Schistosoma mansoni (a Puerto Rican strain) is routinely maintained in the laboratory in the intermediate snail host (Biomphalaria glabrata) and definitive host NMRI (Naval Medical Research Institute) mouse as described previously [31, 32]. Laboratory mice are maintained by a certified person (certificate number CZ 02627) in the laboratories accredited according to the animal welfare laws of the Czech Republic and EU. Free-swimming larvae (cercariae) were shed by light stimulation from infected snails into the water. Adult female mice were infected by immersing their feet and tails into 50 ml water containing approximately 200 cercariae for 45 min. Seven weeks post-infection, mice were killed by intraperitoneal injection of ketamine (Narkamon 5%—1.2 ml/kg body weight) and xylazine (Rometar 2%—0.6 ml/kg body weight) and with heparin to prevent blood clotting. Adult worms were recovered from the mouse hepatic portal system by transcardial perfusion with RPMI 160 medium. Worms in pairs were then transferred into a petri dish containing medium and chilled on ice for male and female separation by gentle prodding with a small brush. Migratory larvae (schistosomula) were prepared from cercariae in vitro and cultivated in Complete Medium 169 according to standard protocols published previously [33,34,35].
Living F. hepatica worms were collected from the liver of infected cattle (Cesky Krumlov district, Czechia) and carefully washed in phosphate-buffered saline (PBS). Worm samples were immediately frozen on dry ice and stored at − 80 °C.
Total RNA from S. mansoni samples (excised snail hepatopancreases with daughter sporocysts, cercariae, 5-day cultivated in vitro transformed schistosomula, adults, eggs, and miracidia) and from F. hepatica adults were isolated according to the TRIzol reagent (Thermo Fisher Scientific) protocol. The final precipitated RNA was air-dried and resuspended in DEPC-treated H2O. Single-stranded cDNA was synthesized from total RNA using SuperScript IV reverse transcriptase (Thermo Fisher Scientific) and an oligo d(T)18 reverse primer according to the manufacturer’s protocol. The resulting cDNA was purified using a QIAquick PCR purification kit (Qiagen) and stored in DEPC-H2O at − 20 °C.
Sample preparation for microscopy
For RNA in situ hybridization and immunohistochemistry, the adults and schistosomula of S. mansoni were freshly collected and fixed in hot 4% formaldehyde heated to approximately 60 °C, but the solution did not boil (90 min for adults/30 min for schistosomula) and was cooled to 25 °C. All fixed samples were dehydrated through progressively concentrated ethanol (25%, 50%, 70%, 90%, 96%, 100% v/v ethanol) for 5 min for each step. A drop of Chromotrope 2R dye (Sigma-Aldrich) was added to the 90% ethanol step. Before embedding in paraffin, S. mansoni adults and schistosomula were incubated in methyl benzoate (Sigma-Aldrich) for 45 min in the case of adults and 20 min for schistosomula. Subsequently, the worms were washed twice for 5 min in benzene (Sigma-Aldrich). Fasciola hepatica adults were fixed in Bouin’s solution (Sigma-Aldrich) for 24 h at RT and then rinsed in PBS containing 0.02% sodium azide until the picric acid was washed off. Fixed samples were dehydrated with increasing concentrations of ethanol as was described for S. mansoni for 30 min for each step.
All tissues were finally embedded in paraffin-Paraplast (Sigma-Aldrich). Sections (S. mansoni 5 µm, F. hepatica 7 µm) were cut using a Shandon Finesse® ME + microtome (Thermo Fisher Scientific) and placed onto X-tra adhesive slides (Leica).
Annotation of M28B metalloproteases
Single genes coding M28B metalloproteases were identified in S. mansoni and F. hepatica based on the sequence similarities from previous sequence submissions: SmM28B as S. mansoni NAALADase L (GenBank XP_018651911) and FhM28B as F. hepatica NAALADase 2 (GenBank THD24162.1). Respective ORFs were amplified, cloned, and verified by sequencing. Fully annotated sequences were deposited into the GenBank as MZ456528 and MZ456529 for SmM28B and FhM28B, respectively.
