The human blood parasite Schistosoma mansoni expresses extracellular tegumental calpains that cleave the blood clotting protein fibronectin

Schistosomes are intravascular, parasitic flatworms that cause debilitating disease afflicting >200 million people. Proteins expressed at the host-parasite interface likely play key roles in modifying the worm’s local environment to ensure parasite survival. Proteomic analysis reveals that two proteases belonging to the calpain family (SmCalp1 and SmCalp2) are expressed in the Schistosoma mansoni tegument. We have cloned both; while highly conserved in domain organization they display just 31% amino acid sequence identity. Both display high relative expression in the parasite’s intravascular life forms. Immunolocalization and activity based protein profiling experiments confirm the presence of the enzymes at the host-parasite interface. Living parasites exhibit surface calpain activity that is blocked in the absence of calcium and in the presence of calpain inhibitors (E64c, PD 150606 and calpastatin). While calpains are invariably reported to be exclusively intracellular (except in diseased or injured tissues), our data show that schistosomes display unique, constitutive, functional extracellular calpain activity. Furthermore we show that the worms are capable of cleaving the host blood clotting protein fibronectin and that this activity can be inhibited by E64c. We hypothesize that SmCalp1 and/or SmCalp2 perform this cleavage function to impede blood clot formation around the worms in vivo.


Results
Schistosoma mansoni tegumental calpains. Proteomic studies revealed the presence of two schistosome calpain homologs in the tegument of Schistosoma mansoni [21][22][23] . We designate these two calpains as SmCalp1 and SmCalp2. In the schistosome DNA sequence database, GeneDB, SmCalp1 (also known as Sm-p80) is annotated as Smp_214190 (previously part of Smp_157500). The coding sequence of this protein has been published 24,25 ; it has a predicted molecular weight of 86,920 Da and its predicted isoelectric point (pI) is 5.24. SmCalp2, annotated in GeneDB as Smp_137410, has not been investigated before. As described in Methods, we used proteomic and genomic data, to design primers which were used to amplify the cDNAs encoding SmCalp1 and SmCalp2. These were sequenced at the Tufts University Core Facility. The predicted molecular weight of the SmCalp2 protein is 90,902 Da and its predicted pI is 7.6. The SmCalp2 cDNA sequence reported here contains an additional 54 amino acids at the N-terminus as well as an additional internal 10 amino acids (I 340 -G 349 ) compared to the currently annotated sequence at NCBI (accession number XP_018648578). Supplementary Figure S1 shows an alignment of SmCalp1 and SmCalp2.
Using the available S. mansoni genome sequence at GeneDB.org and at Parasite.WormBase.org, the genes for both SmCalp1 and SmCalp2 were identified. Figure 1A depicts the exon/intron organization of the two genes; SmCalp1 has 20 exons (shown in red) and extends over 40 kb while SmCalp2 contains 12 exons and is ~22 kb in size. Both genes are located in chromosome 1 (whose current estimated size is 65,476,681 bp) as shown in Fig. 1B; the SmCalp2 gene is found towards the middle of chromosome 1 (position: 33,185,000-33,207,000) while SmCalp1 is more distally located (position: 57,382,000-57,424,000). The domain structure of SmCalp1 and SmCalp2 is depicted in Fig. 1C and is compared with the domain structure of the human classical calpain, CAPN1. Figure 1C shows that both SmCalp1 and SmCalp2, like CAPN1, are predicted to contain all of the domains found in members of this classical calpain family 24 . PC1 and PC2 are the protease core domains 1 and 2 (grey and red in Fig. 1C) which contain conserved active site residues. SmCalp1 has the same conserved residues in its catalytic domain as most classical calpains; they are C 154 , H 313 and N 337 . Whereas in SmCalp2 the conserved active site histidine (H 313 in SmCalp1) is replaced with glutamine (Q 360 in SmCalp2). All members of the platyhelminth Calp2 clade (described below, including SjCalp2 and ShCalp2) share a glutamine (Q) at this site. CBSW (light blue in Fig. 1C) represents the calpain-type beta-sandwich region containing a basic loop and an acidic loop, involved in protein/cell membrane interaction 31 . The penta EF (PEF) hand domain (green in Fig. 1C) contains five Ca 2+ -binding helix-loop-helix structural domains (orange) at the C-terminus. Conservation of all of these domains in SmCalp1 and SmCalp2 clearly place both proteins in the classical calpain family.
