Ethics statement

All mouse procedures performed at Aberystwyth University (AU) adhered to the United Kingdom Home Office Animals (Scientific Procedures) Act of 1986 (project licenses PPL 40/3700 and P3B8C46FD) as well as the European Union Animals Directive 2010/63/EU, and were approved by AU’s Animal Welfare and Ethical Review Body (AWERB). The use of mice at George Washington University Medical School was approved by The Institutional Animal Care and Use Committee (IACUC).

Parasite material

A Puerto Rican strain of S. mansoni (NMRI, Naval Medical Research Institute) was used in this study. Mixed-sex adult worms were perfused from percutaneously infected TO (HsdOla:TO, Envigo, UK), MF-1 (HsdOla:MF1, Envigo, UK) or Swiss Webster (CFW, Charles River Laboratories, USA) mice infected 7 wk earlier with 180 cercariae [26]. These parasites were cultured (10 sex-separated adults or five adult pairs per well in a 48 well tissue culture plate) at 37°C in DMEM (Sigma-Aldrich) supplemented with 10% foetal calf serum, 2 mM L-glutamine and 100 μg/ml penicillin/streptomycin in an atmosphere of 5% CO₂ with a 70% media exchange performed after 24 hr and 48 hr for adult male/adult pairs and female worms respectively. Cultured worms were subsequently used for RNA interference, CRISPR/Cas9 genome editing, total RNA isolation/complementary DNA (cDNA) generation, dual RNA and genomic DNA (gDNA) extraction and enzyme assays.

Multiple sequence alignment and phylogenetic trees

The Pfam identifier PF16499 (melibiase-2) was used to identify S. mansoni, Schistosoma haematobium and Schistosoma japonicum GH family 27 orthologs of H. sapiens α-GAL and α-NAGAL enzymes using WormBase ParaSite [27]. Only those Schistosoma entries that contained >70% of the residues within the melibiase-2 domain identified by Pfam, UniProt and CAZypedia were retained [18,28,29]. These included S. mansoni Smp_170840 (G4VLE1), S. haematobium MS3_10002 (A0A095A3Q8), S. haematobium MS3_10345 (A0A095B3P1), S. mansoni Smp_179250 (G4VLD7), S. mansoni Smp_247760 (A0A5K4F2K9), S. mansoni Smp_247750 (A0A5K4F165), S. haematobium MS3_10001 (A0A095CF43), S. japonicum EWB00_005283 (A0A4Z2D326), S. japonicum EWB00_005285 (A0A4Z2D213), S. japonicum EWB00_005284 (A0A4Z2D211), S. haematobium MS3_11280 (A0A095BSM8) and S. mansoni Smp_089290 (G4VLE3). A multiple sequence alignment of these Schistosoma melibiase-2 domain containing proteins with human α-GAL (H. sapiens, Hs_GAL, P06280), human α-NAGAL (H. sapiens, Hs_NAGAL, P17050), chicken α-NAGAL (Gallus gallus, Gg_NAGAL, Q90744) and C. elegans α-NAGAL (Ce_GANA-1, Q21801) was generated using MUSCLE v3.8 [30]. Amino acids included in the alignment were based on the positions of ligand binding and catalytic aspartic acid (D) residues of the melibiase-2 domains of human α-GAL and α-NAGAL [31–33].

Phylogenetic analysis of melibiase-2 domain containing proteins from a broad range of species across phyla was conducted using Bayesian inference and Maximum Likelihood approaches. MUSCLE v3.8 software was used to align proteins from S. mansoni, S. haematobium, S. japonicum (all noted previously) and additional sequences including H. sapiens Hs_GAL (P06280), H. sapiens Hs_NAGAL (P17050), Drosophila melanogaster Dm_CG7997 (Q7K127), D. melanogaster Dm_CG5731 (Q8MYY3), C. elegans Ce_GANA-1 (Q21801), Arabidopsis thaliana At_AGAL1 (Q9FT97), A. thaliana At_AGAL2 (Q8RX86), A. thaliana At_AGAL3 (Q8VXZ7), Schizosaccharomyces pombe Sp_MEL1 (Q9URZ0), Coffea arabica Ca_GAL (Q42656), G. gallus Gg_NAGAL (Q90744), Rattus norvegicus Rn_NAGAL (Q66H12), Mus musculus Mm_GAL (P51569) and M. musculus Mm_NAGAL (Q9QWR8). The alignment was submitted to Gblocks Server 0.91b which removed non-conserved regions to allow phylogenetic comparisons [34]. In total, 208 amino acid residues were used for this phylogenetic analysis. Bayesian inference analyses were performed using MrBayes v3.2.6 [35], the WAG substitution model [36], 500,000 generations and a sample frequency of 100. As the analysis proceeded, log likelihood values were generated and plotted against the number of generations. The analysis was stopped upon examination of the average standard deviation of split frequencies indicating when the log likelihood values reached a stationary distribution. Maximum Likelihood analyses were performed using MEGA X [37] with the JTT model [38] and 500 bootstrap replicates. The final Bayesian consensus phylogram was generated using FigTree v1.4.3 [39]. The Bayesian inference tree was edited using Adobe Illustrator CS4 software where Bayesian posterior probability support values (outside parentheses) and Maximum Likelihood percentage bootstrap support values (inside parentheses) were superimposed on corresponding nodes.

Homology modelling of Smp_089290

The three-dimensional structure of Smp_089290 was derived by homology modelling using MODELLER [40]. The α-GAL/α-NAGAL template selected for Smp_089290 modelling was the three-dimensional structure of H. sapiens α-NAGAL (Protein Databank identification code 4DO4) [32]. The sequence identity between Smp_089290 and the human α-NAGAL was 40% with a sequence coverage of 97%, hence well within the acceptable range for comparative modelling [41]. The stereochemical quality of the Smp_089290 model was assessed by RAMPAGE (Ramachandran Plot Analysis) [42], ProSA-web (Protein Structure Analysis) [43] and Verify 3D [44].