The amino acid sequences of SmM28B and FhM28B were used for homology modeling. The 3BXM HsGCP2 structure was selected as a modeling template , and Modeler 9.23 software was used to construct the target-template sequence alignment and to generate a set of 3D homology models; the best model for each enzyme was selected based on discrete optimized protein energy (DOPE) scores . DOPE scores for SmM28B and FhM28B homology models were − 82,422 and − 81,303, respectively. The GA341 score of both models was 1. Finally, the SmM28B and FhM28B homology models and HsGCP2 structures were superimposed in PyMol and analyzed by visual inspection.
Cloning and mutagenesis
Codon-optimized genes encoding the SmM28B and FhM28B proteins were custom-made by the Thermo Fisher gene-string synthesis protocol. Coding sequences were PCR amplified with corresponding sets of gene-specific primers (Additional file 1: Table S1), and pD221 donor vectors were constructed using the BP Gateway cloning protocol (Invitrogen). Expression plasmids featuring N-terminal purification His-Strep-HALO tags were prepared by recombining the donor vectors and the in-house expression pDEST320 destination vector (Additional file 2: Fig. S1) using the LR Gateway reaction mix. SmM28B(E439M) and FhM28B(E413M) mutants, harboring inactivating mutations of the putative proton shuttle glutamate, were constructed by Quick-change site-directed mutagenesis using corresponding expression plasmids as templates. To this end, target glutamate residues were mutated to methionine via PCR with mutagenic primers (Additional file 1: Table S1) followed by elimination of the template by DpnI treatment, as described for the HsGCP2 ortholog . Escherichia coli clones transformed by a plasmid containing the modified sequence were verified using Sanger sequencing.
Expression and purification of trematode M28B metalloproteases in HEK-293T cells
For eukaryotic expression, metalloproteases were cloned into the pDEST320 destination vector in frame with the TEV-cleavable His-Strep-HALO tag (Additional file 2: Fig. S1) and expressed and purified essentially as described previously . Briefly, all variants were expressed using HEK-293T/17 cells following transient transfection, mediated by linear polyethyleneimine (PEI) (Polysciences). The suspension culture was grown in Erlenmeyer flasks in FreeStyle F17 medium (Thermo Fisher Scientific) at 110 rpm under a humidified 5% CO2 atmosphere at 37 °C. For large-scale expression, an expression plasmid was diluted in PBS (1 mg in 25 ml of PBS for 1 l of the cell culture). Then 3 ml of the PEI stock solution (1 mg/ml) was added; the mixture was vortexed briefly, incubated for 10 min at room temperature, and then added to the cell suspension (4 × 106 cell/ml). Four hours post-transfection, the cell suspension was diluted with an equal volume of ExCell serum-free medium. Cells were harvested 72 h post-transfection by centrifugation at 500 g for 5 min, and then the cell pellet was frozen in liquid nitrogen and stored at − 80 °C until further use.
The cell pellet was lysed by sonication in ice-cold lysis buffer (100 mM Tris–HCl, 10 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10% glycerol, 0.2% Nonidet P-40, pH 8.0) supplemented with the protease inhibitor cocktail cOmplete EDTA-free (Roche). Following incubation on ice for 30 min, the cell lysate was cleared by centrifugation at 40,000 g for 30 min, and the supernatant was loaded on a Strep-Tactin column (IBA-Lifesciences). The fusion was eluted with the lysis buffer supplemented with 2 mM desthiobiotin. Alternatively, when deemed beneficial, the N-terminal fusion tag was cleaved-off by TEV protease (at a 1:10 TEV to target ratio) at 8 °C overnight. The final purification step comprised size exclusion chromatography on a Superose 6 column (GE Life Sciences) with 20 mM MOPS, 150 mM NaCl, pH 7.4, as a mobile phase. Purified proteins were concentrated at 1 mg/ml and flash-frozen in liquid nitrogen until further use.