While both schistosome calpains exhibit great structural domain conservation as just described, at the amino acid level they are quite divergent, displaying just 31% amino acid sequence identity. Sequence comparisons are shown in supplementary Figure S1. As shown in Fig. 1D, phylogenetic analysis demonstrates that both SmCalp1 and SmCalp2 (red boxes) belong to their own specific platyhelminth calpain clades. For example, SmCalp1 has highest sequence identity with homologs from the other human schistosome species, S. haematobium (95% identity) and S. japonicum (84% identity). Homologs from the trematode Clonorchis sinensis and the cestode Echinococcus granulosus also tree closely with the schistosome SmCalp1 group (with 63% and 50% identify, respectively). In a similar vein, SmCalp2 displays closest similarity with schistosome homologs ShCalp2 (98% identity) and SjCalp2 (92% identity), with homologs from the platyhelminths C. sinensis and E. granulosus more distant. Sequence comparisons of the platyhelminth Calp2 clade proteins are shown in supplementary Figure S2. Both SmCalp1 and SmCalp2 are clearly distant both from each other and from the Drosophila melanogaster calpain CalpB as well as the human calpains CAPN1, CAPN2 and CAPN9.
The S. mansoni tegument external surface has functional calpain. Immunolocalization of schistosome surface calpains. Anti-SmCalp1 and anti-SmCalp2 antibodies were used to immunolocalize SmCalp1 and SmCalp2 in S. mansoni adult sections and in whole, 7-day cultured schistosomula. Figure 2 shows strong SmCalp1 (left panel) and SmCalp2 (central panel) staining predominantly in the tegument in all cases. Images of adult male and female parasites in longitudinal section are shown in Fig. 2A, cross sections of females are shown in Fig. 2B, and whole 7-day cultured schistosomula are shown in Figs 2C and 2D (at higher magnification). A clear "green ring" of tegumental staining around the parasites is revealed especially in the case of the adults. At this resolution, there is no obvious difference between the localization of SmCalp1 versus SmCalp2. Control parasites, exposed to secondary antibody alone (Fig. 2, right panel), do not display signal in the tegument or elsewhere in either adult parasites or schistosomula.
Calpain enzymatic activity assay. After confirming the presence of calpains in the tegument of intravascular life stage schistosomes, we investigated whether the parasites express functional extracellular calpain enzyme activity at the host-parasite interface. In order to determine this, the ability of living parasites to cleave the membrane non-permeable, fluorogenic calpain substrate "Calpain Substrate III" (Calbiochem) was measured. Using this assay, calpain activity was detected in live schistosomula and live adult parasites. As shown in Fig. 3A, calpain activity increases over time as the number of schistosomula included in the experiment increases. Similarly, Fig. 3B shows that calpain activity increases over time for both male and female worms with individual males displaying higher calpain activity compared to individual females. Since the calpain substrate used is cell impermeable, the activity detected derives from extracellular enzyme associated with the surface of the worms.
To investigate the possibility that calpain is released or secreted by cultured parasites, we performed an experiment in which ~1000 schistosomula were first incubated in assay buffer. After 1 hour the buffer was recovered and any calpain activity in the buffer was measured. Separately, complete medium in which 1000 schistosomula had been cultured for 3 days was collected and calpain activity therein was measured. As a positive control, a standard calpain activity assay was conducted using 1000 live schistosomula. Figure 3C shows the results of these analyses which demonstrate that essentially no calpain activity is detected in either the conditioned buffer or in conditioned medium (Fig. 3C, lower lines). Only buffer containing parasites exhibits clear calpain activity (Fig. 3C, upper line (squares)) showing that enzyme remains associated with the external surface of the parasites and, at least within the time frame examined, is not released.
