Novel cholinesterase paralogs of Schistosoma mansoni have perceived roles in cholinergic signaling, glucose scavenging and drug detoxification and are essential for parasite survival

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 – smp_154600 and Smache3 – smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 – smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument and neuromusculature of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. To address the hypothesis that tegumental AChE mediates exogenous glucose scavenging by the parasite, we show that RNAi-mediated knockdown of smache1 and smache3, but not smbche1, significantly reduces glucose uptake by schistosomes. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor DDVP (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP. Author summary Cholinesterases - aceytlcholinesterases (AChE)s and butyrylcholinesterases (BChE)s - are multi-functional enzymes that play a pivotal role in the nervous system of parasites by regulating neurotransmission through acetylcholine hydrolysis. Herein, we provide a detailed characterization of schistosome cholinesterases using molecular, enzymatic and gene-silencing approaches and show evidence for these molecules having roles in glucose scavenging and drug detoxification, in addition to their neuronal function. Further, we demonstrate the importance of these proteins to parasite development and survival through gene knockdown experiments in laboratory animals, providing evidence for the use of these proteins in the development of novel intervention strategies against schistosomiasis.


Abstract 23
Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite 24 neurotransmission but has been poorly defined with respect to the molecules responsible. 25 Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-26 containing genes (Smche)s, which we have shown to encode two functional 27 acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache3 -smp_136690) and a 28 butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of 29 each SmChE localized the proteins to the tegument and neuromusculature of adults and 30 schistosomula and developmental expression profiling differed among the three molecules, 31 suggestive of functions extending beyond traditional cholinergic signaling. For the first time in 32 schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products 33 and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and 34 SmBChE1. To address the hypothesis that tegumental AChE mediates exogenous glucose 35 scavenging by the parasite, we show that RNAi-mediated knockdown of smache1 and 36 smache3, but not smbche1, significantly reduces glucose uptake by schistosomes. Parasite 37 survival in vitro and in vivo was significantly impaired by silencing of each smche, either 38 individually or in combination, attesting to the essential roles of these molecules. Lastly, in the 39 first characterization study of a BChE from helminths, evidence is provided that SmBChE1 40 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to 41 parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor DDVP (DDVP), 42 whereas smbche1-silenced parasites displayed increased sensitivity to DDVP. modeling with AChE from model organisms (H. sapiens and T. californica (Figure S2). All 136 three SmChEs exhibited predicted folding characteristics of the functional globular enzymes 137 as most of the α-helical and β-stranded sheets were tightly aligned. Each predicted SmChE 138 structure consisted of a ChE catalytic domain but, although the core architecture 139 of the catalytic gorges was well aligned, regions that are associated with substrate specificity 140 and catalytic efficiency were disparate. In particular, and in agreement with the sequence 141 alignment, the catalytic triad of SmBChE1 was predicted to be Ser-Gln-Glu instead of the 142 canonical Ser-His-Glu present in the other two paralogs. A phylogenetic tree of the alignment 143 (Figure S3) shows that SmChEs were clustered into three distinct branches, with SmChE1 being 144 phylogenetically distinct from SmBChE1 and SmAChE3. In addition, each SmChE was 145 grouped together with closely related flatworms, including other Schistosoma species. 146 Importantly, as shown in the sequence alignment, SmChEs are divergent from the human 147 homolog. Reflective of the catalytic triad residue difference (Figure S1), trematode BChEs are 148 phylogenetically divergent from nematode and human BChEs (Figure S4). 149 150

Developmental expression analysis of SmChE genes 151
Gene expression patterns of the three SmChE paralogs across different developmental stages 152 were measured using semi-quantitative qPCR  and this data was used to 153 generate a comparative expression heat map of all three genes (Figure 2D). While all smche 154 developmental expression patterns were variable, the transcript levels of all three genes were 155 relatively lower in cercariae compared to the other developmental stages. Overall, the transcript 156 levels of smache1 and smache3 genes in most life stages were higher than that of smbche1. In 157 adult worms, smache1 was expressed at higher levels, specifically in male parasites, followed 158 by sporocysts. 159 160

Immunolocalization of SmChEs 161
To gain insight into the anatomical sites of expression of ChE proteins in S. mansoni, SmChEs 162 were immunolocalized in whole juvenile and sectioned adult parasites. In adults, and consistent 163 with their predicted cholinergic function, all SmChEs were expressed throughout the worms' 164 internal structures (presumably localizing to the neuromusculature) and on the tegument 165 surface. SmAChE1 was the least uniformly distributed of all SmChEs, localizing mostly to 166 the tegument ( Figure 3A). Additionally, anti-SmChE antibodies were able to detect 167 homologous ChEs in adult S. haematobium sections. SmChE proteins were detected in all 168 stages of larval development tested and, as was the case with adult worms, localized to the 169 tegument ( Figure 3B). 170 171