Recombinant production of SmM28B in E. coli
For polyclonal antibody production, the SmM28B antigen was heterologously expressed in a prokaryotic expression system. The codon-optimized sequence encoding SmM28B was cloned into the pEC527 destination vector (a gift from Dominic Esposito; Addgene plasmid #11518) in frame with the TEV-cleavable hexahistidine tag. The recombinant protein was expressed in E. coli BL21(DE3) cells in LB medium at 37 °C upon 1 mM IPTG induction and further purified from inclusion bodies using Ni2+ chelating chromatography under denaturing conditions. Briefly, the cell pellet was disrupted by sonication in PBS, and inclusion bodies (IBs) were isolated by centrifugation at 5000 g for 10 min. IBs were further purified by consecutive sonication-assisted solubilization in PBS + 1 M urea, PBS + 1 M NaCl, and PBS + 1% Triton X-100, where each solubilization step was followed by centrifugation at 5000 g for 10 min. Following the final centrifugation, the IBs were dissolved in an equilibration buffer (10 M urea, 100 mM Tris–HCl, pH 8.0) and centrifuged at 30,000 g for 30 min, and the supernatant was loaded onto a 5-ml Ni–NTA column. His-tagged SmM28B was eluted with the equilibration buffer supplemented with 200 mM imidazole. Fractions containing the purified protein were pooled and used to prepare polyclonal rabbit antiserum.
Expression and purification of human GCP2
Expression and purification of the extracellular part of HsGCP2 (residues 44-750) were carried out as described previously .
Aminopeptidase library screening
The aminopeptidase activities of GCP2-like enzymes were evaluated using the library of proteinogenic amino acids, except cysteine, labeled with a fluorophore, 7-amino-4-methylcoumarin (AMC). The enzymatic assays were carried out in 384-well black plates in a final volume of 50 µl. The reaction mixtures consisted of 5 µM of individual fluorophore-labeled amino acids and 500 nM of enzyme in an assay buffer comprising 50 mM Tris–HCl, 150 mM NaCl, 0.001% C12E8 (dodecyloctaglycol), pH 7.4. The reactions were incubated at room temperature for 60 min, and the release of free AMC was monitored using a CLARIOstar fluorimeter (BMG Labtech GmbH) with excitation/emission wavelengths set at 365 and 440 nm, respectively. The two-fold dilution series of AMC with a starting concentration of 5 µM was used as a positive control and for quantification of the reaction products. The substrate without enzyme was used as a negative control and as a signal background.
Carboxypeptidase library screening
The carboxypeptidase activities of GCP2 enzymes were evaluated using the library of dipeptides. The library was synthesized as 19 mixtures, each containing a defined N-terminal residue and a mixture of 19 proteinogenic amino acids at the C-terminus. Cysteine was not present in the library. Enzymatic assays were carried out in 96-well plates with a final volume of 50 µl. Reaction mixtures consisted of 500 µM peptide mixture and 500 nM enzyme in the assay buffer. Reaction mixtures were incubated at room temperature for 60 min, quenched by the addition of 0.1% trifluoroacetic acid in 5% acetonitrile, and released amino acids were quantified by Waters Corporation AccQ-Tag Ultra chemistry package on RP-HPLC (Shimadzu, HPLC Prominence system) according to the manufacturer's instructions. The complementary peptide library comprising a defined C-terminal residue and a mixture of 19 proteinogenic amino acids at the N-terminus was used for the control experiments.