Total RNA isolation and cDNA generation

S. mansoni total RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research) with slight modifications. Briefly, worms were removed from culture, transferred to 2 ml Eppendorf tubes, and homogenised in QIAZOL reagent (Qiagen) with a 5 mm diameter stainless steel bead (Qiagen) for a total of 6 min (2 x 3 min with 1 min on ice in between homogenisations) at 50 Hz using a TissueLyser LT (Qiagen). Thereafter, the RNA was treated with DNase I to remove contaminating gDNA. RNA was eluted into collection tubes by adding 30 μl of DNase/RNase-free H₂O directly to the Direct-zol column matrix and subsequently centrifuged at 17,000 x g for 60 sec. Yields of total RNA samples were assessed using a NanoDrop ND-1000 UV-Vis spectrophotometer; RNAs were concentrated when necessary in a Concentrator plus (Eppendorf) and re-quantified. Schistosome cDNAs were reverse transcribed from the total RNAs using the SensiFAST cDNA Synthesis Kit (Bioline) according to the manufacturer’s instructions.

cDNA cloning and sequencing of smp_089290

PCR primers used to amplify the full coding sequence of Smp_089290 included forward (5´–ATG GCT ACC GTA CCA CCG– 3´) and reverse (5´–CTA TAA CAA TGT CTG AAA CAG TCC ATC– 3´) pairs. PCR amplification utilised cDNA prepared from total RNA obtained from mixed-sex adult worms [45]. The amplification was performed in 25 μl containing Biomix reaction mix (Bioline) and ultra-stable Taq DNA polymerase (Bioline). Amplicons were subjected to electrophoresis through 1% w/v agarose, stained and visualised with SYBR Safe dye (Thermo Fisher Scientific) under UV light. Products of the predicted size of 1,463 bp were ligated into pGEM-T Easy (Promega) after which α-Select Bronze Efficiency Competent Cells (Bioline) were transformed with the ligation products. Blue/white screening of E. coli colonies was accomplished on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and Isopropyl β-D-1-thiogalactopyranoside (IPTG). Recombinant (white) clones were isolated from a 5 ml High Salt (HS) Luria Bertani (LB) culture containing ampicillin (50 μg/ml) using the QIAprep Spin Miniprep Kit (Qiagen). Inserts in pGEM-T Easy were sequenced at Aberystwyth University’s Translational Genomics Facility and the sequence trace was analysed using FinchTV 1.4.0 software [46]. The full coding sequence of Smp_089290 is deposited under the GenBank accession number MZ508282.

DNA microarray analysis

The S. mansoni long oligonucleotide DNA microarray was designed and constructed by Fitzpatrick and colleagues [45]. The DNA microarray consists of 35,437 S. mansoni oligonucleotide 50-mers as well as 2,195 controls and is deposited in the ArrayExpress database under the accession number A-MEXP-830. S. mansoni lifecycle stages profiled by the DNA microarray included egg, miracidia, mother (2 day) sporocysts, daughter sporocysts, cercariae, 3 hr schistosomula, 24 hr schistosomula, 3 day schistosomula, 6 day schistosomula, 3 wk worms, 5 wk worms, 7 wk worms, male 7 wk worms and female 7 wk worms. Normalised fluorescence intensity values (available via ArrayExpress under the experimental accession number E-MEXP-2094), corresponding to 50-mers representing exons 2 and 3 of smp_089290, enabled the quantification of gene expression profiles across these developmental stages of the schistosome.

RNA-Seq meta data analysis

The RNA-Seq meta database was created by Lu and colleagues [47] by normalising gene expression values derived from RNA-Seq data produced by various publications. Gene expression values for each lifecycle stage were obtained from the following reports: Anderson et al. [48] for egg, Wang et al. [49] for miracidia and sporocysts, Protasio et al. [50] for cercariae, 3 hr and 24 hr schistosomula, Protasio et al. [51] for 21 day juvenile male, 21 day juvenile female, 28 day juvenile male, 28 day juvenile female, 35 day adult male, 35 day adult female, 38 day adult male and 38 day adult female and Lu et al. [52] for 42 day adult male and 42 day adult female. The normalised gene expression values for a gene of interest were obtained by entering the gene ID into the ‘schisto_xyz’ search engine [53] and subsequently plotted as a gene expression profile.

RNA interference (RNAi)

Small interfering RNA (siRNA) duplexes were designed (siSmp_089290: sense strand 5’-CUA AUG AAA UCG UUG CAG A-3’; anti-sense strand 5’-UCU GCA ACG AUU UCA UUA G-3’) based on the cDNA sequence verified smp_089290 amplicon and possessed no significant homology to other gene products (i.e. less than 15 out of 19 nucleotides conserved) in the S. mansoni genome assembly (v7.0) [54,55]. An siRNA duplex designed for firefly luciferase (siLuc) without significant homology to gene products in the S. mansoni genome assembly (v7.0) served as a negative control [56]. Briefly, sets of 10 worms (sex-separated) or five adult pairs were transferred to 0.4 cm pathway electroporation cuvettes (Invitrogen) containing DMEM supplemented with 2 mM L-glutamine and 100 μg/ml penicillin/streptomycin. siRNA duplexes (5 μg) were subsequently added and worms were electroporated with a single pulse at 125 V (LV mode) for 20 ms using an ECM 830 Square Wave Electroporation System (BTX, Harvard Apparatus, Holliston, MA). Worms were subsequently transferred to a 48 well tissue culture plate and cultured for up to seven days.