Expression and ChE activity of fSmChEs 172
Soluble, functionally active proteins were expressed in P. pastoris, purified via IMAC and 173 tested for ChE activity. Both fSmAChE1 and fSmAChE3 demonstrated significantly stronger 174 hydrolase activity when AcSCh was used as a substrate, compared to fSmBChE1 and, 175 conversely, fSmBChE1 hydrolyzed BcSCh to significantly higher levels compared to 176 fSmAChE1 and fSmAChE3 (Figure 4A). All paralogs exhibited Michaelis-Menten kinetics 177 (Table 1) when hydrolyzing their designated substrate, with fSmAChE1 having a substrate 178 affinity approximately twice that of fSmAChE3. In addition, preferred substrate activity of both 179 fSmAChE1 and fSmAChE3 was inhibited by DDVP, an AChE inhibitor, whereas iso-OMPA, 180 a specific inhibitor of BChE, only inhibited SmBChE1 activity (Figure 4B

BChE and secreted ChE activity in schistosomes 187
Although the presence of nonspecific ChE activity has long been known in schistosomes [33], 188 the identity of the gene product and its function remain unknown. Prompted by the 189 identification of SmBChE1 as a BChE, based on its substrate preference and enzymatic 190 inhibition by iso-OMPA, we sought to investigate the distribution of BChE activity in juvenile 191 and adult schistosomes. Extracts from S. mansoni schistosomula had higher BChE activity 192 compared to S. mansoni adult worms (Figure 5A), and that activity was significantly greater in 193 S. mansoni compared with S. haematobium adults ( Figure 5B). Varied amounts of AChE 194 activity were detected in ES from all developmental stages tested. ES products from adult males 195 had double the AChE activity of adult female ES products (P < 0.001), while cercariae ES 196 exhibited the highest activity (at least ten-fold more than male ES products (P < 0.0001)) and 197 egg ES had the lowest ( Figure 5C). Availability of ES precluded the measurement of secreted 198 BChE activity from all developmental stages but, of those tested, activity in schistosomula ES 199 products was the highest -twice as high as that of adult (P < 0.01) and cercariae (P < 0.01) ES 200 ( Figure 5D). SmChEs were purified from ES products of S. mansoni adult worms using 201 edrophonium-sepharose affinity chromatography. Purification resulted in an activity increase 202 of more than 200-fold relative to crude ES (Figure 5E). Resolution of the purified proteins by 203 SDS-PAGE resulted in a doublet with a major band migrating at 70 kDa under denaturing and 204 reducing conditions ( Figure 5F). The identity of purified, secreted SmChEs was substantiated 205 by in-gel LC-MS/MS analysis with the peptide data generated used to interrogate the S. 206 mansoni proteome (predicted from the S. mansoni genome -207 http://www.genedb.org/Homepage/Smansoni). The false discovery rate was set at <1% and 208 only proteins with at least two unique peptides having significant Mascot identification scores 209 (P < 0.05) were considered. The top protein hits were identified as SmAChE1 (Smp_154600) 210 and SmBChE1 (Smp_125350); SmAChE1 had a relative abundance of more than 40-fold that 211 of SmBChE1 (supplementary table 3). 212 213

Suppression of SmChE activity 230
Suppression of AChE activity was seen in smache1 and smache3 siRNA-treated parasites from 231 5 and 3 days after electroporation, respectively (Figure 7A), compared to the luc control, while 232 schistosomula treated with smbche1 siRNA did not show any significant reduction in BChE 233 activity, even 7 days after electroporation (Figure 7B). Parasites electroporated with a cocktail 234 of all three smche siRNAs showed significant decreases in AChE activity at 3 days (62% 235 reduction, P ≤ 0.001), 5 days (67% reduction, P ≤ 0.001) and 7 days (71% reduction, P ≤ 0.001) 236 after treatment ( Figure 7C). BChE activity was not measured in the cocktail siRNA treatment 237

group. 238
Individual silencing of smache1 or smache3 genes and combined silencing of all three smche 239 genes reduced glucose uptake in schistosomula by 24.9% (P ≤ 0.001), 32.34% (P ≤ 0.001) and 240 38.61% (P ≤ 0.001) at 48 h post-treatment, respectively, relative to the luc control. However, 241 smbche1-silenced parasites showed no significant changes in glucose uptake at the same 242 timepoint and there was no difference in the glucose consumed by the smache1 or smache3 243 siRNA-treated groups compared with the cocktail siRNA-treated group (Figure 7D). 244 Transcript levels of the glucose transporters, sgtp1 and sgtp4, were neither decreased nor 245 significantly increased in individual or cocktail smche-silenced parasites ( Figure S5). 246 247