In Fig. 3D, the surface calpain activity is compared with the total calpain activity detected in a homogenate of a single male. This experiment shows that the surface calpain activity makes up about 30% of the total detectable. Finally, live, single male worms of the three major schistosome species that infect humans: S. mansoni, S. haematobium and S. japonicum were compared for surface calpain activity and Fig. 3E shows the results. Extracellular calpain activity is detected in each case with the highest level being seen in S. mansoni.

Characterizing live schistosome tegumental calpain activity.
To determine if the enzymatic activity exhibited by living parasites requires Ca 2+ (as is characteristic of other calpain enzymes 32 ), the activity assay was conducted with living worms in the presence or absence of Ca 2+ . Figure 4A shows that removing Ca 2+ from the assay buffer effectively shuts down activity (p < 0.0001). Similarly, to determine if the enzymatic activity exhibited by living parasites could be blocked by known calpain inhibitors, worms were incubated with the membrane non-permeable inhibitor E64c or the permeable inhibitors calpastatin or PD150606. The calpain activity displayed by living schistosomula (Fig. 4B) and adult males (Fig. 4C) is effectively blocked by the presence of any of these inhibitors (p < 0.001). Worms treated in culture for 1-2 days with E64c exhibit no morphological differences compared to controls. However, after prolonged incubation in the presence of E64c (7 days), treated worms show ~10% lower viability compared to untreated controls (p < 0.001). In contrast, incubation of parasites with a membrane-permeable form of E64c (known as E64d) results in 100% worm killing within 24 h (Fig. 4C).
SmCalp1 and SmCalp2 are highly expressed in intravascular life stages. The relative expression of SmCalp1 and SmCalp2 in different schistosome life stages was measured using RT-qPCR and Fig. 5 shows the results of this analysis. Both SmCalp1 (A) and SmCalp2 (B) are relatively highly expressed in intravascular life stages particularly in schistosomula and males. For both genes, lowest relative expression is seen in cercaria. Unlike SmCalp2, the relative expression of SmCalp1 is high in eggs. Figure 5C shows that the expression of SmCalp1 is ~20 fold higher relative to that of SmCalp2 in adult males. SmCalp1 and SmCalp2 are present at the tegumental surface. Activity based protein profiling. We have shown that SmCalp1 and SmCalp2 are both expressed in the schistosome tegument and that living worms display clear extracellular calpain activity. In order to determine whether SmCalp1 or SmCalp2 (or both) could be , compared to that seen in either conditional buffer (i.e. buffer that had contained 1,000 schistosomula for 1 h, up triangles) or in conditional medium (i.e. medium that had contained 1,000 schistosomula for 3 days, down triangles). (D) Calpain activity detected in individual living adult male parasites (squares) compared to that detected in total lysates of individual males (triangles). (E) Calpain activity in individual adult males of Schistosoma mansoni, S. japonicum, and S. haematobium (as indicated). All data are presented as relative fluorescence units (RFU, mean+/−SD, n ≥ 3). Calpain activity is given in relative fluorescence units (RFU). Fluorescence is generated following substrate cleavage and is measured at excitation/ emission of 320/480. responsible for the activity detected, we employed activity based protein profiling. For this we incubated live parasites with biotin-labeled E64c. This compound (like non-biotinylated E64c) can effectively block the extracellular calpain activity of live worms, as shown in Fig. 6A. Immunostaining using a biotin-binding, streptavidin-Alexa Fluor ® 488 conjugate also confirms the presence of biotinylated E64c bound on the parasite surface, as shown in Fig. 6B. A sharp "green ring" on the surface of live schistosomula is evident. In some schistosomula, we additionally see staining in the caecum, probably due to ingestion of the biotinylated reagent.
