*3.1. The F. hepatica Genome Contains Developmentally Regulated Cytosolic and Extracellular SODs*

Analysis of the *F. hepatica* genome identified three superoxide dismutase genes, corresponding to the three types of SOD molecules identified within helminth parasites. Here, we report the two SODs that are known to be secreted by *F. hepatica* and, therefore, act at the host-parasite interface; a cytosolic SOD referred to herein as FhSOD1; and an extracellular SOD identified according to the presence of an N-terminal signal peptide sequence referred to herein as FhSOD3 (Figure 1). FhSOD1 corresponds to the previously reported cytosolic SOD [15,16].

**Figure 1.** Comparative analysis of *F. hepatica* and *F. gigantica* SOD1 and SOD3 amino acid sequences. Metal binding sites are highlighted in grey and predicted signal peptides in blue. The arginine residue responsible for guiding the superoxide anion into the active site is indicated in green and the cysteine residues involved in disulphide bond formation are shown in red. Gaps are indicated by dashes. Amino acid conservation is shown below the alignment.

The two SOD genes display different transcriptional profiles across the developmental parasitic stages found within the mammalian host (Figure 2A). FhSOD1 is constitutively expressed at low levels across all the life cycle stages analysed. In contrast, FhSOD3 displays a markedly higher level of transcription by the infective stage metacercariae and the NEJ, which then drops to comparable levels to FhSOD1 expression within the immature and adult flukes.

**Figure 2.** *Cont*.

**Figure 2.** Expression profile of FhSOD1 and FhSOD3 throughout the *F. hepatica* life cycle within the mammalian host. (**A**) Graphical representation of the stage-specific transcription of the FhSOD1 and FhSOD3 genes displayed as transcripts per million (TPM) extrapolated from the transcriptome study of the metacercariae (Met), newly excysted juveniles at 1, 3, and 24 h post-excystment (NEJ 1 h, NEJ 3 h, NEJ 24 h), immature and adult liver flukes by Cwiklinski et al. [5]. (**B**) Graphical representation of the FhSOD1 and FhSOD3 proteins secreted within the ES protein fraction by the NEJ, immature, and adult parasites, represented by the exponentially modified protein abundance index (emPAI). The adult secretome data are displayed as the EV-depleted fraction (Adult\_ES), and the microvesicle (Adult\_15K) and exosome (Adult\_120K) sub-fractions of the EV component recovered following centrifugation at 15,000× *g* and 120,000× *g*, respectively. (**C**) Graphical representation of the FhSOD1 and FhSOD3 protein abundance within the somatic proteome of the metacercariae (Met); newly excysted juvenile (NEJ) 3, 24, and 48 h post-excystment (NEJ 3 h; NEJ 24 h; NEJ 48 h); and immature liver flukes. The proteomic data for the NEJ, immature, and adult parasites are extrapolated from Cwiklinski et al. [6], Cwiklinski et al. [7], and Murphy et al. [30], respectively.

Despite the relatively lower level of gene transcription, proteomic analysis revealed that FhSOD1 is more abundantly secreted than its extracellular counterpart, FhSOD3, predominantly by the NEJ and adult parasite stages (Figure 2B). Analysis of the adult secretome revealed that, in addition to being secreted within the protein soluble fraction, FhSOD1 is also found within extracellular vesicles (EVs), specifically the microvesicles that are known to be released by the gastrodermal cells of the gut. This is consistent with other *F. hepatica* proteins that lack a signal peptide sequence for classical secretion, highlighting that the parasite uses non-classical routes of secretion to release multiple molecules that interact with its host [31]. FhSOD3 is abundantly secreted by the NEJ parasites consistent with its gene transcription profile, with low levels secreted by immature flukes and no protein detected within the adult stages (Figure 2B). The somatic proteome profile for both FhSOD proteins is also consistent with that observed at the gene level, with comparable levels of FhSOD1 being detected across all the life cycle stages analysed, in comparison to FhSOD3, which displays a higher protein abundance within the metacercariae and NEJ 24 h post-excystment (Figure 2C).

#### *3.2. Cytosolic and Extracellular SODs Are Distinct Yet Highly Conserved in Trematodes*

Interrogation of available Platyhelminth genomes revealed a number of homologous SOD1 and SOD3 genes (SOD1: n = 14; SOD3: n = 19) from parasitic flatworms of class Trematoda and free-living flatworms of class Turbellaria. Phylogenetic analysis separates the SOD1 and SOD3 sequences into two distinct clades (Figure S2). The *F. hepatica* SOD1 and SOD3 sequences cluster with homologous sequences from F. gigantica and Echinostoma caproni, as is expected in these closely related parasite species. The majority of the SOD genes are present within their respective genomes as single copy genes, with the exception of Paragonimus westermani, which appears to contain two SOD1 sequences, while several *Schistosoma* spp. appear to contain two SOD3 sequences that cluster into distinct sub-clades (Figure S2). The SOD1 sequence from the free-living flatworm *Schmidtea mediterranea* does not cluster with its parasitic counterparts within the SOD1 clade.

All the SOD1 sequences analysed were comparable with that observed for the *F. hepatica* and *F. gigantica* SOD1 sequences, in that they all lacked an N-terminal signal peptide for classical secretion in line with the predicted cytosolic function of these proteins (data not shown). The majority of the Platyhelminth SOD3 sequences contain a signal peptide implying a common extracellular role for these proteins. Three Platyhelminth SOD3 sequences were missing the signal peptide sequence in the current respective genome assemblies (*E. caproni* SOD3, *T. regenti* SOD3, *S. rodhaini* SOD3; Table S1), with further investigation required to confirm their annotation.

Alignment of FhSOD1 with FhSOD3 revealed 34.64% sequence identity and 63.28% sequence similarity (ClustalO/uniprot) (Table S2). Comparison with the predicted *F. gigantica* SOD1 and SOD3 amino acid sequences shows a high level of conservation between the two species, with 98.70 and 100.00% identity and similarity, respectively, between the SOD1 sequences, and 96.61 and 99.44% identity and similarity, respectively, between the SOD3 sequences (Table S2). FhSOD1 has an average sequence identity of 69.81% to other Platyhelminth SOD1 sequences, and an average sequence identity of 59.42% with the four selected mammalian (*O. aries*, *B. taurus, B. indicus*, and *H. sapiens*) SOD1 sequences (Table S2). The trematode SOD1 sequences showed an average similarity of 90.20%, whereas the SOD3 sequences were more diverse, with an average similarity of 68.40% (Table S2). Collectively, this information suggests that FhSOD1 is more closely related to the human cytosolic enzyme than its own extracellular enzyme. Alignment against the respective *O. aries*, *B. taurus*, *B. indicus*, and *H. sapiens* SOD1 and SOD3 sequences shows 100% conservation of predicted copper and zinc metal binding sites (His69, His71, His86, His94, His103, Asp106, His143) (Figure S1; residue numbering relative to FhSOD3) [11,14,16].