Preparation of CRISPR/Cas9 plasmid constructs

Two CRISPR target sites (single guide RNA; sgRNA sequence) for Cas9-catalysed gene editing for smp_089290 were designed by Breaking-Cas [57] with default parameters compatible for the protospacer adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (NGG) [58, 59]. Two sgRNAs targeting the coding regions exons 1 (SmNAGALX1: 5´–CUA CCG UAC CAC CGA UGG GU– 3´) and 2 (SmNAGALX2: 5´–UUG UAA UCU AUG GCG UAU GC– 3´) were used in this study. The sgRNAs contained >40% GC-content, no self-complementarity and no off-target sites against the S. mansoni genome assembly (v7.0) as predicted by Breaking-Cas software. A 20 nucleotide (nt) ‘Scramble’ sgRNA designed with low homology to the S. mansoni genome assembly (v7.0) and lack of an adjacent PAM site (necessary for Cas9 function) served as a non-targeting control (5´–GCA CUA CCA GAG CUA ACU CA– 3´). CRISPR/Cas9 plasmid constructs were assembled using the pLenti-Cas-Guide construction Kit (GE100010, OriGene, Maryland, USA) and each sgRNA was ligated into the pLenti-Cas-sgRNA backbone as per the manufacturer’s instructions. Expression of the sgRNA (SmNAGALX1, SmNAGALX2 or Scramble containing plasmids) was driven by the mammalian U6 promoter and expression of Cas9 from S. pyogenes with nuclear localisation signals was driven by the human cytomegalovirus (CMV) promoter. Each CRISPR/Cas9 plasmid construct was independently transformed into DH5-α Chemically Competent Cells (GoldBio) with chloramphenicol (25 μg/ml) as drug selection. The chloramphenicol resistant transformants were confirmed for corrected orientation of sgRNA in the vector backbone by Sanger direct sequencing. To amplify the plasmid DNA, the transformants were first cultured in a 10 ml HSLB culture containing chloramphenicol (25 μg/ml) in a shaking incubator at 37°C, 225 rpm for approximately 7 hr. Thereafter, 10 ml of the culture was added to 240 ml of fresh HSLB culture containing chloramphenicol (25 μg/ml) and cultured overnight, as above. Plasmid DNA was recovered from 150 ml of this overnight culture using the GenElute HP Plasmid Midiprep Kit (Sigma-Aldrich) as per the manufacturer’s instructions except for a modified elution step; here, accomplished with 800 μl of nuclease-free H₂O. Plasmid DNAs were quantified using the NanoDrop ND-1000 UV-Vis spectrophotometer (Thermo-Fisher Scientific), after which the DNA was precipitated and subsequently dissolved in Opti-MEM Reduced Serum Medium (Thermo-Fisher Scientific) and stored at -20°C.

CRISPR/Cas9 mediated genome editing

A mixture of 24 μg of CRISPR/Cas9 plasmid DNA (either SmNAGALX1, SmNAGALX2 or Scramble) reconstituted in a total volume of 400 μl Opti-MEM Reduced Serum Medium was dispensed into a 0.4 cm pathway electroporation cuvette (Invitrogen). Five pairs of worms were transferred to the cuvettes, which had been chilled on wet ice, and subjected to square wave electroporation with a single pulse of 125 V (LV mode) for 20 ms (BTX, Harvard Apparatus). Worms were subsequently transferred to a 48 well tissue culture plate and cultured for up to seven days at 37°C in an atmosphere of 5% CO₂. Upon completion of the seven day experiment, RNA and gDNA was extracted from adult male and female worms using both RNAzol reagent (Sigma-Aldrich) and DNAzol reagent (Invitrogen) based on the manufacturer’s instructions. One ml of RNAzol reagent was added to the parasite material, then subsequently homogenised four times with a 5 mm diameter stainless steel bead (Qiagen) for 3 min at 50 Hz using a TissueLyser LT (Qiagen) with 1 min incubation on ice in between homogenisations. Once the tissues of the schistosomes were completely disrupted, the protocol steps were followed until completion. RNA samples were immediately used for cDNA synthesis, as above. gDNA samples were amplified by PCR using primers encompassing the programmed double-strand break (DSB) site at exon 1 (Forward: 5´–CTT ATA GGT GTG CCA TAT TAA CGA T– 3´, Reverse: 5´–ATG CAC TAC ATT CGA AAG ACA– 3´) or exon 2 (Forward: 5´–AGT GTT CTC ATG CAG TTA TCC T– 3´, Reverse: 5´–TCC ATG TCA GCT GAG ATC A– 3´) of smnagal. Correct amplification was verified by 1% w/v agarose gel electrophoresis. Thereafter, amplicons were subjected to the QIAseq 1-Step Amplicon Library Kit (Qiagen) for Illumina compatible next generation sequencing (NGS) library construction with GeneRead DNAseq Targeted Panels V2 (Qiagen) as per the manufacturer’s instructions. Amplicon size of each NGS library was verified using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). The NGS libraries were quantified using the GeneRead Library Quant Kit (Qiagen). NGS libraries were pooled at the Genewiz NGS facility (Genewiz, NJ) and processed with a MiSEQ configuration of 2x250 bp. The demultiplexing data generated by the Genewiz NGS facility was exported as Fastq files (.qz format). The mutations around the cut sites were analysed by CRISPResso2 software using default parameters [60,61]. Background mutations (i.e. mutations not attributable to genome editing) were inferred by identifying mutations present in the control samples and the treatment samples. The unique mutations around the programmed DSB sites only reported in the high quality sequence reads of the target sample were designated as CRISPR/Cas9-induced mutations. The software analysed and reported the percentage of indels (insertions and deletions) and substitutions in the target sample (inferred by the number of reads that are modified in comparison to the total reads), the percentages of each mutation type and a list of each specific mutation showing which nts have been altered. Sequence reads from the NGS libraries are available at the Sequence Read Archive (SRA) under BioProject ID PRJNA743897.

