In silico characterisation of the complete Ly6 protein family in Fasciola gigantica supported through transcriptomics of the newly-excysted juveniles

Abstract

Fasciola gigantica is one of the aetiological trematodes associated with fascioliasis, which heavily impacts food-production systems and human and animal welfare on a global scale. In the absence of a vaccine, fascioliasis control and treatment is restricted to pasture management, such as clean grazing, and a limited array of chemotherapies, to which signs of resistance are beginning to appear. Research into novel control strategies is therefore urgently required and the advent of 'omics technologies presents considerable opportunity for novel drug and vaccine target discovery. Here, interrogation of the first available F. gigantica newly excysted juvenile (NEJ) transcriptome revealed several protein families of current interest to parasitic flatworm vaccine research, including orthologues of mammalian complement regulator CD59 of the Ly6 family. Ly6 proteins have previously been identified on the tegument of Schistosoma mansoni and induced protective immunity in vaccination trials. Incorporating the recently available F. gigantica genome, the current work revealed 20 novel Ly6 family members in F. gigantica and, in parallel, significantly extended the F. hepatica complement from 3 to 18 members. Phylogenetic analysis revealed several distinct clades within the family, some of which are unique to Fasciola spp. trematodes. Analysis of available proteomic databases also revealed three of the newly discovered FhLy6s were present in extracellular vesicles, which have previously been prioritised in studying the host-parasite interface. The presentation of this new transcriptomic resource, in addition to the Ly6 family proteins here identified, represents a wealth of opportunity for future vaccine research.

Fig. 1. Multiple sequence alignment of domain region protein sequences (C₁XXC₂ motif to GPI anchor, N-terminal signal peptide and C terminal hydrophobic region removed) for the twenty novel FgCD59 members. Reference sequences from S. mansoni (SmLy6B and SmLy6G) and F. hepatica (FhCD59-1.1) are included as denoted by the black bar to the left of the alignment. Conserved motifs are indicated above the alignment, with the ten cysteines highlighted by the black boxes. GPI anchors are denoted in grey. Predicted cysteine bonds are indicated by the grey brackets above the alignment. Alignments produced in CLUSTAL Omega and annotated in Jalview.

Fig. 2. De novo tertiary structural predictions for the twenty novel FgLy6s, produced using Rosetta. Beta strands, loops and helices are represented in yellow, green and red, respectively. Note the conserved central fold and presence of a defined ‘three finger’ motif, primarily characterised by three protruding loops at the top of the protein structure. C and N terminals are also annotated, where visible. The strand breaks visible in FgLy6-B, -I and -J (blue boxes) are artefacts of uniform snapshot scaling and are not present in the full model.

Fig. 3. Phylogenetic analysis of all known CD59 family members of F. gigantica, F. hepatica and S. mansoni. Sequences were processed to remove the N-terminal sequence prior to the C₁XXC₂ motif and C-terminal post GPI anchor before alignment using Clustal Omega. SmLy6D, a double-domain CD59, was further divided into two (D1 and D2). Maximum likelihood analysis was performed in MegaX to 2000 bootstraps with JTT substitution. Life stage-specific expression profiles are indicated by boxes in addition to in vitro expression data where applicable. Clades were defined with an ancestral support cut-off of ≥ 40.