**5. Conclusions**

We propose that *F. hepatica* exploits two distinct SODs to defend against host-generated ROS during early invasion and infection. We have shown that these proteins have unique expression profiles and secretory pathways, and are, thus, likely to play divergent roles in the development and growth of the parasite in its mammalian host. Going forwards, it will be imperative to define how these proteins interact with each other and with the slew of other antioxidant proteins secreted by the parasite during early invasion, and thus work in concert to detoxify ROS intracellularly and in the extracorporeal environment.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11101968/s1, Table S1. Trematode and mammalian amino acid sequence details for phylogenetic analysis. Table S2. Similarity and identity of trematode and mammalian SOD1 and SOD3 amino acid sequences. Table S3. Significance of *F. hepatica* NEJ survival rates when co-cultured with enzymatically generated superoxide dismutase in vitro. Figure S1. Alignment of mammalian and trematode SOD1 and SOD3 amino acid sequences for phylogenetic analysis. Selection of conserved blocks from the complete SOD alignment was determined by Gblocks 0.91b [18,19], with 41% of the positions conserved. Metal binding sites are indicated by grey bars. Gaps are shown as dashes. Variable residues are highlighted in red. *Fasciola hepatica* and *Fasciola gigantica* sequences are highlighted in bold. The first amino acid is numbered 67 (relative to FhSOD3—

see Figure 1 in main text). Figure S2. Phylogenetic analysis of trematode and mammalian SOD amino acid sequences. Phylogenetic analysis was carried out using the maximum likelihood method and the Whelan and Goldman model [21]. The tree with the highest log likelihood (−3031.18) is shown. The percentage of trees in which the associated taxa clustered together (out of 1000 iterations) is shown below the branches (Bootstrap support values less than 50% are omitted). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Figure S3. Expression and enzyme activity of rFhSOD1 and rFhSOD3. (A) Recombinant proteins expressed in *E. coli* ClearColi cells and purified by affinity column, resolved in a 4–20% SDS-PAGE gel and stained with Biosafe Coomassie. (B) Recombinant proteins were electro-transferred onto a nitrocellulose membrane and probed with mouse monoclonal antibodies to poly-histidine (1:10,000). P; *E. coli* pellet, S; purified supernatant. M; molecular weight in kDa (Precision Plus Protein Dual, BioRad). (C) Activity of recombinant FhSOD1 and FhSOD3 compared to native bovine erythrocyte SOD (BS). Both rFhSOD1 and rFhSOD3 exhibited a specific activity of ~400 U/mg relative to the standard curve, where one unit is the amount of enzyme required to exhibit 50% dismutation of the superoxide radical. Figure S4. The predicted 3D structure of (A) FhSOD1 and (B) FhSOD3. Structure predicted by the AlphaFold Protein Structure Database [25,26]. The colour of each amino acid indicates the per-residue confidence score (pLDDT) where dark blue resembles very high confidence (> 90 pLDDT), light blue resembles confident (90 > pLDDT > 70), yellow indicates low confidence (70 > pLDDT > 50). Figure S5. Specificity of polyclonal and anti-peptide antibodies against recombinant *F. hepatica* SODs. (A) Purified recombinant (0.05 ug/well) FhSOD1 and FhSOD3 were resolved in a 4–20% SDS-PAGE gel and stained with Biosafe Coomassie. Lane 1: BS; lane 2: rFhSOD1; lane 3: rFhSOD3. (B–F) Western blot analysis of rFhSOD1 and rFhSOD3. Lane 1: BS; lane 2: rFhSOD1; lane 3: rFhSOD3. Immuno blots were probed with (B) rabbit pre-immune sera (negative control), (C) anti-FhSOD1 peptide antibodies, (D) anti-FhSOD3 peptide antibodies, (E) anti-rFhSOD1 polyclonal antibodies, and (F) anti-rFhSOD3 polyclonal antibodies raised in rabbit. Figure S6. SOD and catalase inhibit the lethal effects of superoxide and hydrogen peroxide on *F. hepatica* NEJ in vitro. (A) Principal of the assay demonstrating the step-wise reduction of hypoxanthine into xanthine and superoxide by xanthine oxidase and the subsequent production of hydrogen peroxide and uric acid as a by-product of the reaction. (B) Enzyme activity of rFhSOD1 and rFhSOD3 vs. BS prior to their inclusion in the NEJ assay at 0.01 mg/mL (rFhSODs) and 0.001 mg/mL (BS) (C) Killing of *F. hepatica* NEJ using enzymatically generated superoxide is prevented via the addition of catalase (CAT) and BS in a dose-dependent manner. PBS; negative control, Vehicle; positive control (superoxide generated in vitro). (D) The impact of superoxide and hydrogen peroxide on *F. hepatica* NEJ after 24 h of co-culture is shown. Black scale bars; 100 μM, white scale bar; 50 μM, ns; not significant.

**Author Contributions:** Conceptualization, N.E.D.C. and J.P.D.; methodology, N.E.D.C., C.D.M.V., H.L.J., K.C., A.F. and J.P.D.; formal analysis, N.E.D.C. and K.C.; investigation, N.E.D.C.; resources, J.P.D.; data curation, N.E.D.C. and K.C.; writing—original draft preparation, N.E.D.C.; writing review and editing, N.E.D.C., C.D.M.V., K.C. and J.P.D.; visualization, N.E.D.C., H.L.J. and K.C.; supervision, J.P.D.; project administration, N.E.D.C.; funding acquisition, J.P.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Science Foundation Ireland (SFI, Ireland) Research Professorship grant 17/RP/5368.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The transcriptome data sets used to extrapolate the FhSOD gene transcription were previously reported by Cwiklinski et al. [5] and are available in the European Nucleotide Archive repository, PRJEB6904; http://www.ebi.ac.uk/ena/data/view/PRJEB6904 (30 August 2022). The mass spectrometry proteomics data analysed as part of this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following data set identifiers (a) NEJ specific datasets Cwiklinski et al. [6]: PXD007255, PXD016561; (b) immature fluke Cwiklinski et al. [7]: PXD021221; (c) adult ES and EV datasets Murphy et al. [30]: PXD002570 and PXD016561.

**Acknowledgments:** The authors would like to thank Siobhán Gaughan for her invaluable administrative assistance in her role as Project Manager of the Molecular Parasitology Laboratory at the University of Galway, Ireland.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; nor in the decision to publish the results.