Quantitative reverse transcription (RT)–PCR (qRT-PCR) analysis

cDNAs synthesised from freshly perfused, siRNA treated or CRISPR/Cas9 plasmid treated schistosomes were used as templates for qRT-PCR to analyse transcript abundance. smp_089290 transcript levels in freshly perfused schistosomes were quantified relative to Ras-Related GTP-binding protein D (smrgbd) and Tubulin-specific chaperone D (smtscd); these two genes were identified by Normfinder [62] as being constitutively expressed across nine S. mansoni lifecycle stages, including the adult [63]. smp_089290 transcript levels in siRNA treated and CRISPR/Cas9 plasmid treated schistosomes were quantified relative to α-tubulin (smat1). qRT-PCR was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) and SensiFAST SYBR Hi-ROX mix (Bioline). Total reaction volume was 10 μl with 150 nM of each primer, 5 μl of SensiFAST SYBR Hi-ROX mix, 2 μl of cDNA template and 2.7 μl of nuclease-free H₂O. qRT-PCR primers for smp_089290 were designed based on the sequence verified smp_089290 amplicon and included forward (5’-CAC GAC TGA TGG TGG TGG-3’) and reverse (5’-CTC GAT ACA TCA TTA TCC CGC T-3’) pairs. smrgbd primers included forward (5’-CGG CTT TAA CTC GCC TAC AC-3’) and reverse (5’-CAT TCG ACG GTT GTT CAC AC-3’) pairs. smtscd primers included forward (5’-GCC AAC AAA TTT CGT GGT CT-3’) and reverse (5’-TTC ACC ATT TCG GTC GTA CA-3’) pairs. smat1 primers included forward (5’-CTT CGA ACC AGC AAA TCA GA-3’) and reverse (5’-GAC ACC AAT CCA CAA ACT GG-3’) pairs. To determine smp_089290 knockdown (KD) in adult worms following RNAi and CRISPR/Cas9 programmed knockout, smp_089290 transcript levels in siRNA treated samples (at 48 hr post electroporation) and CRISPR/Cas9 plasmid treated samples (at seven days post electroporation) were quantified relative to smat1 as described previously [64]. The Geometric mean method was used to quantify smp_089290 transcript levels in freshly perfused male and female schistosomes as described previously [65,66]. Melting curves (dissociation curves) were generated for each qRT-PCR analysis to verify the amplification of one product only. A two-tailed student’s t-test was used to test for significance between siRNA treatments. A Kruskal-Wallis ANOVA with Dunn post-hoc comparisons was used to test for significance between CRISPR/Cas9 plasmid treatments. A two-tailed student’s t-test was used to test for significance between freshly perfused adult male and female worms.

Whole mount in situ hybridisation (WISH)

The full coding sequence of Smp_089290 was amplified using forward (5´–ATG GCT ACC GTA CCA CCG– 3´) and reverse (5´–CTA TAA CAA TGT CTG AAA CAG TCC ATC– 3´) primer pairs. The PCR products were subsequently cloned into the pJC53.2 vector [67] using standard cloning methods as mentioned previously. These recombinant plasmids were subsequently used to generate digoxigenin-labelled riboprobes using the Riboprobe System (Promega) with SP6 or T3 RNA polymerases and digoxigenin-labelled Uracil triphosphate (Roche) [68]. Antisense treatment riboprobes (generated by SP6 polymerase) and sense control riboprobes (generated by T3 polymerase) were processed as described [68] and stored at -20°C until needed for the WISH staining protocol. Riboprobes were used within 2 wk of their initial storage at -20°C.

Adult male and female worms were relaxed and separated by incubation (15 min) in a 0.25% solution of the anaesthetic ethyl 3-aminobenzoate methanesulphonate (Sigma-Aldrich) dissolved in DMEM [69]. Relaxed parasites were subsequently killed in a 0.6 M solution of MgCl₂ (1 min) and fixed for 4 hr in 4% formaldehyde in PBSTx (PBS + 0.3% Triton X-100). Fixed parasites were dehydrated in MeOH and stored at -20°C until needed. Parasite samples were rehydrated in 1:1 MeOH:PBSTx (5–10 min) followed by incubation in PBSTx (5–10 min). Rehydrated parasite samples were bleached in formamide bleaching solution (0.5% v/v de-ionised formamide, 0.5% v/v SSC and 1.2% w/w H₂O₂, brought to a final volume of 10 ml with diethyl pyrocarbonate H₂O) for 90 min, rinsed twice in PBSTx, treated with proteinase K (10 μg/ml, Invitrogen) for 45 min at room temperature and post-fixed for 10 min in 4% formaldehyde in PBSTx. Parasite samples were processed as previously described [67,68] with several modifications. Antisense treatment riboprobes and sense control riboprobes were mixed with hybridisation solution (50% v/v de-ionised formamide, 10% w/v dextran sulphate, 1.25% v/v SSC, 1 mg/ml yeast RNA, 1% v/v Tween-20, brought to a final volume of 40 ml with diethyl pyrocarbonate H₂O) and hybridised overnight at 52°C. Parasite samples were transferred to fresh colorimetric developing solution consisting of alkaline phosphatase buffer (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl₂, 0.1% v/v Tween-20, brought to a final volume of 10 ml with 10% polyvinylalcohol solution) supplemented with 4.5 μl/ml NBT (Roche) and 3.5 μl/ml BCIP (Roche). All parasite samples treated with the antisense treatment riboprobe were developed at room temperature in the dark until the desired level of purple signal was reached (male samples = 2 hr, female samples = 45 min). In parallel, all parasite samples treated with the sense control riboprobe were developed for the same length of time. Once the desired level of signal was reached, the colorimetric developing solution was removed and worms were rinsed twice in PBSTx to stop any further development from occurring. Worms were dehydrated in 100% ethanol for 5 min and subsequently submerged in 80% glycerol in 1x PBS and incubated overnight at 4°C. Thereafter, stained worms were mounted onto microscope slides and examined with a light microscope (Leica LMD6000 Laser Microdissection Microscope).

Single-cell RNA-Seq (scRNA-Seq) analysis

Localisation of smp_089290 (smnagal) found within the 68 adult worm clusters generated from available scRNA-Seq data [70] was depicted as uniform manifold approximation and projection (UMAP) plots using Seurat V3 [71].

α-GAL/α-NAGAL enzymatic activity measurements

Soluble worm antigen preparation (SWAP) was derived from worms (freshly perfused or electroporated with siRNAs) removed from culture after 1 wk. Worms were homogenised with a 5 mm diameter stainless steel bead (Qiagen) for 4 min at 50 Hz in 100 μl of 0.15 M McIlvaine buffer pH 4.6 [72] with EDTA-free protease inhibitors (Sigma-Aldrich) using a TissueLyser LT (Qiagen). After homogenisation, tubes were centrifuged at 21,100 x g for 30 min at 4°C and SWAP was collected and quantified by the Bradford method (Sigma-Aldrich).

