Schistosomiasis, a disease caused by parasitic trematodes of the genus Schistosoma, is second only to malaria as the most socioeconomically devastating parasitic disease, with an estimated 252 million people infected worldwide in 2015 (Disease et al., 2016; Mortality and GBD 2015 Mortality and Causes of Death Collaborators, 2016). Larval digenean trematodes share a common feature in the use of snails (gastropod mollusks) as intermediate hosts for transmission to a vertebrate host (Esch et al., 2002). The snail Biomphalaria glabrata transmits several species of trematode, including Schistosoma mansoni, the predominant causal species of intestinal schistosomiasis (Pila et al., 2016a). The molecular interactions between B. glabrata and S. mansoni have been studied extensively towards better understanding this essential life cycle stage of an important human parasite (Adema, 2015).

Various genetically determined resistance phenotypes exist with respect to S. mansoni infection in B. glabrata (Richards, 1975a; Richards, 1975b). Some B. glabrata snails (such as BS-90 strain) naturally resist S. mansoni infection. Susceptible B. glabrata (such as M-line strain) can develop transient levels of acquired resistance following previous exposure to incompatible or radiation-attenuated parasites with modest cross protection to related digenean species (Lie and Heyneman, 1975; Lie et al., 1975; Sullivan et al., 1982). Previous research has focused on identifying the mechanisms responsible for determining resistance profiles. Using in vivo and in vitro models of B. glabrata snails, it has been demonstrated that the killing of S. mansoni larvae is associated with a haemocyte-mediated cytotoxic mechanism, and passive transfer of natural resistance to S. mansoni has been successfully accomplished when haemocytes from a susceptible B. glabrata strain are incubated in cell-free haemolymph (plasma) from a resistant strain (Bayne et al., 1980a; Bayne et al., 1980b; Granath and Yoshino, 1984; Loker and Bayne, 1982). Thus, haemocytes from resistant or susceptible B. glabrata strains do not appear to differ a priori in their cytotoxic capabilities, but their response requires activation by some humoral factor(s) (Bayne et al., 1980b; Granath and Yoshino, 1984; Vasquez and Sullivan, 2001), for proper recognition of S. mansoni and enhancing haemocyte cytotoxicity (Hahn et al., 2001).

Researchers have long sought immune determinants present in resistant B. glabrata plasma that specifically activate haemocytes to encapsulate and destroy S. mansoni sporocysts. Identified in B. glabrata, a large family of soluble lectins termed fibrinogen-related proteins (BgFREPs) attracted widespread interest because of their unique structure (Adema et al., 1997; Gordy et al., 2015), capacity for somatic diversification (Zhang et al., 2004), and immune function (Hanington et al., 2010; Hanington and Zhang, 2011). BgFREPs are composed of a C-terminal fibrinogen (FBG) domain and either one or two N-terminal immunoglobulin superfamily (IgSF) domains linked to the FBG domain by an interceding region (ICR) (Léonard et al., 2001; Zhang et al., 2001). Numerous studies have implicated BgFREP3 as playing a central role in B. glabrata resistance to digenetic trematodes (Hanington et al., 2010; Hanington et al., 2012; Hanington and Zhang, 2011; Lockyer et al., 2012; Lockyer et al., 2008; Pila et al., 2017b). BgFREP3 contains two tandem IgSF domains whereas BgFREP2 has one (Léonard et al., 2001). The expression of BgFREP2 in BS-90 snails is dramatically up-regulated (57-fold) after exposure to S. mansoni, whereas M-line snails do not feature such a drastic upregulation, thereby suggesting a role for BgFREP2 in resistance (Hertel et al., 2005). Interestingly, the time course for heightened BgFREP2 expression overlaps the interval within which sporocysts are encapsulated and killed, and only occurs in BS-90 haemocytes (Dinguirard et al., 2018; Hertel et al., 2005). Furthermore it was reported that BgFREP2 is reactive with S. mansoni polymorphic mucins (SmPoMucs) found on the tegumental surface of larval S. mansoni (Moné et al., 2010).

