Fascioliasis is primarily an infection of the livestock caused by F. hepatica and F. gigantica. It is a severe food-borne neglected tropical disease (NTD) that causes massive health and economic loss in the developing countries of the tropics and subtropical areas (1-4). Fasciola spp. has a worldwide distribution and it infects livestock, such as cattle, sheep, and goats resulting in an estimated economic loss of more than 3 billion USD per annum worldwide (4-6). Humans become accidental hosts through consumption of watercress or other fresh aquatic vegetation or by drinking water contaminated with viable parasite metacercariae. Once the larvae are ingested, they develop into adult flukes inside the host and start feeding on the liver tissues. As a result, the liver becomes dysfunctional, and the pathology is characterized by severe damage to the liver tissues and bile ducts (7). According to the World Health Organization (WHO) estimates, at least 2.4 million people are infected worldwide, and 180 million are at risk of infections (8, 9). Fascioliasis is principally treated with a single WHO-approved drug triclabendazole (TCBZ), but recent studies report increasing cases of TCBZ-resistant flukes in Europe, Australia, and in more than 70 countries across the world (10-14). Therefore, the development of new therapeutics against these platyhelminthic parasites is one of the most important challenges for researchers.

Signal transduction through the integration of protein phosphorylation and dephosphorylation by specialized enzymes are essential to respond to certain extracellular signals that help maintain homeostasis as well as other complex cellular adaptation in parasites. Various protein functions, such as cell division, cellular metabolism, growth, differentiation, survival, and apoptosis are regulated through phosphorylation by protein kinases (PKs) that cause alterations in the protein structure by covalent modifications, and hence, facilitates the formation of hydrogen bonds among specific amino acid residues for mediating intracellular signals (15). Protein kinases catalyse the transfer of γ-phosphate from high energy molecules like ATP or GTP to its polypeptide substrates, thereby, modulating their structures and functions. The action of PKs is reversed by phosphatases that remove phosphoryl moieties from their substrates. Structurally, most PKs contain a catalytic domain responsible for binding and phosphorylating target proteins, and a regulatory domain for maintaining a catalytically inactive state to prevent constitutive substrate phosphorylation (16). Protein kinases are either auto phosphorylated or get phosphorylated by other PKs (17). The catalytic core of the PKs consists of a Gly-rich ATP/GTP binding domain in the N-terminus and a conserved Asp residue in the centre (18). Substrate recognition by PKs employs two types of interactions: (i) recognition of the consensus phosphorylation sequence by the active site residues and (ii) distal interactions between the PK and the substrate mediated by regions away from the phosphorylation site in the substrate and regions in PK located distally from its active site. Protein kinases are classified into two superfamilies: (i) the eukaryotic or conventional protein kinases (ePKs), and (ii) the atypical protein kinases (aPKs). The sequences of the aPKs are different than those of ePKs but show kinase activity (18). Based on the phosphate acceptor site in the substrate, ePKs are classified into Ser/Thr kinases (STKs) and Tyr kinases (TKs). Kinases that specifically phosphorylate both Ser/Thr and Tyr are also found (19). The ePKs are grouped according to their homologous catalytic domains, which generally consist of 250-300 amino acid residues (20, 21). According to KinBase (19), ePKs have been classified into eight groups: AGC (cAMP-dependent Protein Kinase or Protein Kinase A/Protein Kinase G/Protein Kinase C), CAMK (Calcium/Calmodulin regulated Kinases), CK1 (Cell Kinase I), CMGC (Cyclin-dependent Kinases and other close relatives), RGC (Receptor Guanylate Cyclases), STE (Sterile Ser/Thr Kinases), TK (Tyr Kinase), and TKL (Tyr Kinase-Like). In addition, a ninth group called ''Other kinases'' exists, consisting of several kinases that cannot be classified into any of the above families (22). Many PK inhibitors have been developed and approved for the treatment of different human diseases, including infections with parasitic helminth, like Schistosoma mansoni (the blood fluke) (23, 24).

Recent sequencing of the F. gigantica genome (25) has provided a crucial platform for further exploration of the biochemistry of the parasite. Understanding the structure and function of proteins encoded by the genome of this platyhelminthic parasite will provide information for structure-based drug design and development. Therefore, the study of the ePKs of F. gigantica is a major step towards these goals. Alteration in the functionality of these enzymes may result in a cascade of pathological signs that might contribute to the mortality of the parasite. Thus, the present work aims at analysing the F. gigantica genome by combined computational approaches, including Hidden Markov Models (HMMs) and phylogenetic analyses to identify all the ePKs encoded in the genome; and to identify potential kinases having a special role in the signalling process of the parasite to utilize them as a novel drug target against F. gigantica. The schematic representation of the methodology is shown in Figure 1.

Figure 1

Schematic representation of the methods used for prediction and classification of the F. gigantica ePKs.

More than 90% of all the cellular proteins undergo phosphorylation at some point or other, which is primarily dependent on the balance between protein kinases and phosphatases. Any perturbation in this equilibrium may result in important cellular processes in an organism. In the present study we identified and classified 455 ePKs encoded in F. gigantica genome, which represents ~2% of the genes encoded in its genome, indicating the importance of protein phosphorylation for regulating the signal transduction processes of the parasite. F. gigantica represented kinases from each major ePKs group. Certain FgePKs have unique domains, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. For this, the FgePKs anticipated to have essential function in parasite survival can be selected followed by heterologous recombinant expression, purification and characterization of the ePKs. Nowadays, libraries based on the inhibitory scaffold structure of PKs are also available. Inhibitor library screening will allow identification and optimization of the predicted hits. Co-crystallization of the hits with the target ePKs can provide detailed view of the several interactions between the hit and the target ePKs (70). Reverse genetics approach has been utilised to characterise various parasitic kinomes (70, 71). Moreover, from this analysis of the F. gigantica genome, 115 kinases were derived that showed <35% query coverage when compared to human ePKs (Table 5). This phylogenetic distance between ePKs of F. gigantica parasite and their human host highlights significant divergences in their respective kinomes, further providing a platform for novel structure-based drug designing. Also, this information can be used to identify many of the associated proteins that regulate ePKs activity. ePKs has been validated as potential targets for drug design and development as they play an essential role in several parasites. Furthermore, latest successful drugs bind ePKs near to the ATP binding site and block ATP access to the kinase to compromise enzyme activity (24). Thus, ePKs of F. gigantica with high sequence similarity to host proteins can also be used as targets since the inhibitor binds in non-conserved residues outside the ATP binding site. Also, the unusual domains found in FgePKs can be used for designing more specific F. gigantica inhibitors. Since ePKs currently represents one of the most attractive drug targets, we are performing studies on the biochemical and structural analysis of FgePKs that we will address in further studies.

The present study provides a useful resource for the selection of high-priority candidates for functional genomic and biological studies in F. gigantica. Methods, such as RNA interference, can now be used for the functional validation of ePKs-encoding genes in F. gigantica. In addition, immunological methods can be used for the identification of ePKs ligands and their localization in flukes. Using these tools, future insights into the roles of ePKs in these parasites could provide a path to understanding the molecular biology, biochemistry and parasite-host interactions of these flukes, and might help in the design of novel interventions. Evidently, this study provides information on ePKs for F. gigantica that will assist future investigations on both fundamental and applied levels. Improved annotation of ePKs from other flukes might also trigger broader comparative investigations.

Mr. Kanhu Charan Das and Dr. Parismita Kalita contributed equally to this paper. The authors declare that there are no competing interests. KCD and PK carried out the experiments. KCD, PK and TT analysed the data, conceived the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript.