoni), CsSmad4 (Clonorchis sinensis), OvSmad4 (Opisthorchis viverrini), EgSmadD (Echinococcus granulosus), EmSmadD (Echinococcus multilocularis), BtSmad4 (Bos taurus), BbSmad4 (Bubalus bubalis), and HsSmad4 (Homo sapiens).
Phylogenetic relationships of FgSmad4
The phylogenetic relationships of FgSmad4 with other Co-Smad proteins were assessed by constructing a phylogenetic tree based on sequences in the MH1 and MH2 domains. The tree incorporated sequences of FgSmad4 and other Co-Smad proteins from various organisms, including mammalian hosts (human, cattle, buffalo), free-living nematodes (C. elegans), parasitic nematodes (H. contortus), free-living flatworms (Planaria) and parasitic flatworms (trematode, cestode).
In the MH1 domain analysis, FgSmad4 exhibited its closest relationship with homologues of Smad4 from parasitic trematodes, supported by a high bootstrap value. Notably, in comparison to other organisms, FgSmad4 demonstrated a closer relationship to mammalian hosts such as HsSmad4, BbSmad4, and BtSmad4 than to those of cestodes and nematodes (Figure 5A).
Figure 5. Phylogenetic relationships of FgSmad4 and Co-Smad proteins from related organisms based on the MH1 domain (A) and MH2 domain (B). The phylogenetic trees were inferred by maximum likelihood using 65 and 106 amino acid sequences of the MH1 and MH2 regions, respectively. The DAD sequence from Drosophila melanogaster [BAA22841.1] served as the outgroup for both MH1 and MH2 phylogenetic analyses. The FgSmad4 sequence identified in our study [PP856693] is highlighted in red bold font. Bootstrap values greater than 50% are displayed at the nodes of the phylogenetic trees (1000 replicates).
In the phylogenetic analyses of the MH2 region, Co-Smad proteins were categorised into three major branches according to taxonomic classification: Mammalian hosts, Nematodes, and Platyhelminthes. FgSmad4 formed a distinct subgroup within the Platyhelminthes branch, positioning itself with the trematode Smad4 proteins and separating from free-living flatworms and cestodes (Figure 5B).
Predicted two-dimensional and three-dimensional structures
Structural analysis of FgSmad4 protein conducted via the online Phyre2 tool revealed its secondary structure composition, with 14% beta strands, 12% alpha helices, and 2% transmembrane helices. The majority of the core structure was predicted to be disordered.
The three-dimensional model of FgSmad4 (Figure 6) was visualised using Chimera software. This modelled protein was generated based on 216 amino acids (75% identity) from the conserved Smad domain of H. sapiens Smad4 (d1dd1a), with a maximum confidence score of 100%.
Figure 6. Modelled three-dimensional (3D) structure of FgSmad4. The 3D structure prediction was generated using Phyre2 homology modelling with reference to the template model of Homo sapiens Smad4 (d1dd1a). Chimera was utilised for visualizing the protein model. The colour scheme in the models indicates blue for the N-terminus and red for the C-terminus.
Life stage expression of FgSmad4
Quantitative reverse transcription was performed to evaluate the expression of the FgSmad4 gene in different developmental stages of F. gigantica, including unembryonated egg, embryonated egg, miracidium, cercaria, metacercaria, NEJ, and adult. For normalisation, FgGAPDH was chosen as a reference gene due to its consistent expression levels throughout the entire lifecycle.
The expression of FgSmad4 was detected in all examined stages of F. gigantica. The highest expression was observed in the NEJ stage, followed by unembryonated egg, metacercaria, miracidium, and redia, respectively. In contrast, the expression of FgSmad4 was lowest in the adult liver fluke. Notably, a significant disparity in expression levels between NEJ and adult was observed, with NEJ exhibiting a 100-fold higher expression. Both miracidium and redia displayed similar levels of FgSmad4 expression, which were nearly 10 times higher than in adult stage. Among the larval stages of F. gigantica, NEJ exhibited the highest level of FgSmad4 expression, which was approximately 61 times greater than in cercaria (Figure 7).
Figure 7. Quantitative expression analysis of FgSmad4 across various life cycle stages of F. gigantica. The relative mRNA expression levels of FgSmad4 throughout the parasite’s life stages were determined via quantitative PCR, with FgGAPDH serving as the reference gene. The data represent the mean ± standard error of the mean from three independent biological replicates.
