**4. Discussion**

Superoxide dismutases (SODs) are amongst a collection of antioxidants that play essential roles in parasite defence against oxygen free radicals generated physiologically by cellular metabolism and externally by innate immune cells, such as macrophages and neutrophils, during invasion and infection [4,7,32]. Similar to many trematodes, the liver fluke *F. hepatica* possesses a complex system involved in host immuno-modulation and immuno-evasion wherein a repertoire of proteins is excreted/secreted to simultaneously defend against and distract the host's immune response [30,31]. By interrogating existing genomic, transcriptomic, and proteomic analysis of various intra-mammalian life stages of *F. hepatica,* we identified several antioxidants in the protein cocktail deployed by the parasite during the early invasive and migratory processes, including both a cytosolic (FhSOD1) and an extracellular (FhSOD3) SOD [5–7]. Identified and characterised in *F. hepatica* for the first time herein, we demonstrate that FhSOD3 is NEJ-associated and, thus, likely to play a key role in invasion of the mammalian host; for example, the penetration of the intestine

and liver tissue. FhSOD1 on the other hand, which is released into host tissues through alternative secretion pathways, may function simultaneously in parasite metabolism and defence against exogenous ROS. Through a series of immuno-localisation, serological, and functional in vitro experiments, we propose that FhSOD1 and FhSOD3 play distinct roles in the development of *F. hepatica* and its interaction with mammalian definitive hosts.

As is observed with other proteins excreted/secreted by *F. hepatica* throughout its life cycle, such as the cathepsin L and B proteases, the transcription and expression of FhSOD1, and FhSOD3 are tightly regulated [6,7]. FhSOD1 is constitutively transcribed across all *F. hepatica* life stages at relatively low levels. FhSOD3, however, is highly expressed in the infective metacercariae and early NEJ, before declining to transcription levels, similar to FhSOD1 in immature and adult worms. The lower level of FhSOD1 transcription in metacercariae and NEJ compared to FhSOD3 is in contrast to the relative protein abundance in these early stages and suggests that the parasite produces and stores FhSOD1 prior to excystment in the small intestine. Both FhSOD1 and FhSOD3 are at their most abundant in *F. hepatica* E/S and somatic extract early in the life cycle, which implies that they are vital during early invasion of the mammalian host. Macrophages and neutrophils play an essential role in the early innate immune response against invading pathogens via the production of ROS, and thus it is reasonable to suggest that *F. hepatica* NEJ, which are vulnerable and living on limited glycogen stores, would have a pre-prepared store of antioxidants to defend against this onslaught of ROS [33]. The abundance of both FhSODs in metacercariae and NEJ may also reflect a defence against ROS arising from normal metabolic processes occurring in the most environmentally robust stage of the parasite.

Of the two proteins, only FhSOD3 possesses an N-terminal signal peptide and is, thus, transported to the extracellular environment via the classical secretory pathway. Fh-SOD1, on the other hand, is found in both the microvesicular fraction and the EV-depleted supernatant of adult *F. hepatica* E/S. This is an interesting finding given that cytosolic SOD enzymes are thought to act solely on endogenous ROS produced during cellular metabolism, and not interact with host-derived ROS [32]. Although the mechanism(s) by which this compartmentalisation of FhSOD1 occurs remains unknown, the presence of FhSOD1 in different vesicular and non-vesicular fractions of the E/S suggests distinct methods of delivery to host immune cells and, thus, different defensive or immuno-modulatory roles. Given the observed homology between the cytosolic SOD of *F. hepatica* and other trematode parasites, it is worth investigating if these findings are unique to this species or if they are indicative of an as-of-yet unexplored generalised helminth defence mechanism. Contrary to the cytosolic SOD, the diversity observed between the signal-peptide containing extracellular SOD, including the apparent duplication of this isoform within *Schistosoma* spp., could indicate unique host-specific adaptations in these worms.