Extracts of the biotinylated E64c-treated, non-biotinylated E64c-treated and control worms were resolved by SDS-PAGE, blotted to PVDF membrane and subjected to western blot analysis. Probing the blot with a streptavidin-conjugated detection reagent reveals that the extracts of the biotinylated E64c-treated parasites contain proteins of the expected size of both SmCalp1 (Fig. 6C,  Schistosomes cleave fibronectin. To investigate whether schistosomes could cleave the blood-clotting protein fibronectin, we incubated parasites in the presence of commercially-obtained, pure fibronectin and examined their impact on that protein at 2, 6 and 24 h thereafter. Since the fibronectin used was biotinylated, we detected the full-length protein and any cleavage products at high sensitivity by blotting the samples and probing the blot with a streptavidin conjugate. Figure 7A shows that fibronectin is indeed cleaved in the presence of adult schistosomes. A band running at ~180 kDa (arrow, Fig. 7A left panel) appears beneath the full-length 220 kDa fibronectin monomer and becomes more intense the longer the incubation continues. Longer exposure of this blot reveals an additional cleavage product running at ~40 kDa (Fig. 7A, right panel, arrow) and this product too is generated only in the presence of the parasites. (The longer exposure also reveals that the fibronectin used contains multiple additional biotinylated moieties of diverse molecular weight that can be seen in all lanes, even at 0 h.) To determine if the cleavage detected could be mediated by surface calpain activity, the experiment was repeated in the presence of the membrane-impermeable calpain inhibitor E64c. As shown in Fig. 7B, it is clear that cleavage is blocked in the presence of the inhibitor ("+" lanes) and that the characteristic cleavage products at ~180 (seen following short exposure, arrow, left panel) and at 40 kDa (seen following long exposure, arrow, right panel) are not detected.  To determine if the parasites can cleave fibronectin in plasma (and not just purified fibronectin), worms were incubated in murine plasma for 6 hours. Aliquots were then resolved by SDS-PAGE, blotted to PVDF membrane and murine fibronectin was detected with a polyclonal anti-fibronectin antibody. In Fig. 7C the pattern obtained is compared with that seen using control plasma that was incubated in the absence of worms. The characteristic ~40 kDa band is only detected in the plasma sample that contained the worms (Fig. 7C, arrow). (Intense staining of high molecular weight moieties prevented the clear detection of the ~180 kDa band in the sample containing worms.)

Discussion
In this work we focus on the characterization of schistosome tegumental proteases that are expressed at the host-parasite interface. Calpains comprise calcium-dependent cysteine proteases that are widely expressed in nature. Proteomic studies have revealed the presence of two schistosome calpain homologs in the tegument of Schistosoma mansoni [21][22][23] . We designate these as SmCalp1 and SmCalp2 and we have cloned cDNAs encoding these proteins in this work. The proteins encoded by these cDNAs are predicted to be very similar in size (SmCalp1 is 87 kDa and SmCalp2 is 91 kDa) and the predicted domain structure of both proteins is highly conserved. All domains characteristic of classical calpains are found in SmCalp1 and SmCalp2. These include the protease core domains (PC1 and PC2), the membrane association domain (CBSW) and the C-terminal Ca 2+ -binding penta-EF (PEF) domain. Despite this strong domain conservation, at the amino acid level SmCalp1 and SmCalp2 share just 31% identity. Phylogenetic analysis reveals that each belongs within its own distinct platyhelminth-specific clade. For both SmCalp1 and SmCalp2 we find close homologs in other schistosome species (S. haematobium and S. japonicum), in the trematode C. sinensis and in the cestode E. granulosus. It is noteworthy that all members of the calp2 clade have a distinctive catalytic triad, "C, Q, N", while most all other typical calpains (including the calp1 clade members discussed here) have the traditional catalytic triad "C, H, N" 18 . The presence of glutamine (Q) instead of histidine (H) in the calp2 catalytic triad may be a signature sequence for clade 2 calpains, suggestive of different substrate specificities and inhibition profiles of the different clades. The organization of the SmCalp1 and SmCalp2 genes are also quite distinct. The SmCalp1 gene contains 20 exons and spans over 42 K at the distal end of S. mansoni chromosome 1; the SmCalp2 gene contains 12 exons spread over 22 K more centrally on chromosome 1.