Effects of smche silencing on schistosomula viability in vitro and development in vivo. 248
Parasites treated with smache1, smbche1 or smache3 siRNAs showed significant decreases in 249 viability at days 3, 5 and 1 after treatment, respectively, compared to luc controls. At days 5 250 and 7 post-treatment, the most significant decrease in parasite viability was seen in the group 251 which received the cocktail siRNA treatment, compared to luc controls. Furthermore, viability 252 in this group was also significantly lower than it was for any individual treatment at these two 253 time points ( Figure 8A). 254 To examine whether RNAi-mediated smche suppression reduced parasite viability in vivo, 255 mice were infected with smche-silenced parasites and worm burdens were measured after three 256 weeks. From two independent experiments, there was an average 88.15%, 55.15%, 75.95% 257 and 88.35% decrease in adult fluke burdens from mice injected with smache1-, smbche1-, 258 smache3-and smche cocktail-silenced schistosomula, respectively, compared to mice infected 259 with luc-treated parasites (Figures 8B and C). All worm burden decreases were significant 260 and there was no significant difference in fluke burdens between mice injected with luc-261 treated parasites and non-electroporated control parasites. All mice had been successfully 262 infected with parasites, as serum from necropsied mice contained parasite-specific antibodies 263 (data not shown). Compared to luc-treated parasites, worms recovered from smche-treated 264 parasites showed no difference in smche transcript levels (data not shown). 265 266

Bio-scavenging of carboxylic esters by SmBChE1 267
The hypothesis that SmBChE may act as a molecular decoy in schistosomes and detoxify the 268 effects of organophosphorus AChE inhibitors was examined by testing whether ( Cholinesterase (ChE) activity in S. mansoni was first described by Bueding in 1952 [33] and 283 was well characterized biochemically in the four decades succeeding this discovery. The assembly has identified two additional ChE-encoding genes that are paralogs to Smp_154600 292 (which we have termed SmAChE1); Smp_125350 (SmBChE1) and Smp_136690 (SmAChE3). 293 In this current study, we have provided a more in-depth characterization of the previously 294 documented SmAChE1 and described two novel ChEs from S. mansoni: SmAChE3 -an AChE 295 not previously reported; and SmBChE1 -a BChE which, to the best of our knowledge, has 296 never been documented in the helminth literature. 297 298 All SmChEs share a modest level of identity which is consistent with their divergence over 299 evolutionary time, an occurrence that is possibly due to a series of gene duplications due to the 300 phylogenetic distance between the relative clades. This divergence between SmChEs and, also, 301 ChEs of other organisms, provides evidence for the increasing reports of non-cholinergic 302 functions of ChEs in the literature. Additionally, the relative lack of sequence identity between 303 SmChEs and human ChEs suggests potential scope for the development of intervention 304 strategies targeting schistosome ChEs that will not affect the host. Despite the diversity 305 between ChEs, all enzymes analyzed herein would appear to be enzymatically active as they 306 possessed a catalytic triad with an active site serine, the amino acid responsible for ester 307 hydrolysis [32]. It is interesting to note, however, the catalytic triad His -Gln substitution in 308 SmBChE1 (and the other platyhelminth BChE1 homologs); while this change is not a hallmark 309 of model BChEs, that it occurs within an entire parasite lineage is noteworthy and will be 310 investigated further. 311