In addition to BgFREPs, other soluble immune effector factors which are involved in both the direct killing of sporocysts and preparation of haemocytes to mount a cell-mediated response have been characterized (Pila et al., 2017a). These factors include B. glabrata thioester-containing proteins (BgTEPs) (Bender et al., 1992; Moné et al., 2010; Portet et al., 2018) and Biomphalysin (Galinier et al., 2013). TEPs are key components of invertebrate and vertebrate immune responses, aiding in melanization, opsonization, and killing of invading pathogens (Baxter et al., 2007; Blandin et al., 2008; Levashina et al., 2001; Pila et al., 2017a; Portet et al., 2018; Povelones et al., 2013; Yassine and Osta, 2010). BgTEP was first characterized as an alpha macroglobulin proteinase inhibitor, and has recently re-emerged as a molecule of interest due to its association with BgFREP2 and SmPoMucs, and the capacity to bind to various invading pathogens (Bender et al., 1992; Moné et al., 2010; Portet et al., 2018). Biomphalysin is a cytolytic protein in B. glabrata belonging to the β pore-forming toxin (β-PFT) superfamily (Galinier et al., 2013). Biomphalysin binds to the surface of S. mansoni sporocysts in the absence of plasma, while its cytolytic activity is drastically increased when plasma is present, suggesting that other factor(s) within the plasma may mediate the conversion of the oligomeric pre-pore to a functional pore (Galinier et al., 2013). Although the functional mechanisms of these factors are not thoroughly understood, studies suggest that these factors function as key determinants in the final outcome of S. mansoni challenge of B. glabrata (Galinier et al., 2013; Moné et al., 2010).

While studies have implicated BgFREP3 in resistance to S. mansoni, the underlying mechanism of BgFREP3 function; how it binds to S. mansoni sporocyst surfaces, and then how recognition is translated into haemocyte engagement, activation, and ultimately parasite encapsulation, is still unknown (Hanington et al., 2010). Here, we report an association between BgFREP3, BgTEP1, Biomphalysin (UniProtKB/TrEMBL: A0A182YTN9 and A0A182YTZ4) and BgFREP2 (UniProtKB/TrEMBL: A0A2C9L9F5). BgFREP3 associates with BgTEP1 and Biomphalysin in both M-line and BS-90 strains, but only in BS-90 strain uniquely interacts with BgFREP2 and other versions of BgFREP3 (a variant of BgFREP3.3), providing us with an important insight into why BS-90 strain is refractory to S. mansoni infection. In this study, we demonstrate that BgFREP3 binds to S. mansoni sporocysts without any other soluble plasma factors, yet binding of BgFREP2 relies on BgTEP1. However, BgFREP3 still interacts with BgTEP1 to form unique immune complexes, which significantly enhance the ability of haemocytes and plasma from S. mansoni-susceptible B. glabrata (M-line) to kill S. mansoni sporocysts. A more striking finding is that the combination of BgFREP3, BgTEP1 and BgFREP2 renders M-line haemocytes capable of killing S. mansoni sporocysts at nearly the same level as S. mansoni-resistant BS-90 haemocytes. Reactive oxygen species (ROS) are shown to play an important role during this haemocyte-mediated killing of S. mansoni sporocysts. These results provide insight into how the numerous previously characterized immune factors known to be important in the anti-S. mansoni immune response to B. glabrata are acting in concert to defend the snail host.

In 1984, Granath and Yoshino successfully achieved passive transfer of resistance to S. mansoni from a refractory B. glabrata strain to susceptible snails via injection of cell-free haemolymph (plasma). Similar passive transfer of resistance had previously been shown using an in vitro model (Bayne et al., 1980a; Bayne et al., 1980b; Loker and Bayne, 1982). Although the oxidation of haemoglobin, which is the main protein component (97% of total protein) in B. glabrata plasma was later found to be toxic to S. mansoni sporocysts in long-term cultures (Bender et al., 2002), and thus likely explained the passive transfer study results, further studies suggest that specific anti-parasitic factors must be present at or above a certain threshold level in S. mansoni-resistant snail plasma to stimulate a haemocyte-mediated destruction of sporocysts within 24–48 hr post-infection (Dinguirard et al., 2018). Although many important immune factors have been identified from B. glabrata plasma/haemocytes, such as BgFREPs (Adema et al., 1997), BgTEP1 (Moné et al., 2010), Biomphalysin (Galinier et al., 2013), Toll-like receptors (BgTLRs) (Pila et al., 2016b), granulin (BgGRN) (Pila et al., 2016a) and macrophage migration inhibitory factor (BgMIF) (Baeza Garcia et al., 2010), it is still not known which factors ultimately underpin B. glabrata immunity to S. mansoni. Here, we provide insight into the broader interactions that take place between B. glabrata immune factors that leads to development of an S. mansoni-resistant phenotype.