Discussion
The Smad proteins are essential components of TGF-β and BMP signalling transductions with Co-Smad being a central mediator in these processes. In the present study, we identified and characterised a Co-Smad homologue from F. gigantica termed FgSmad4. From our results, it appears that FgSmad4 represented only one type of the Co-Smad observed in F. gigantica genome. This aligned with similar observations in other trematodes, such as S. mansoni, where a single Co-Smad has been identified in the genome (Osman et al. Reference Osman, Niles and LoVerde2004). In the case of cestode, Echinococcus granulosus and E. multilocularis genomes were found to contain only a single Co-Smad known as EgSmadD and EmSmadD, respectively (Zavala-Góngora et al. Reference Zavala-Góngora, Derrer, Gelmedin, Knaus and Brehm2008; Zhang et al. Reference Zhang, Wang, Wang, Pu, Yang, Li, Wang, Lü, Lu, Zhang, Vuitton, Wen and Lin2014). Our findings are in agreement with the recent study of Wu et al. (Reference Wu, Kong, Zhang and Di2023) who documented a similar finding in the number of Co-Smad in F. gigantica. However, we have here provided further details on the characteristics of FgSmad4.
The specific number of Co-Smad proteins and their significance can vary not only between phyla but also within species and genera. Research conducted by Masuyama et al. (Reference Masuyama, Hanafusa, Kusakabe, Shibuya and Nishida1999) on Xenopus spp. revealed the existence of two Co-Smads, XSmad4α and XSmad4β, sharing a 70% similarity in amino acid sequences, the author suggested that these proteins may have overlapping but distinct functions. In most mammals, the presence of a single Co-Smad highlights a notable contrast to the diversity observed in Xenopus spp., emphasising the variability in Co-Smad configurations across different organisms. Invertebrates, with their simpler anatomical structures compared to mammals, tend to have less complex TGF-β signalling pathways. Notably, both free-living and parasitic flatworms, similar to mammals, possess only one Co-Smad. In contrast, parasitic and free-living nematodes, such as C. elegans (Ce-SMA-4 and Ce-DAF-3) and Haemonchus contortus (Hc-DAF-3), have been reported to contain multiple Co-Smad molecules, each specialised for their specific biological processes (Di et al. Reference Di, Liu, Zhang, Li, He, Wang, Ahmad, Hassan, Fang and Hu2019). This diversity may reflect the evolutionary adaptations and functional requirements of various organisms in their respective ecological niches.
The FgSmad4 sequence obtained from 5’ and 3’ RACE was found to encode 771 amino acids, which aligned closely with the entry sequence FGIG_03163 in the F. gigantica genome database (PRJNA230515). Our result suggests that FgSmad4 likely has a single transcript variant, given that both our 5’ and 3’ RACE results encoded identical protein isoform sizes. Additionally, the exon–intron structure derived from our sequence closely matches the pattern observed in FGIG_03163, which comprises nine exons and eight introns.
The length of the Co-Smad protein appears to be relatively conserved across animal species with the range of 400–500 amino acids (Makkar et al. Reference Makkar, Metpally, Sangadala and Reddy2009). In our study, FgSmad4 was approximately 218–219 amino acids longer than the mammalian Co-Smad proteins (553 amino acids in sheep, 552 amino acids in human). Notably, the Smad4 proteins in trematode and cestode exhibit a larger size compared to Smad4 in mammals and other reported animals. This observation aligned with Osman et al. (Reference Osman, Niles and LoVerde2004), who noted that the Smad4 homologue of S. mansoni (738 amino acids) possessed an additional 200 amino acids compared to other known Co-Smad proteins. Likewise, Zhang et al. (Reference Zhang, Wang, Wang, Pu, Yang, Li, Wang, Lü, Lu, Zhang, Vuitton, Wen and Lin2014) and Zavala-Góngora et al. (Reference Zavala-Góngora, Derrer, Gelmedin, Knaus and Brehm2008) documented that EgSmadD in E. granulosus and EmSmadD from E. multilocularis comprise 719 amino acids, providing additional evidence that supports the idea of an enlarged size in Co-Smad proteins within parasitic flatworms. The function or relevance of the increased length of these Co-Smad proteins remains unknown.
Classically, Smad proteins are characterised by two key conserved domains: the MH1 situated at the N-terminus, crucial for specific DNA binding, and the MH2 located at the C-terminus, essential for protein–protein interactions. These domains are linked by a proline-rich non-conserved intermediate region (Liu Reference Liu2003; Liu et al. Reference Liu, Hata, Baker, Doody, Cárcamo, Harland and Massagué1996). FgSmad4 exhibited similarity with Co-Smad proteins from various animal species. Within the MH1 domain, the highest degree of similarity was observed among trematode species, exceeding 80.00%, whereas the observed similarity to nematode and cestode species was comparatively lower at 50.00%–68.80% and 66.67%, respectively. Remarkably, the MH1 sequences of FgSmad4 and those of mammalian hosts exhibited a significant similarity of 80.00%. For the MH2 domain, FgSmad4 displayed significant conservation, aligning well with taxonomic classifications. FgSmad4 exhibited the greatest similarity within the platyhelminth group, particularly showing the most similarity to other trematodes, ranging from 83.73% to 96.77%. The observed similarity extended to 74.80% for cestodes, whereas relatively lower similarities were noted compared to nematodes (34.96%–43.90%) and mammalian hosts (65.45%). Our findings revealed that both MH1 and MH2 sequences share the greatest similarity with trematode species, with MH1 exhibiting a notable similarity to mammalian hosts. This observation was consistent with the findings in SmSmad4, where the MH1 domain showed a higher similarity (81%–87%) to the MH1 domain of Smad4 from mammals, whereas the MH2 domain displayed a lower level of similarity (Osman et al. Reference Osman, Niles and LoVerde2004). These findings underscore the conserved nature of the MH1 domain, particularly among trematodes, and suggest a potential co-evolutionary relationship between host and parasite.