The enzymatic activity of SWAP derived from freshly perfused adult male and female worms, siLuc treated and siSmp_089290 treated adult male and female worms was measured using 4-Nitrophenyl α-D-galactopyranoside (α-GAL colorimetric substrate) and 4-Nitrophenyl N-acetyl-α-D-galactosaminide (α-NAGAL colorimetric substrate) in separate reactions. Differing concentrations of human α-GAL (Fabrazyme, kindly provided by the Leiden University Medical Centre, LUMC) and commercially sourced α-NAGAL cloned from Chryseobacterium meningosepticum and expressed in E. coli (New England Biolabs, Ipswich, MA) were measured with corresponding substrates.

Enzyme assays were performed in standard flat-bottomed 96 well plates (STARLAB). α-GAL/α-NAGAL assays consisted of 100 μl of α-GAL or α-NAGAL substrate dissolved in 0.15 M McIlvaine buffer (pH 4.6) with the addition of differing concentrations of α-GAL or α-NAGAL and 0.15 M McIlvaine buffer pH 4.6 to give a final volume of 125 μl per well. Concentrations of α-GAL used to generate a standard curve of activity were as follows: 0.95, 0.475, 0.19, 0.0475, 0.0095, 0.00475 and 0.0019 μg/ml. Concentrations of α-NAGAL used to generate a standard curve of activity were as follows: 20, 15, 10, 5, 2 and 1 μg/ml. SWAP reactions were set-up in the same way but α-GAL/α-NAGAL enzymes were replaced with different quantities of sample specific SWAP and incubated with both α-GAL and α-NAGAL substrates. For untreated adult male and female worms, 5 μg of SWAP was used per well to enable comparison between gender. For siLuc treated and siSmp_089290 treated adult male and female worms, 6.45 μg and 1.08 to 2.44 μg of SWAP was used per well to enable comparison between siRNA treatments, respectively. Reaction proceeded for 60 min at 37°C and terminated by the addition of 70 μl of 0.4 M glycine (pH 10.4) [72]. Final absorbances were quantified at 410 nm using a POLARstar Omega microplate reader (BMG Labtech). Absorbance values produced from different SWAP treatments were compared to α-GAL/α-NAGAL standard curves to calculate α-GAL and α-NAGAL activities (μg/ml). A two-tailed student’s t-test was used to test for significance between siRNA treated samples or between genders.

Motility analysis quantified by WormAssayGP2 and adult worm scoring matrix

The digital image processing-based system known as WormAssayGP2 was derived from Marcellino et al. [73] and implemented by us as previously described [74]. Individual wells containing up to 10 adult worms were recorded for 60 sec each day and analysed by the Lucas-Kanade algorithm. Once recording was completed, the data were quantified and stored as a .csv file which was further processed to calculate the mean motility for the control and treatment groups. In parallel, adult worm motility was also scored using the WHO-TDR scoring matrix [75]. All worms were scored daily from two days after electroporation until seven days after electroporation. Worms were ranked between 4–0 based on their motility; 4 = normal active/paired up, 3 = full body movement but slowed activity, 2 = minimal activity, occasional movement of head and tail only, 1 = movement in the suckers only or slight contraction of the body and 0 = total absence of motility. For both motility analyses, a General Linear Mixed-Effects Model was fitted to each dataset (‘NLME’ package) and statistical differences were determined by performing pairwise comparisons of the estimated marginal means of each group per time point (‘EMMEANS’ package) in R. Video footage of adult worms was captured using a NexiusZoom stereo microscope (Euromex) and edited with ImageFocus 4 software (Euromex).

Enumeration of vitellocytes and egg volume measurements

The eggs released by adult female worms from RNAi and CRISPR/Cas9 genome editing experiments were collected and fixed in 1 ml 10% neutral buffered formalin solution (Sigma-Aldrich) for 24 hr at 4°C. Thereafter, eggs were enumerated using a Sedgewick Rafter Counting Chamber [76]. Prior to visualisation by laser scanning confocal microscopy (LSCM), stored eggs from RNAi experiments only were immersed in PBS supplemented with DAPI (4’,6-diamidino-2-phenylindole, 2 μg/ml). Fluorescence images (10 eggs per treatment) were captured on a Leica TCS SP8 super resolution laser confocal microscope fitted with a 63x (water immersion) objective using the Leica Application Suite X (LAS X). Green (egg autofluorescence) fluorescence was visualised with an argon or diode-pumped, solid state (DPSS) laser at 488 nm. DAPI was visualised using a 405 nm blue diode laser. The number of vitellocytes (DAPI⁺ cells) and overall volume (mapped by the green autofluorescence) for individual eggs were calculated using IMARIS 7.3 software (Bitplane). A two-tailed student’s t-test was used to test for significance between siRNA treated samples with regard to the total number of eggs produced and individual egg volume. A Kruskal-Wallis ANOVA with Dunn post-hoc comparisons was used to test for significance between CRISPR/Cas9 plasmid treated samples with regard to the total number of eggs produced. IMARIS 7.3 software (Bitplane) was also used to create a video showing the 360° horizontal rotation of a representative egg from each siRNA treatment.

Vitelline droplet staining and diphenol oxidase localisation

To visualise vitelline droplets, whole siRNA treated-adult worms were stained with 1% Fast Blue BB as described previously [77]. Diphenol oxidase activity was localised in whole siRNA treated-adult worms by incubating intact, previously frozen parasites (-80°C) in a 0.2 M KH₂PO₄ (pH 6.7) buffer containing 4 mM 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH, Sigma) and 1 mM l-3,4-dihydroxyphenylalanine (L-DOPA, Sigma) as described previously [78]. After incubation for 30 min at 30°C, the reaction was stopped with 1% acetic acid. Thereafter, all worms were mounted onto microscope slides and examined with a light microscope (Zeiss Axio Imager 2 Microscope).

Schistosoma mansoni contains a single α-N-acetylgalactosaminidase (SmNAGAL)

α-N-acetylgalactosaminidase (α-NAGAL) is a member of the glycosyl hydrolase (GH) 27 family and contains both a melibiase-2 (PF16499) and a melibiase-2 C-terminal (PF17450) domain. As all enzymatically important (ligand binding and catalytic) amino acids are conserved within the melibiase-2 domain [33], we focused our interrogation of the S. mansoni genome (v7.0) for the presence of putative homologs that contained >70% of the residues within this domain. Our analysis revealed the presence of five schistosome members that met this criterion: Smp_170840, Smp_179250, Smp_247760, Smp_247750 and Smp_089290 (Fig 1).