Previous studies have shown that BgFREPs are capable of binding to digenean trematode sporocysts and are up-regulated in response to larval infection (Adema et al., 1997; Wu et al., 2017; Zhang et al., 2001). BgFREP3 has been shown to be a central part of the anti-S. mansoni immune response (Hanington et al., 2010). BgFREP3 is up-regulated based on three characteristics (age, strain, and acquired resistance) of resistance of B. glabrata to infection with S. mansoni or Echinostoma paraensei (Hanington et al., 2010). Knock-down of BgFREP3 in resistant snails using siRNA-mediated interference altered the phenotype of resistant B. glabrata by increasing susceptibility to E. paraensei infection (Adema, 2015; Hanington et al., 2010). Nevertheless, we have little knowledge of the underlying mechanism by which BgFREP3 interacts with other snail plasma factors in order to signal and trigger an immune response after recognizing the pathogen. In order to explore this mechanism, we conducted a series of pull-down experiments that allowed us to discover the interaction between BgTEP1, Biomphalysin and BgFREP2 with BgFREP3 (Figure 1).

Proteomic analysis of B. glabrata plasma revealed that BgFREP3, BgTEP1 and BgFREP2 display affinity for S. mansoni sporocyst tegumental membrane proteins (Portet et al., 2018; Wu et al., 2017). BgTEP1 was previously reported to play a role in BgFREP2 recognition of S. mansoni sporocyst SmPoMucs, (Moné et al., 2010; Portet et al., 2018). Our immunofluorescence results illustrate that BgFREP3 independently recognizes S. mansoni sporocysts, whereas BgFREP2 must be involved with the participation of BgTEP1 (Figure 2). We speculate that this may be due to their structural differences: BgFREP3 possesses two IgSF domains, yet BgFREP2 has only one; BgFREP3 exists as a multimer in its natural state (Figure 3), whereas BgFREP2 does not (Zhang et al., 2008). Alternatively, it is possible that the targets they recognize are different, so the immunoaffinity reactions that occur are also fundamentally different. A recent report suggests that BgFREPs are likely to form multimers through the coiled-coil region of the ICR. However, this has not been supported by experimental evidence and does not completely rule out the possibility that BgFREP multimer formation is mediated by the FBG or IgSF domain (Gorbushin, 2019).

BgTEP1 and BgFREP3 form a complex without any S. mansoni molecules present (Figure 3). We do not yet know exactly how BgTEP1 is activated during infection. It is possible that BgFREP3 is stabilizing BgTEP1 like the leucine-rich repeat immune protein (LRIM)/Anopheles Plasmodium-responsive Leucine-rich repeat protein 1 (APL1C) complex that has been found to stabilize TEP in Anopheles gambiae, while also guiding it to the sporocyst’s surface (Fraiture et al., 2009). Additionally, our LC-MS/MS results seem to indicate that BgFREP3 interacted with a full-length form of BgTEP1, while previous work has only found the C-terminal portion of BgTEP1 associating with SmPoMucs (Moné et al., 2010). This may indicate that once brought in association with the parasite, BgTEP1 becomes cleaved/activated. BgFREP3 does not require BgTEP1 to recognize pathogens, this suggests BgTEP1 is more likely to play a role in the downstream immune response initiated by BgFREP3. Following the addition of rBgFREP3 and rBgTEP1, plasma demonstrated an increased capacity to kill sporocysts compared to controls (Figure 4A). Toxicity of haemoglobin oxidation to S. mansoni sporocysts was ruled out as a possible mechanism underpinning sporocyst killing because the B. glabrata plasma used in these tests had haemoglobin removed. These studies also demonstrated that recombinant BgFREP3 and BgTEP1 alone were not able to increase sporocyst killing, but showed that the addition of cell-free plasma with both recombinants was required, thereby implying the necessity of another factor(s). Based on our pull-down experiments, we suggest that this factor could be a Biomphalysin. Biomphalysin possesses many similarities to members of the β-PFT superfamily (Howard and Buckley, 1985; MacKenzie et al., 1999; Wilmsen et al., 1992; Xu et al., 2014), and has been shown to have cytolytic activity that is mediated by a plasma factor(s) that remains to be identified (Galinier et al., 2013). Our results suggest a scenario in which BgFREP3 and/or BgTEP1 could be the factors associating with Biomphalysin to promote plasma-mediated sporocyst killing. BgFREP3 and/or BgTEP1 might activate Biomphalysin in an unknown manner, assist in the oligomerization of Biomphalysin while it forms its heptameric channel, or mediate the conversion of the oligomeric pre-pore to a functional pore. Further investigation is warranted to illustrate the role of BgFREP3 and BgTEP1 in the activation of Biomphalysin.