In life stage expression analyses, the presence of the FgSmad4 transcript was observed in all examined stages of F. gigantica. This pattern aligns with the findings in SmSmad4, where the SmSmad4 gene expression was documented throughout the entire life cycle of S. mansoni (Osman et al. Reference Osman, Niles and LoVerde2004). Notable variation in the expression levels of FgSmad4 was observed throughout the life cycle of F. gigantica, highlighting a significantly high transcript level in developing life cycle stages, including unembryonated egg, redia, and NEJ. In contrast, embryonated egg, cercaria, and adult exhibited relatively low levels of FgSmad4 transcript, indicating a potentially diminished significance of this gene in these specific stages.
The transcript level of FgSmad4 in unembryonated eggs was nearly 10 times higher than in embryonated eggs, underscoring its essential role in initiating developmental processes during early embryogenesis in F. gigantica. In the NEJ stage, FgSmad4 expression showed a dramatic increase, exceeding levels in cercaria by more than 60-fold and adult stages by more than 100-fold. This notable increase in expression, along with previously observed high FgTLM levels in NEJ (Japa et al. Reference Japa, Prakhammin and Flynn2022), indicates that FgSmad4 may be critical for preparing the juvenile parasite to adapt to the host environment, likely by promoting growth, driving essential developmental changes, and supporting survival.
We observed a striking 13-fold increase in FgSmad4 expression in the metacercaria stage compared to cercaria, suggesting a critical role for FgSmad4 during this transitional phase. This elevated expression aligns with previously reported high levels of FgTLM in metacercaria (Japa et al. Reference Japa, Prakhammin and Flynn2022). Although traditionally considered a dormant stage, metacercaria actively maintains essential biological processes to ensure survival and viability until reaching a mammalian host. Zhang et al. (Reference Zhang, Cwiklinski, Hu, Zheng, Sheng, Zhang, Elsheikha, Dalton and Zhu2019) identified key regulatory functions within the transcriptional profile of F. gigantica metacercaria, including gene transcription, protein phosphorylation, and signal transduction, which collectively support critical processes such as metabolic regulation, nucleotide synthesis, pH balance, and endopeptidase activity. Taken together, our findings and Zhang’s observations suggest that FgSmad4 likely acts as a key mediator in TGF-β signalling pathways, coordinating a range of biological and metabolic processes essential for the metacercaria’s resilience and readiness for host transmission. Further studies are needed to elucidate the specific biological processes involving FgSmad4 at this stage.
In this study, real-time PCR analysis of FgSmad4 expression across developmental stages revealed stage-specific patterns, indicating distinct regulatory functions at each phase. These functions likely include regulating growth, supporting developmental transitions, and enabling the parasite to adapt to varying environmental conditions. To validate and expand on these findings, further protein-level analyses, such as western blot quantification and localisation studies through immunohistochemistry or in situ hybridisation, could provide deeper insights into FgSmad4’s role in mediating stage-specific developmental processes and adaptive mechanisms, clarifying its essential role in the biology of F. gigantica.
Conclusion
This study provides a detailed characterisation of FgSmad4, the sole identified Co-Smad protein in F. gigantica. FgSmad4 exhibits the typical molecular features of the Co-Smad family, including the defining MH1 and MH2 domains. Expression analysis revealed significant variation in FgSmad4 transcript levels across developmental stages, with the highest expression observed in the NEJ stage and the lowest in mature flukes. These findings underscore the conserved structure and potentially crucial role of FgSmad4 as a signalling mediator in F. gigantica. Given that Co-Smad participates in numerous biological pathways, further exploration of FgSmad4’s functions could yield valuable insights into the TGF-β signalling pathway in this important parasite, enhancing our understanding of host-parasite interactions. This signal transduction pathway may represent a promising target for future drug development and vaccine initiatives.
Acknowledgements
The authors are grateful for the technical assistance provided by Ms. Wannapha Kanameung and Ms. Thanawadee Chaiphutorn.
Author contribution
O.J. and R.J.F. conceived and designed the study. O.J., C.P., and K.K. collected and maintained the parasites. O.J. performed the experiments, data collection, and bioinformatics analyses. O.J. and K.P. analysed and interpreted the data. O.J. and R.J.F. drafted and revised the manuscript. All authors read and approved the final manuscript.
Financial support
This research project was supported by the Thailand Science Research and Innovation Fund and the University of Phayao (Grant No. FF66-RIB070).
Competing interest
The authors declare that they have no competing interests.