Immunofluorescence analysis confirms that SmCalp1 and SmCalp2 are both found in the tegument of the intravascular life stages examined. We see clear peripheral staining in sections of adult male and female worms and in schistosomula.
In order to assess whether the tegumental calpains might be involved in host-parasite interaction (and not simply in internal tegumental metabolism) we incubated live parasites (adult males and females as well as schistosomula) with the non-cell-permeable synthetic peptide "calpain substrate III, Fluorogenic". All life stages tested were found to be able to cleave this substrate and, given that it is not membrane permeable, we conclude that the activity detected is driven by enzyme that faces the exterior of the parasites. We have no evidence that intravascular schistosomes release or secrete calpains since no activity is detected in either buffer or medium in which worms were previously incubated. Activity is only detected in the presence of living parasites. The activity displayed by a living adult male is ~30% of that detected in an adult male total lysate. This demonstrates that, in addition to being present in the tegument, calpains, not surprisingly, are also well expressed in the internal tissues of the worms. Indeed, the genome of S. mansoni contains several calpain homologs beyond SmCalp1 and SmCalp2 15 . Like S. mansoni, live adult male worms belonging to the two other schistosome species that are parasitic to humans (S. japonicum and S. haematobium) also display similar external calpain activity.
The calpain activity displayed by living parasites can be blocked by removing calcium from the reaction buffer or by adding one of several known calpain inhibitors. The inhibitors E64c, calpastatin and PD150606 can each essentially eliminate the surface calpain activity in schistosomula and in adult males. E64c is a non-cell-permeable and irreversible cysteine protease inhibitor 33 that effectively blocks schistosome surface protease activity but, at least in the short term (1-2 days), has no impact on the morphology of cultured parasites. This suggests that the external calpain does not perform an especially essential function for the parasites in culture. It is only after prolonged exposure to the inhibitor (for 7 days) that schistosomula viability is impacted somewhat, with ~10% greater mortality versus controls. However, we speculate that the schistosome external calpain does play an important role for the parasites within an infected animal, potentially cleaving key host proteins, as described later. Incubating parasites with the related, but membrane permeable, irreversible inhibitor E64d kills all parasites within 24 hours. This shows, not surprisingly, that blocking calpain activity more broadly within schistosomes is catastrophic for the worms.
Developmental expression analysis reveals that both SmCalp1 and SmCalp2 are relatively highly expressed in the parasite's blood stages, especially in schistosomula and adult males and both are relatively poorly expressed in the cercarial life stage. This suggests that these calpains are of especial importance for the worms in the intravascular environment.
While proteomic analysis of the S. mansoni tegument reveals the presence of SmCalp1 and SmCalp2 there, no single proteomic study finds both calpains. SmCalp1 (but not SmCalp2) is reported as being removed from parasites by brief treatment with trypsin (a process called trypsin shaving) 21 and SmCalp2 (but not SmCalp1) is identified as being available for parasite surface biotinylation 23 . The fact that these proteomic approaches (trypsin shaving and surface biotinylation) detect SmCalp1 and SmCalp2 suggest that these calpains are outward facing and host interactive. To further test this notion here, we utilized activity based protein profiling in which worms were incubated with a biotinylated form of the non-cell-permeable, irreversible inhibitor E64c. This treatment (like treatment of worms with regular, non-biotinylated, E64c) effectively blocks the calpain activity of living parasites. Localization of biotinylated E64c on the treated worms, reveal the reagent bound to their surface. We hypothesize that this surface binding is to external schistosome calpains. Indeed, proteins with the molecular mass of both SmCalp1 and SmCalp2 are biotinylated following this treatment, as revealed by probing western blots of parasite extracts with a (biotin-binding) streptavidin conjugate. This result strongly suggests that both SmCalp1 and SmCalp2 are host exposed in the outer tegument of intravascular schistosomes. This finding is striking since calpains are invariably described as being intracellular 15,16,34 and our data show that schistosomes display unique, constitutive, functional extracellular calpain activity.