Within 2–3 hr of S. mansoni entry into B. glabrata, haemocytes surround the developing sporocysts forming multi-layered cellular capsules that kill encapsulated larvae within 24–48 hr post-infection (Dinguirard et al., 2018; Loker and Bayne, 1982). This occurs in both susceptible and resistant snail strains and has been replicated in our in vitro functional studies, where only resistant snails can eventually destroy the sporocysts without external assistance. Dinguirard et al. conducted a comparative proteomic analysis on the responses of haemocytes participating in S. mansoni sporocyst encapsulation reactions from susceptible and resistant B. glabrata strains, showing a striking differences in proteins expressed, with susceptible snail haemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed proteins involved immunity (e.g., BgFREP2) and ROS production compared to resistant snails (Dinguirard et al., 2018). This could explain why BS-90 haemocytes alone can kill S. mansoni sporocysts under normal in vivo conditions. After challenge, the transcript abundance of numerous BgFREPs increases by 3-fold or greater in BS-90 compared to M-line B. glabrata (Adema et al., 1997). Noteworthy in the context of our current study is that following challenge BS-90 snails with S. mansoni, transcript abundance for BgFREP2 increases over 50-fold by 1 day post-exposure (Dinguirard et al., 2018; Hertel et al., 2005), BgFREP3 increases around 4-fold by 1 day post-exposure (Hanington et al., 2010), and BgTEP1 which increases by 2- to 3.5-fold from 6 to 24 hr post-exposure (Portet et al., 2018). These dramatic increases in the abundance of immune-relevant transcripts occurring over the first 12–24 hr post exposure to S. mansoni sporocysts in BS-90 snails aligns closely with our results that demonstrate sporocyst killing by BS-90 haemocytes rapidly increases after 12 hr post-exposure (Figure 4).

Numerous studies have demonstrated the production of ROS by haemocytes in B. glabrata, especially H₂O₂, which plays a vital role in anti-schistosome defense (Galinier et al., 2013; Hahn et al., 2000; Hahn et al., 2001). Haemocytes from S. mansoni-resistant snails produce significantly more ROS than susceptible snails (Bender et al., 2005; Bender et al., 2007; Goodall et al., 2004). In our study, ROS was critical in BS-90 haemocyte-mediated S. mansoni sporocyst killing, as this ability was significantly reduced after pre-incubation with the ROS inhibitors NAC and DPI (Figure 4E). However, according to the phenomenon of passively transferred resistance, the cytotoxic potential of susceptible haemocytes may not be fundamentally different from resistant snails (Granath and Yoshino, 1984). We infer that M-line plasma may lack some factors or may not achieve sufficient thresholds of certain factors to stimulate haemocytes to produce ROS. It is also possible that M-line haemocytes are lacking specific receptors to receive signals that upregulate ROS to effective levels able to kill S. mansoni sporocysts. Previously, our team identified a toll-like receptor (BgTLR) from haemocyte surface of B. glabrata snails, which differed in abundance between M-line and BS-90 strains (Pila et al., 2016b). It has been repeatedly demonstrated in other organisms that activation of Toll-like receptors is associated with increased intracellular ROS (Asehnoune et al., 2004; Tsung et al., 2007; Wong et al., 2009).