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Fig 1. Schistosoma mansoni contains five GH27 family members, but only Smp_089290 contains all residues necessary for α-NAGAL substrate binding and cleavage.

A concatenated multiple sequence alignment of α-GAL and α-NAGAL proteins from H. sapiens (Hs), G. gallus (Gg), C. elegans (Ce), S. japonicum (EWB), S. mansoni (Smp), and S. haematobium (MS3) throughout the melibiase-2 domain (PF16499). Numbers located at the beginning of each sequence represent the amino acid position in the protein sequence. Ligand binding residues are highlighted green whereas non-conserved amino acids in the same position in other sequences are white. Catalytic aspartic acid (D) residues are highlighted light blue whilst non-conserved amino acids in the same position in other sequences are highlighted red. Amino acid residues which are missing from ‘Ce_GANA-1’, ‘Smp_170840’ and ‘MS3_10001’ are indicated with — whereas N/A is used for the amino acid position.

https://doi.org/10.1371/journal.ppat.1009828.g001

Among these five S. mansoni gene products, only one (Smp_089290, highlighted yellow, Fig 1) contained all 13 ligand binding residues (green shaded amino acids, Fig 1) as well as both catalytic aspartic acid residues (light blue Ds, Fig 1) critical for α-NAGAL activity [32]. MS3_11280 (S. haematobium homolog) and EWB00_005284 (S. japonicum homolog) also shared these diagnostic characteristics. Furthermore, while two additional S. japonicum homologs (EWB00_005283 and EWB00_005285) contained both catalytic aspartic acid residues, they did not possess all 13 ligand binding residues. These five schistosome proteins, containing the essential catalytic aspartic acid residues, clustered into a separate clade discrete from other putative schistosome GH27 family members (Fig 2).

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Fig 2. Schistosoma melibiase-2 domain containing proteins with conserved catalytic aspartic acid residues cluster in a distinct clade of GH27 family members.

Phylogenetic analyses were conducted using a concatenated multiple sequence alignment of Schistosoma proteins that contained >70% of the residues within the melibiase-2 domain and α-GAL/α-NAGAL protein types across phyla. Proteins were analysed using both Maximum Likelihood and Bayesian inference approaches. Branch lengths (indicated by scale bar) represent distance among different taxa as predicted by the Bayesian inference approach. Node labels outside parentheses represent Bayesian posterior probability support values whilst those within parentheses represent percentage bootstrap support values from Maximum Likelihood analysis. Schistosoma proteins conserving catalytic aspartic acid residues are highlighted in the blue dashed box. The phylogram includes protein sequences from S. mansoni (Smp), S. haematobium (MS3), S. japonicum (EWB), A. thaliana (At), C. arabica (Ca), C. elegans (Ce), D. melanogaster (Dm), G. gallus (Gg), H. sapiens (Hs), R. norvegicus (Rn), M. musculus (Mm) and S. pombe (Sp).

https://doi.org/10.1371/journal.ppat.1009828.g002

The predicted three-dimensional structure of Smp_089290 was derived by homology modelling and passed all stereochemical quality assessments (S1 Fig). Homology modelling of Smp_089290 as a monomer (HsNAGAL functions as a homodimer [32]) revealed the positioning of these 13 ligand binding residues and two catalytic residues around the putative active site (purple, Fig 3). Despite the melibiase-2 C-terminal domain (red, Fig 3) not possessing any residues involved in ligand binding and substrate cleavage mechanisms, the predicted Smp_089290 model and H. sapiens α-NAGAL structure are consistent across this region. Both the predicted Smp_089290 model and H. sapiens α-NAGAL structure possess eight anti-parallel β-strands (Fig 3) [33]. Overall, the homology modelling analysis predicted that Smp_089290 likely possesses folding topology and spatial arrangements that closely resemble typical α-GAL and α-NAGAL proteins.

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Fig 3. Comparisons of the catalytic active site pockets found in the Smp_089290 homology model and the crystal structure of human α-NAGAL.

The grey dashed boxes depict a close-up view of the catalytic active site pocket within the Smp_089290 homology model’s and H. sapiens α-NAGAL crystal structure’s melibiase-2 domain (purple). The close-up views label all 13 ligand binding residues (green) and two catalytic Asp residues (light blue, underlined with double line) with their corresponding amino acid positions in each of the protein sequences shown. The red arrows show the location of the eight anti-parallel β-strands found near the C-terminus of the Smp_089290 homology model’s and the H. sapiens α-NAGAL crystal structure’s melibiase-2 C-terminal domain (red). Black arrows show the locations of the N and C-termini of the model/structure.

https://doi.org/10.1371/journal.ppat.1009828.g003

Together, these data suggest that the S. mansoni genome encodes a single gene product (Smp_089290) containing all essential catalytic amino acid residues for hydrolysis and release of α-N-acetylgalactosamine from glycosylated substrates. Therefore, this putative schistosome α-N-acetylgalactosaminidase (SmNAGAL, Smp_089290) was taken forward for further transcriptional, enzymatic, and functional genomics studies.

Smnagal is developmentally regulated, female-enriched and localised to vitellaria, mature vitellocytes and parenchymal cells

To begin deciphering SmNAGAL function, both transcriptomic- and enzymatic-based approaches were initiated (Fig 4). Meta-analysis of historical DNA microarray data across the S. mansoni lifecycle [45] was facilitated by two 50-mer oligonucleotide probes that retained 100% base-pair complementarity to exon 2 (CONTIG7235) and exon 3 (CONTIG6265), respectively, of smnagal (Fig 4A). For each of these two oligonucleotides, similar patterns of smnagal abundance were deduced. While smnagal expression was low in eggs, it increased in miracidia only to wane as schistosome development (sporocysts—cercariae) continued in the molluscan host (Fig 4B). Upon early intra-mammalian schistosome maturation (3 hr—3 day schistosomula), smnagal transcription remained invariably low, until day six post schistosomula transformation. At this point and extending into more developmentally mature lifecycle forms (3 wk–7 wk schistosomes), smnagal expression increased, reaching peak abundance in 7 wk old schistosomes. Here, female-enriched smnagal expression was clearly observed in adult schistosomes, which was independently confirmed by qRT-PCR (Fig 4C). Additionally, female-biased expression of smnagal was observed when interrogating S. mansoni RNA-Seq meta data (S2 Fig) [47]. Further support for female biased expression of smnagal was obtained from the analysis of α-N-acetylgalactosaminidase activity in adult worm protein extracts (Fig 4D). In a direct comparison, female protein extracts contained significantly higher levels of α-NAGAL activity when compared to males. This female biased trend was also observed with α-GAL activity measured in the same extracts (Fig 4D).

