Coordination of humoral immune factors dictates compatibility between Schistosoma mansoni and Biomphalaria glabrata

Essential revisions:

1) Physical evidence that rFrep3 alone, but not rFrep2 or rTep1 alone, can bind to sporocysts is provided by ICC using fixed parasites. Assuming, as stated, that recomb. proteins bind to the outer tegumental of larvae, the localization pattern of Frep3 and Frep2/Tep1 binding do not appear to reflect such a surface binding pattern. This raises the question regarding the specificity of Frep2/3/Tep reactivity with the tegument of living sporocysts, which is at the center of this paper. Performing similar ICC experiments, initially treating live sporocysts to recombinant proteins (suggest shorter exposure times of 4-6 hrs), washing and then fixing, should provide a more accurate representation of Frep/Tep binding interactions with the unaltered sporocyst surface.

Thank you for the advice. Initially, we did not use confocal microscopy to evaluate tegumental binding. In our revised submission, we confirm tegument binding by clearly demonstrating a ring of recognition around the sporocyst. We approached this study as suggested, incubating live sporocysts with rBgFREP3, rBgFREP2, rBgFREP2+rBgTEP1 and rBgTEP1 along with controls. As is now indicated in the Materials and methods, live primary sporocysts of S. mansoni were incubated with recombinant proteins at 26°C, then washed and fixed, and the subsequent steps were consistent with previous immunofluorescence staining experiments. We also replicated the method of our initial study, adding therBgFREP3+rBgTEP1 treatment to further validate our pull-down results that suggest that FRE3 and TEP associate without immune stimulation, and assessed using a confocal microscope. These repeated studies also yielded clear surface binding. To replace the original images, we chose to use fixed sporocysts because the fluorescence intensity with fixed sporocysts was stronger than that with live sporocysts. We have included the images of the sporocysts that were incubated with the treatments while alive (as was suggested) as Figure 2—figure supplement 1.

2) Biomphalysin, a pore-forming protein, is speculated as the mechanism responsible for plasma-mediated killing of sporocysts in vitro (Figure 4A). However, an alternative mechanism may be the presence of ROS (e.g., H₂O₂) naturally occurring in the plasma of BS-90 compared to M-line snails. To resolve this question, an experiment in which a ROS scavenger enzyme (e.g., catalase) is added to BS-90 plasma (and M-line plasma treated Frep/Tep) prior to sporocyst exposure in the CMC assay could be performed to eliminate an ROS-mediated mechanism.

We hadn’t considered the possibility that BS-90 plasma may contain naturally-occurring ROS that was underpinning plasma-mediated killing. We undertook the recommended study to utilize a ROS scavenger specific to H₂O₂, added in advance to the BS-90 plasma. A ROS scavenger (catalase) was pre-incubated with cell-free BS-90 plasma and then the rest of the study was performed following the previously established method. With the exclusion of H₂O₂, BS-90 plasma was still able to kill S. mansoni sporocysts, thereby strengthening the likelihood of a non-ROS mediated mechanism by which humoral killing is mediated. The new results are presented in a revised Figure 4—figure supplement 1B.

3) "These pull-down results" Also with interaction taking place without involvement of parasite products, is this the same for BS90 and M snails? How is this regulated in absence of pathogen to avoid aspecific continuous immune activation? Does FREP3 transport TEP to recognized parasite antigens, resulting in activation of TEP to facilitate downstream activation?

Regarding the rBgFREP3 pull-down results, we identified BgTEP1 in both BS-90 and M-line snails without the involvement of parasite products. Unfortunately, we do not know whether these interactions differ regarding their frequency within the different strains. Given that we have found BgTEP1 levels to be similar between strains (Figure 1—figure supplement 3), the noticeable difference in BgFREP3 levels, as evidenced by our Western Blots of snail plasma (Figure 1B), may in fact result in higher circulating levels of a BgTEP/BgFREP3 complex.

The reviewers raise an excellent point in questioning the mechanism of BgTEP1 activation. Although BgFREP3 appears to interact with BgTEP1 in the plasma of both snail strains, as is demonstrated by the rBgFREP3 pull-down results, we also show that rBgFREP3 specifically interacted with BgFREP2 and other versions of rBgFREP3 only in BS-90 snail plasma. These differing results between strains suggests that specific immune complexes that associate with BgTEP1 may be differentially activated and/or regulated depending on the specific BgFREPs present. To date, we are not sure of the exact mechanism by which BgTEP1 is activated, nor do we know how this activation is regulated in the absence of pathogen. In pursuit of a resolution to this query, we hypothesized that BgTEP1 may operate in a similar manner to TEP1 of the Anopheles gambiae, in which a pair of Leucine-rich repeat containing molecules stabilize AgTEP, prior to its binding to the surface of Plasmodium ookinetes (Fraiture et al., 2009). We thought that BgFREP3 may function analogously to the LRR molecules LRIM1 and APLC, serving to stabilize BgTEP1, while also guiding it to specific targets recognized by the BgFREP on the parasite surface. Further protein pull-down investigations with a focus on determining whether BgTEP1 cleavage products were observed reflected our past results, suggesting that BgFREP3 interacts with a full-length form of BgTEP1. This contrasts the observations from the Anopheles model and also those of Mone et al. which suggest that the C-terminal portion of BgTEP1 associates with S. mansoni polymorphic mucins (SmPoMucs). This suggests that once brought in association with the parasite, BgTEP1 becomes cleaved/activated, but that activation (via cleavage into the N and C-terminal domains) does not occur without some pathogen-related stimulation. We have added clarification of this in the text of our Discussion.