A clue to the normal function of the schistosome extracellular calpain may be the observation that renal ischemia in mice leads to the pathological release of normally intracellular murine calpain into the external milieu where it acts primarily to cleave fibronectin and promote healing 35 . One function of schistosome calpain might be to similarly degrade fibronectin -a 220 kDa protein that plays a central role in generating stable blood clots 36 . Weakening such clots (through calpain-mediated fibronectin cleavage) may be one way that schistosomes prohibit thrombus formation in their vicinity. Certainly, thrombus formation is not detected around the worms in vivo 5,7 and ex vivo the parasites can severely impede the ability of blood to clot 37 . Results reported here show that the parasites in culture can, in fact, cleave added fibronectin to generate ~180 kDa and ~40 kDa fragments. These cleavage fragments are only seen in the presence of parasites but are not generated in the presence of inhibitor E64c. Parasites incubated in murine plasma likewise cleave plasma fibronectin and we conclude that SmCalp1 and/or SmCalp2 is responsible for the fibronectin cleavage observed. In this manner, we hypothesize that schistosomes modulate fibronectin function to limit its ability to contribute to blood clot formation and this permits the worms more unrestricted movement within the vasculature.
Other pathogens are reported to bind fibronectin [37][38][39][40] and culture supernatants of one (the fungus Cryptococcus neoformans) is reported capable of cleaving the protein 41 . Our data are the first to show that a metazoan parasite (S. mansoni) can likewise target fibronectin for cleavage.
As noted above, our immunolocalization data confirms that both SmCalp1 and SmCalp2 are highly expressed in the intravascular schistosome tegument. These findings corroborate earlier work demonstrating the presence of calpains throughout the tegumental syncytium in S. mansoni 42 and in the tegument of S. japonicum 43 . Recently, cultured S. mansoni adult worms and schistosomula have been shown to release extracellular vesicles and one of the many protein components found therein is SmCalp1 44,45 . SmCalp1 was also shown by proteomic analysis to be among the proteins released during skin invasion by S. mansoni cercariae 46 . Likewise the S. japonicum homolog, SjCalp1, was demonstrated by immunolocalization to be present in the S. japonicum cercarial penetration glands and to be secreted from cercariae 43 . Since fibronectin, in addition to being found in blood, is a key component of the extracellular matrix, it is possible that one function of the secreted SmCalp1 of invading cercariae is to cleave this protein as an aid in the migration of the infecting parasites through the subdermal tissues to the vasculature where the parasites seek to establish a patent infection.
While schistosome tegumental calpains may have more substrates than just fibronectin, one potential explanation for the protective effect of vaccination with SmCalp1 (Sm-p80) is that the immune response generated may block calpain function and prevent the worms from efficiently cleaving fibronectin. Thus migration of cercariae within connective tissue, and migration of blood stage schistosomula and adults within the vasculature, may be severely impeded in vaccinated animals and this may trap and debilitate the worms. Whatever the precise mechanism of action, if an anti-SmCalp1 immune response generates protective immunity, we speculate that targeting the second tegumental calpain, SmCalp2, by vaccination may be additionally beneficial. Since our work confirms that both calpains are accessible to the exterior of the parasites, we suggest that a vaccine targeting SmCalp1 and SmCalp2 together may be an optimal formulation.