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Fig 4. smnagal (smp_089290) expression and α-NAGAL/α-GAL activities are female-enriched.

(A) Diagrammatic representation of smnagal/smp_089290 gene structure with 50-mer oligonucleotides mapped. Exons are depicted as red boxes, which are linked by lines representing introns. Numbers written inside each exon represent their position in the gene sequence. Numbers written below exons and above introns represent their length in base pairs. The 5′ and 3′ ends are shown above exon 1 and 3, respectively. The positions of oligonucleotide 50-mers corresponding to CONTIG7235 and CONTIG6265 are shown above exon 2 and 3 respectively. (B) DNA microarray analysis of smp_089290 expression across 15 lifecycle stages. DNA microarray gene expression profile consisted of normalised mean fluorescence intensities of smnagal/smp_089290 transcript abundance derived from oligonucleotides CONTIG7235 and CONTIG6265 as described previously [45]. (C) smnagal/smp_089290 transcript levels in untreated adult male and female were quantified relative to smrgbd and smtscd to validate normalised mean fluorescence intensities produced in 7 wk male and 7 wk female schistosomes. Statistical significance is indicated (Student’s t-test, two tailed, unequal variance, ** = p<0.01). (D) Equal quantities of SWAP (5 μg per well) were used for both sexes and measured for α-NAGAL and α-GAL activity on diagnostic α-NAGAL and α-GAL substrates. Statistical significance is indicated (Student’s t-test, two tailed, unequal variance, * = p<0.05 and ** = p<0.01).

https://doi.org/10.1371/journal.ppat.1009828.g004

Expanding these temporal investigations of smnagal abundance and enzymatic activities to spatial localisation, by WISH and scRNA-Seq, in adult worms revealed additional gender-specific traits. An antisense RNA probe spanning the full length coding sequence of smp_089290 was used for each WISH localisation experiment (Fig 5) [68]. A negative control was prepared using a sense smp_089290 probe (S3 Fig); no specific staining was observed. While the ovary (containing mature and immature oocytes) was not a rich source of smnagal expression, the vitellarium (and mature vitellocytes passing through the vitello-oviduct) was highly enriched for this putative α-NAGAL gene product (Fig 5A). These observations were supported by scRNA-Seq approaches, which showed prominent smnagal abundance within the vitellaria and mature vitellocytes and no expression within female gametes (S4 Fig). In addition, low levels of smnagal expression were found within the parenchyma, which was more clearly observed in the female scRNA-Seq plots (S4 Fig). Moderate smnagal expression was also observed in some clusters of neuronal cells, tegument lineage cells and muscle cells when inspecting the female scRNA-Seq plots. No appreciable expression was found in any other female tissue examined. The WISH analysis of males revealed that smnagal expression was predominantly localised to parenchymal cells widely distributed throughout the body (Fig 5B), which was confirmed by the male scRNA-Seq expression profile plots (S5 Fig). A lack of staining was additionally observed throughout the primary reproductive organs (testes), which was supported by scRNA-Seq profiles. WISH analyses also showed that cells lining the tegument and intestine lacked intense smnagal expression in both genders, although this was easier to deduce in males. Similar to females, small yet noticeable levels of smnagal expression were evident in some neuronal cell clusters when inspecting male scRNA-Seq plots (S5 Fig). Appreciable expression was not observed elsewhere in any other male tissue examined.

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Fig 5. smnagal expression is concentrated in the vitellarium, mature vitellocytes and parenchymal cells.

Micrographs of the anterior, mid-section and posterior (10x magnification) of (A) female and (B) male schistosomes as well as anterior images with a higher magnification (40x magnification, area depicted by black dashed box). Structures labelled include egg (E), ovary (O), vitellarium (V), vitello-oviduct (VOD), intestine (I), oral sucker (OS), oesophagus (OES), ventral sucker (VS) and testes (TES). Black scale bars = 200 μm and red scale bars = 50 μm.

https://doi.org/10.1371/journal.ppat.1009828.g005

α-N-acetylgalactosaminidase activity is required for worm motility, egg production and development

The localisation of smnagal to adult parenchymal cells, neuronal clusters and mature vitellocytes as well as the well-documented neuromuscular defects characteristic of Schindler/Kanzaki disease (due to α-nagal deficiencies, [23,24]) implicated key roles for this gene product in schistosome motility, oviposition and development. Therefore, to assess whether these processes were dependent upon α-NAGAL activity, functional genomics investigations of smnagal/smp_089290 were conducted in adult schistosomes (Fig 6). RNA interference (RNAi) of smnagal/smp_089290, using small interfering RNAs (siRNAs), led to a highly significant knockdown (92%) of smnagal in adult male worms when compared to controls (Fig 6A). In parallel, genome editing approaches were implemented with CRISPR/Cas9 plasmid constructs and used to complement RNAi experiments. The CRISPResso2 pipeline was utilised to quantify NHEJ-associated mutations within aligned MiSEQ library sequencing reads from genome-edited worms (S1 Table). Oligonucleotide primers designed for MiSEQ library generation were designed to encompass the programmed DSB sites for each sgRNA used (S6 Fig). All genome edited samples showed detectable levels of genome editing (0.25–0.31%) with substitutions being the most frequently observed mutation (S2 Table). Further analyses of modified sequence reads revealed all CRISPR/Cas9 plasmid treatment samples displayed NHEJ-associated mutations predicted to introduce frameshifts at the smnagal locus (likely resulting in the translation of substantially truncated proteins) or to ablate smnagal transcription (S7 Fig). Similar to RNAi, CRISPR/Cas9 genome editing led to significant knockdowns (46–60%) of smnagal in mixed-sex adult worms when compared to controls (Fig 6B). Reassuringly, smnagal depletion in siRNA-treated worms significantly reduced α-NAGAL activity in SWAP derived from both males and females when compared to siLuc controls (Fig 6C). However, RNAi-mediated smnagal knockdown did not result in a significant reduction in SWAP-derived α-GAL activity when compared to siLuc treated schistosome samples (S8 Fig).