Our results indicate that the BgFREP3-BgTEP1 complex plays a role in raising ROS levels in M-line haemocytes (Figure 4F). We found that BS-90 haemolymph contains more BgFREP3 protein than M-line (Figure 1B). Moreover, the BS-90 haemocytes significantly increased the expression level of BgFREP2 during encapsulating S. mansoni sporocysts compared with susceptible snail haemocytes (Dinguirard et al., 2018). These suggest that BgFREP3 and BgFREP2 may play a crucial role in inducing haemocytes which take part in encapsulating sporocysts to generate ROS. We suppose that BgTEP1 also plays an opsonin role in the B. glabrata snail haemolymph, which interacts with molecules on the surface of haemocytes, thereby promoting the phagocytosis of pathogens.

Our data suggests that there are fundamental differences between the BgFREP3-mediated immune response between BS-90 and M-line B. glabrata. Protein Pull-down experiments yielded two obvious and unique protein bands that were present in BS-90 but not M-line pull-downs. We identified the bands to be a variant of BgFREP3.3 and BgFREP2 (Figure 1C), showing different immune factor interactomes between different strains. This suggests that BgFREP3 variants and BgFREP2 in BS-90 plasma are more prone to rBgFREP3 binding, and the combination of different versions of BgFREPs seems to play an important and unknown role in snail resistance. Both BgFREP2 and BgFREP3 are characterized by high diversity, our results imply that the versions of BgFREP2 and BgFREP3 in the two snail strains are different. After exposure to S. mansoni, the expression of BgFREP2 in resistant snails is superior to that of susceptible snails (Dinguirard et al., 2018; Gordy et al., 2015; Hertel et al., 2005). This again suggests that BgFREP2 is important in determining the outcome of the compatibility of B. glabrata against S. mansoni. Our study implies that BgFREP3 seems to play an ‘all or none’ switching effect in determining B.glabrata-S. mansoni compatibility, while BgFREP2 seems to play an additive role.

It has been decades since the discovery and initial characterization of BgFREPs in the B. glabrata immune response to S. mansoni. While numerous studies since have implicated BgFREP3 and BgFREP2 along with a suite of other immune factors, as being important elements of the snail immune response to S. mansoni, how the response is coordinated has remained elusive. Our study presents a model in which many of the known determinants of snail resistance to S. mansoni work in concert to protect the snail host against infection. We map a mechanism for the interaction of BgFREP3, BgTEP1, BgFREP2 and Biomphalysin with S. mansoni sporocysts in B. glabrata snail haemolymph that ultimately leads to sporocyst killing (Figure 5). Central to this response is BgFREP3, which is able to recruit unique representatives of both BgFREP2 and BgFREP3 in S. mansoni-resistant BS-90 B. glabrata. With this new-found understanding of the B. glabrata immune response comes the ability to now undertake comprehensive assessments of how modifications to both the snail host and the parasite influence the underpinning immunological drivers of compatibility in this important model for studying human schistosomes.

Figure 5

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All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Hongyu Li

   1. Ocean College, Beibu Gulf University, Qinzhou, China
   2. School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Jacob R Hambrook

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Emmanuel A Pila

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Abdullah A Gharamah

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Jing Fang

   1. Ocean College, Beibu Gulf University, Qinzhou, China
   2. School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Xinzhong Wu

   1. Ocean College, Beibu Gulf University, Qinzhou, China
   2. College of Animal Sciences, Zhejiang University, Hangzhou, China

   Contribution

   Funding acquisition, Investigation, Methodology, Writing - review and editing

   For correspondence

   wuxzqinzhou@163.com

   Competing interests

   No competing interests declared

7. Patrick Hanington

   School of Public Health, University of Alberta, Edmonton, Canada

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Project administration, Writing - review and editing

   For correspondence

   pch1@ualberta.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3964-5012

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