Multiplex substrate profiling by mass spectrometry (MSP-MS)
The MSP-MS assays were performed as previously described . Briefly, protease samples were incubated in triplicate reaction tubes with a mixture of 228 synthetic tetradecapeptides (0.5 μM each) in the reaction buffer. Then, 20 μl of the reaction mixture was removed from each replicate after a set incubation time (15, 60, 120, 240, and 1200 min of incubation), and enzyme activity was quenched by adding 40 μl of 6.4 M guanidinium chloride (GuHCl). Samples were flash frozen immediately at − 80 °C. A control reaction consisted of enzyme pre-treated with GuHCl prior to peptide library exposure. All samples were subsequently thawed, acidified to pH < 3.0 with 1% formic acid, desalted with C18 LTS tips (Rainin), injected into a Q Exactive Mass Spectrometer (Thermo) equipped with an Ultimate 3000 HPLC (Thermo), and analyzed as previously described .
RT-qPCR analysis of S. mansoni gene expression
RT-qPCR involved specific primers designed and selected for gene targets encoding SmM28B, S. mansoni cathepsin B1.1 (SmCB1.1, GenBank AJ506157), and S. mansoni cytochrome C oxidase I (SmCOX I, GenBank AF216698) as the sample reference gene transcript (Additional file 3: Table S2). Primer3 software (http://frodo.wi.mit.edu/ ) was used to design specific novel primers for the gene encoding SmM28B. Primers were evaluated by serial dilutions of both the primers and the cDNA template as described [33, 42, 43], while SmCB1.1 primers were adopted from our previous studies [33, 43]. The cDNA from various life stages was generated using the mRNA isolation protocol described above and previously [33, 43]. Reactions containing LightCycler 480 SYBR Green I Master (Roche) were prepared in final volumes of 25 μl in 96-well plates and carried out as described previously . PCR reactions were performed in triplicate with at least one biological replicate. Analysis using the reference gene transcript and the 2−CT method was as previously published [33, 43] to measure transcript levels . The whole experiment was repeated when CT values of technical replicates fluctuated by 0.5 or more. The resulting transcript levels were calculated as a percentage of the nonspecific control (mCherry dsRNA for RNAi) or the highest transcript level that was set at 100%.
Probes design for RNA in situ hybridization
The specific PCR products were amplified by polymerase chain reaction (PCR) from the first-strand cDNA of adults S. mansoni and F. hepatica using gene-specific primers (Additional file 4: Table S3). The PCR fragments of approximately 450 bp were ligated into the pGEM-T Easy Vector (Promega), and cloned sequences were verified by DNA sequencing. Verified constructs were linearized and used as a template for the production of sense/antisense RNA probes according to the manufacturer’s instructions (DIG RNA labeling kit SP6/T7, Roche).
RNA in situ hybridization (ISH)
Sections (7 µm F. hepatica/5 µm S. mansoni) of adult worms were de-paraffinized in xylene, rehydrated, and washed in diethyl-pyrocarbonate (DEPC, Sigma-Aldrich)-treated water. The S. mansoni sections were incubated in 0.01 M sodium citrate buffer, pH 6.0, in a boiling water bath for 15 min and were then cooled for 30 min. In the case of F. hepatica, the sections were treated with proteinase K (final concentration 2 µg/ml, Roche) for 5 min at 37 °C. Hybridization was performed for 16 h at 42 °C (S. mansoni) and 55 °C (F. hepatica) with RNA probes diluted to 1:200 in a hybridization mixture (5 × SSC, 1 × PBS, 0.1% Torula yeast RNA (Sigma-Aldrich), 50% formamide, 10% dextran sulfate molecular weight 4000 (Sigma-Aldrich), 1% Tween 20) according to the modified protocols [45, 46]. After hybridization, slides were washed and incubated in blocking solution as described [45, 46]. Subsequently, final detection in the tissue of F. hepatica was carried out using Anti-DIG-AP antibodies (Roche) at a 1:500 dilution and SIGMAFAST™ Fast Red TR/Naphthol AS-MX tablets (Sigma-Aldrich); results were visualized with a Nikon ECLIPSE Ni-E microscope. In the case of the significantly smaller S. mansoni, hybridized probes were labeled with Anti-DIG-HRP antibodies (Roche) at a 1:500 dilution, and signal amplification was performed using the Tyramide Signal Amplification (TSA) system with the Cyanine Plus 5 Tyramide Reagent fluorescence system (Perkin Elmer) according to the manufacturer’s protocol. Sections were embedded in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific) and visualized with an Olympus IX83 microscope equipped with PCO edge 5.5. camera and CoolLED pE-4000 LED illumination system. As negative controls, sections were incubated under the same conditions with sense probes and without any probe. Images from both S. mansoni and F. hepatica slides were processed using ImageJ software version 1.52 u .