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Fig 6. RNAi and CRISPR/Cas9 approaches lead to smnagal knockdown and siRNA-treated adult worms contain reduced α-NAGAL activity.

(A) 7 wk old adult male schistosomes were electroporated with 5 μg siRNA duplexes targeting luciferase (siLuc) and smp_089290 (siSmp_089290). After 48 hr, total RNA was isolated and used to generate cDNA, which was subjected to qRT-PCR. Percent knockdown (KD) is indicated. Statistical significance is indicated (Student’s t-test, two tailed, unequal variance, ** = p<0.01). (B) 7 wk old adult male and female schistosomes were electroporated with either lentiviral CRISPR/Cas9 plasmid constructs targeting exon 1 (SmNAGALX1) or exon 2 (SmNAGALX2) of smnagal. Additionally, both exons were targeted by electroporating a mixture of both plasmid constructs (SmNAGALX1/X2). Electroporations with CRISPR/Cas9 plasmid DNA containing a Scramble sgRNA were used as a control. After seven days, total RNA was isolated and used to synthesise cDNA for qRT-PCR analysis. Percent knockdown (KD) is indicated. Statistical significance is indicated (Kruskal-Wallis ANOVA with Dunn post-hoc comparisons, * = p<0.05). (C) α-NAGAL activity was measured in SWAP derived from siLuc treated and siSmp_089290 treated adult male and female worms (6.45 μg for males and 2.44 μg for females) as described above. Statistical significance is indicated (Student’s t-test, two tailed, unequal variance, * = p<0.05 and ** = p<0.01).

https://doi.org/10.1371/journal.ppat.1009828.g006

Having established that smnagal encodes a functional α-NAGAL and given that RNAi and CRISPR/Cas9 depleted this transcript from intracellular RNA pools, motility and egg-laying phenotypes of siSmnagal treated schistosome pairs were subsequently examined and quantified. Regardless of the quantification metric used (WormAssayGP2 [73,74] or WHO-TDR standards [75]), a clear motility defect was observed in both male and female schistosomes when smnagal was depleted by RNAi (Figs 7A and S9 and S1 Video). This motility defect was apparent by day two post RNAi and maintained until day 7, when the assay was terminated. Upon completion of the RNAi assays (day seven), eggs were collected from in vitro cultures and quantified for number, volume and retention of mature vitellocytes. Here, a significant reduction in the quantity of deposited eggs was associated with smnagal deficiency (Fig 7B). The CRISPR/Cas9 genome editing approach further supported these observations with all smnagal-edited worms displaying a significantly reduced number of deposited eggs when compared to controls (Fig 7C). Notably, eggs derived from siRNA-treated female worms were also significantly smaller than those collected from wells of siLuc treated worms (Fig 7D) and contained abnormally spaced vitellocytes (Figs 7E and S10 and S2 Video).

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Fig 7. Reductions in smnagal/smp_089290 affect adult worm motility and egg production processes.

(A) Motility of siLuc treated and siSmnagal treated adult male and female worms was analysed daily for up to seven days after electroporation using WormAssayGP2 as described in the Materials and methods. Statistical significance is indicated (General Linear Mixed-Effects Model, NLME and EMMEANS R packages, * = p<0.05 and ** = p<0.01). (B) The total number of eggs produced by siLuc treated and siSmnagal treated adult female worms were collected seven days after electroporation and enumerated. Statistical significance is indicated (Student’s t-test, two tailed, unequal variance, * = p<0.05). (C) The total number of eggs produced by adult worm pairs electroporated with SmNAGALX1 CRISPR/Cas9 plasmid, SmNAGALX2 CRISPR/Cas9 plasmid and SmNAGALX1/X2 CRISPR/Cas9 plasmid were counted seven days after electroporation. Electroporations with lentiviral CRISPR/Cas9 plasmid constructs containing a Scramble sgRNA served as a control. Statistical significance is indicated (Kruskal-Wallis ANOVA with Dunn post-hoc comparisons, * = p<0.05). (D) Volumes of eggs produced by siLuc treated and siSmnagal treated adult female worms were calculated as described in the Materials and methods. Statistical significance is indicated (Student’s t-test, two tailed, unequal variance, ** = p<0.01). (E) Representative images of fluorescence in eggs collected from wells of siLuc and siSmp_089290 treated worm pairs. Blue = DAPI⁺ cells (405 nm blue diode laser) and white scale bars = 20 μm. Average number of DAPI⁺ cells per egg calculated by IMARIS 7.3 software for each siRNA treatment is shown below each image. DAPI⁺ cells in eggs derived from wells of siSmp_089290 treated worm pairs could not be determined and are labelled as ‘nd’.

https://doi.org/10.1371/journal.ppat.1009828.g007

Mature vitellocytes contain two types of large cytoplasmic inclusions known as vitelline droplets and lipid droplets, which both coalesce to assist in eggshell formation [79,80]. Furthermore, eggshell formation also requires phenol oxidase-mediated protein cross-linking (quinone tanning) facilitated by tyrosinase activity originating from mature vitellocytes [77,79,81–83]. To further investigate the potential role of SmNAGAL in vitellaria biology and egg-laying, siLuc treated and siSmnagal treated adult female worms were either stained with Fast Blue BB (to visualise vitelline droplets in mature vitellocytes) or used for diphenol oxidase localisation (to assess tyrosinase activity in mature vitellocytes) assays and imaged by bright-field microscopy. In both assays, no differences were observed between siRNA treatments (S11 Fig).