Material and Methods
Parasites and mice. Schistosoma mansoni-infected Biomphalaria glabrata snails (strain NMRI) were obtained from the Schistosomiasis Resource Center, at the Biomedical Research Institute (BRI, Cat. No. NR-21962) Rockville MD. Larval schistosomes (cercariae, strain NMRI) were obtained from the infected snails and schistosomula were prepared 47 . Adult male and female parasites were recovered by perfusion from Swiss Webster mice that were infected with 120 cercariae (S. mansoni) or 25 cercariae (S. japonicum) at BRI, 7 weeks previously. Adult S. haematobium were recovered by perfusion of Golden Syrian hamsters that had been infected with 350 cercariae at BRI, 12 weeks previously. All parasites were cultured in complete DMEM/F12 medium supplemented with 10% heat-inactivated fetal bovine serum, 200 U/ml penicillin and 200 µg/ml streptomycin, 0.2 µM Triiodo-L-thyronine, 1 µM serotonin and 8 µg/ml human insulin and were maintained at 37 °C, in an atmosphere of 5%CO 2 48 . Parasite eggs were isolated from infected mouse liver tissue. All protocols involving animals were approved by the Institutional Animal Care and Use Committees (IACUC) of Tufts University under protocol G2015-113. All experimental procedures were carried out in accordance with approved guidelines of the IACUC.
Cloning SmCalp1 and SmCalp2. Guided by published sequence 24,25 (for SmCalp1) and by S. mansoni genome sequence online at http://www.genedb.org/Homepage/Smansoni (for SmCalp2), the following primers, that flank the predicted start and stop codons of the calpain cDNAs, were synthesized (IDT Inc, Coralville, IA, USA) and used in a PCR with adult worm cDNA as template: SmCalp1Fw (5′-AAACGCTGTTAAATTGGGTGAACTTTT-3′) and SmCalp1Rv (5′-CTACAGATGCAACCATACTACGACAT-3′), SmCalp2Fw (5′-CTGATCAACCAGGTGAATTTTTATTACGTT-3′) and SmCalp2Rv (5′-AGTT GCAGCTAAACCACTAAGTTCT-3′). PCR conditions were as follows: 50 °C for 2 min, then 95 °C for 10 min; cycling stage (40 cycles), 95 °C for 15 sec, then 60 °C for 2 min. The resulting amplified products were gel purified and sequenced at the Tufts University Core Facility. As described, our SmCalp2 sequence (GenBank accession number: MF590064) differs at the amino terminus compared to that predicted at NCBI (XP_018648578). While a clear SmCalp2 homolog had been annotated in the S haematobium genome (accession no: XP_012791984), this was not the case for S. japonicum. However, by BLASTing the SmCalp2 coding sequence against the S japonicum genome at SchistoDB, one contig (sjc_S000186) was identified. The full SjCalp2 coding DNA was then amplified by PCR using specific primers designed following genome sequence analysis (Accession no: MF590065).
Anti-SmCalp1 and anti-SmCalp2 antibody production. The peptides NH2-CDGSPQWREISEQEKKN-COOH and NH2-YRLPAGANPPMPRGFFETN-COOH (derived from SmCalp1 and SmCalp2 respectively) were synthesized by Genemed Synthesis, Inc. (San Antonio, TX, USA) and conjugated to bovine serum albumin (BSA). Approximately 500 µg of each peptide-BSA conjugate in Freund's complete adjuvant was used to immunize two New Zealand white rabbits subcutaneously. The rabbits were boosted with 100 µg of peptide alone in incomplete Freund's adjuvant 20, 40, and 60 d later. Ten days following this, serum was recovered from the rabbits and anti-SmCalp1 and SmCalp2 antibodies were affinity-purified and dialyzed against phosphate buffered saline (PBS, pH 7.2) 49 .
To assess the ability of parasites to cleave fibronectin in plasma, the experiment just described was repeated using fresh plasma obtained from Swiss Webster mice. Mouse blood was recovered from the tail vein into a collecting tube containing heparin, centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatant (plasma) was used in this assay. Mouse fibronectin was detected by western blotting using a polyclonal anti-fibronectin antibody (ab23750, Abcam, Cambridge, UK).

Statistical analysis.
For RT-qPCR data, one way analysis of variance (ANOVA) was used and for calpain activity assays, two-way ANOVA was used. P values were considered significant at <0.05. Statistical analyses were performed using GraphPad Prism 5 (La Jolla, CA, USA).