Polyclonal antibody production and immunohistochemistry (IHC)
Purified recombinant SmM28B (BL21 E. coli) and FhM28B (HEK-293FT source, with the N-terminal HALO tag removed) were used to produce rabbit polyclonal antibodies at the service facility of the Institute of Microbiology, Czech Academy of Sciences. Rabbit polyclonal antibodies against major S. mansoni gut protease SmCB1 were raised against recombinantly produced protein  and kindly provided by Dr. Daniel Sojka, from the Biology Centre, Czech Academy of Sciences. Sample fixation, deparaffinization, rehydration, and antigen retrieval followed the same protocol as was described in the RNA in situ hybridization section below [49, 50]. After antigen retrieval with sodium citrate buffer, pH 6.0, sections were washed three times in 1 × PBS. Then, sections were permeabilized in fresh PBS, pH 7.5, supplemented with 0.25% (v/v) Triton X-100 (PBS-Tx) for 20 min followed by overnight incubation in the blocking buffer composed of PBS-Tx + 2% BSA. Finally, the sections were washed once in antibody diluent (AbD; PBS-Tx + 1% BSA) and subsequently probed with respective immunized sera at a dilution of 1:50. Slides were incubated in the wet chamber at 4 °C overnight. Respective controls included primary sera and no sera to exclude non-specific reactions of sera or secondary antibodies, respectively. After three washes in (PBS-Tx) for 10 min each, slides were incubated with goat anti-rabbit IgG H&L Alexa Fluor Plus 647 secondary antibodies (ThermoScientific) at a dilution of 1:500, followed by 45 min of incubation at 37 °C in the wet chamber. Slides were again washed 3 × in PBS-Tx for 10 min each followed by a final 10 min wash in PBS. Slides were mounted in ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) and visualized by Nikon ECLIPSE Ni-E fluorescence microscope. Images were processed with ImageJ software version 1.52u.
RNA interference in S. mansoni
As templates for dsRNA synthesis, PCR products of targeted SmM28B and positive control S. mansoni cathepsin B1.1 (SmCB1.1, GenBank AJ506157) were PCR amplified from cDNA prepared from 5-day-old in vitro-cultivated S. mansoni schistosomula as described previously . DNA coding mCherry protein was used as a nonspecific control dsRNA. PCR products were approximately 500 bp in size (Additional file 5: Table S4). Synthesis of dsRNA from gel-purified PCR templates was accomplished using the T7 RiboMAX Express RNAi System (Promega) as described previously . Schistosomula transformed from cercariae or adults perfused from the infected mice [33,34,35] were incubated in 24-well plates containing 1 ml of Complete Medium 169 with 5% FBS , and dsRNA 30 µg/ml at 37 °C and 5% CO2 . (i) For monitoring of gene silencing levels, parasites were collected after 5 days of cultivation, washed three times in PBS, and resuspended in 50 ml of Trizol reagent (Thermo Fisher Scientific). RNA isolations and evaluations of gene expression levels by RT-qPCR followed previous protocols [33, 43] as described for the reaction design above. (ii) For observing phenotypic changes, dsRNA-treated parasites were monitored regularly for 14 days.