In the current study, neither rFhSOD1 nor rFhSOD3 were immunogenic in experimentally infected sheep. Our findings reflect those previously observed when both experimentally and naturally infected buffalo sera were probed for antibodies to FgSOD1 [17]. Similarly, there has been a lack of detectable antibodies when sera from experimentally infected rats were probed with recombinant serine protease inhibitors (serpins; rFhSrp1/2), and when sera from experimentally infected sheep was probed with recombinant cathepsin B1 (rFhCB1) [28,29]. FhSOD1 is highly homologous to its mammalian counterpart, with an average similarity of 87.34% compared to SOD1 sequences from *O. aries, B. taurus, B. indicus*, and *H. sapiens*. It is possible that the parasite exploits these similarities as yet another strategy of immune evasion, wherein their high host homology and, thus, low immunogenicity allows them to operate undetected and unimpeded. Indeed, this host mimicry has been observed in *Schistosoma mansoni*, another trematode parasite of humans, leading to concerns that this high homology with host molecules may induce autoimmune responses during vaccine trials [34].

Unlike *F. hepatica*, the cytosolic SOD of *S. mansoni* (SmCT-SOD) is expressed at the highest levels in adult worms, leaving the larval stages susceptible to immune elimination via host-generated superoxide [34]. In these parasites, SmCT-SOD was localized to the

tegument and gut epithelium of adults. This contrasts with the findings of the current study, which showed that FhSOD1 and FhSOD3 localise to the tegument and gut of NEJ and show a marked decline in FhSOD-specific immuno-localisation in adult parasites. The differential life-stage expression of cytosolic SOD between *F. hepatica* and *S. mansoni* may reflect different host migration routes and tissue tropism between these parasites. *S. mansoni* infects mammalian hosts via penetration of the skin before eventually residing in the mesenteric venules, where they would be more available to host-generated ROS than *F. hepatica*, which is located within the bile ducts and gallbladder upon maturation [35]. Nevertheless, in both parasites, immuno-localisation occurs at the host:parasite interface and supports the theory that these proteins are employed in defence of exogenous superoxide.

Previous studies have shown that *F. hepatica* NEJ are resistant to killing by superoxide produced in vitro chemically or by peritoneal lavage cells isolated from Indonesian Thin-Tailed (ITT) sheep [36,37]. ITT sheep are resistant to infection by *F. gigantica* but have been shown to be susceptible to infection with *F. hepatica* [38]. It was suggested that this susceptibility is related to the higher gene expression of SOD1 mRNA in *F. hepatica* NEJ compared to *F. gigantica* [36]. In the current study, we demonstrated robust in vitro killing of *F. hepatica* NEJ by enzymatically generated ROS. Interestingly, ROS-associated killing of NEJ was partially prevented via the addition of rFhSOD3 only, but completely inhibited via the introduction of catalase. Given that all three SODs were utilized at concentrations of equal enzyme activity, these results suggest that rFhSOD1 and rFhSOD3 do not behave similarly under physiological conditions. Further, the production of hydrogen peroxide in our assay results from the (a) two-step reduction of hypoxanthine into xanthine and superoxide and (b) the reduction of superoxide by SOD. It is well known, however, that the production of superoxide by macrophages during the oxidative burst is part of a cascade of highly damaging reactive oxygen and nitrogen species; thus, our data imply that *F. hepatica* needs to employ an array of antioxidant proteins to defend against cell-mediated immune responses [9,32]. Helminths do not express catalase, but rather exploit a thiol-independent antioxidant system to detoxify hydrogen peroxide wherein thioredoxin/glutathione reductase (FhTGR) reduces thioredoxin (FhTrx), which then reduces and activates peroxiredoxin (FhPrx), all of which are up-regulated in *F. hepatica* NEJ [7,8,39]. In our in vitro assay, we utilized catalase instead of FhPrx, and thus circumvented this complex cascade and provided proof-of-principal evidence that *F. hepatica* NEJ exploit a series of antioxidants to defend against host ROS during early invasion. Future work will focus on the complex interactions between each of these antioxidant proteins and their collective role in combatting damaging ROS.