312
The transcript levels of each smche varied among parasite developmental stages and this is 313 likely a response to the differing cholinergic and cholinesterase-independent requirements of 314 the parasite throughout its lifecycle. For example smache1 is expressed at a higher level in 315 adult males than females, probably due to the more "muscular" roles of attachment and 316 movement orchestrated by the male compared to the female, which remains sedentary once 317 inside the gynecophoric canal of the male [34]. Expression of smbche1 was highest in the egg 318 stage; there is evidence for BChE involvement in chicken embryo neurogenesis and 319 development, independent of its enzymatic function [35], which suggests that SmBChE1 could 320 play a role in parasite embryogenesis. The miracidium and sporocyst stages had the highest 321 levels of smache3 expression, in agreement with Parker-Manuel et al [36]. Full-length and functional SmChEs were expressed in P. pastoris. SmAChE1 had preferred 341 substrate specificity for AcSCh over BcSCh, albeit at a three-fold lower affinity than previously 342 reported for SmAChE1 expressed in Xenopus laevis oocytes [15]. SmAChE3 also had a 343 substrate preference for AcSCh and an affinity twice that of SmAChE1. Extremely low enzyme 344 activity was observed with SmBChE1 when AcSCh was used as a substrate, but enzymatic 345 activity significantly increased with the use of BcSCh as the substrate. Although sequence 346 alignment of SmBChE1 with the other two SmChEs revealed a single amino acid substitution 347 in the peripheral anionic site (Glu -Trp), acyl binding pocket (Val -Leu) and catalytic triad 348 (His -Gln), it was unclear whether these changes alone were enough to classify SmBChE1 as 349 a BChE; based on the significant difference in substrate preference, however, this classification 350 would appear valid. Cloning of a recombinant BChE from S. mansoni is consistent with our 351 observations of BChE activity in parasite extracts, and S. mansoni schistosomula exhibited 352 significantly more activity than adults, as did S. mansoni compared to S. haematobium adults. 353 It has been reported that S. mansoni is more sensitive to the BChE inhibitor, iso-OMPA, than 354 S. haematobium [39] and it may be due to the increased BChE activity in S. mansoni. Indeed, 355 this relationship has been documented between AChE and metrifonate (precursor of DDVP 356 used in this study); S. haematobium is more sensitive to the inhibitor than S. mansoni because 357 of the greater amount of AChE on the worm's surface [39]. 358 359 For the first time, we document the presence of secreted SmChE activity in schistosomes and 360 AChE activity was highest in cercarial ES products. Of the intra-mammalian stages tested, 361 AChE activity was highest in schistosomula and adults and may be acting to bind and neutralize 362 exogenous AChE inhibitors [40] (thus protecting tegumental and somatic AChE) or host-363 derived ACh to mitigate the immunomodulatory effects of this molecule. Extending this 364 hypothesis, ES products from cultured female worms had lower AChE activity than males and 365 could be due to females worms having less of a requirement for this defensive mechanism as 366 they reside in the relative shelter of the gynecophoric canal. BChE activity was present in the 367 ES products of adults, schistosomula and cercariae and was significantly higher in the intra-368 mammalian larval stage than the other two stages. The SmChE molecules present in ES were 369 isolated by purification on edrophonium (a reversible ChE inhibitor) sepharose and, consistent 370 with the class of activity observed in ES, identified by mass spectrometry as SmAChE1 and 371 SmBChE1; the former being forty-fold more abundant than the latter. 372 373 RNAi-mediated silencing of smache1 and smache3 in schistosomula showed decreases in 374 AChE activity, consistent with reductions in transcript and protein expression levels. 375 Moreover, inhibition of this biochemical activity was greater in schistosomula treated with the 376 smche siRNA cocktail than parasites receiving any of the individual treatments, further 377 evidence suggestive of simultaneous silencing of all smache paralogs. AChE activity inhibition 378 in smache3-silenced parasites was more pronounced than in smache1-silenced parasites, which 379 was inconsistent with protein level reductions and this may be due to the increased AChE 380 activity reported for SmAChE1 (Vmax = 5.57 nmol/min/mg, Km = 5.83 mM) compared to 381 SmAChE3 (Vmax = 5.59 nmol/min/mg, Km = 10.87 mM). It is also possible that there may 382 not be a direct correlation between AChE activity and protein expression, given that additional, 383 non-cholinergic functions have been ascribed to ChEs [5,22,41]. This may also be the reason 384 why no significant decrease in BChE activity was observed in smbche1-silenced parasites, 385 despite significant reductions in transcript and protein expression levels. genes. Individual gene knockdown of smache1 and smache3 suppressed glucose uptake in 392 schistosomula, implying that both genes were involved in regulation of this mechanism. 393 Tegumental AChE is speculated to mediate glucose uptake by limiting the interaction of ACh 394 with tegumental nicotinic ACh receptors which is thought to decrease the amount of glucose 395 uptake through surface glucose transporters. The fact that both molecules are localized to the 396 tegument and can hydrolyze ACh therefore provides evidence for their role in this pathway. 397 Silencing of smbche1 in schistosomula did not show any difference in glucose uptake and is 398 probably reflective of the molecule's limited role in ACh hydrolysis. Transcript levels of sgtp1 399 and sgtp4 were not significantly changed in smache1-and smache3-silenced parasites, 400 suggesting that SmAChEs may facilitate glucose uptake in a manner which does not directly 401 involve glucose transporters. Indeed, at least in nematodes, AChEs have been proposed to be 402 involved in altering the permeability of surrounding host cells, allowing nutrients (such as 403 glucose) to leak into the parasite niche and be uptaken [42].   6 well plates. Media containing ES products was initially collected after 3 h for schistosomula 516 or 24 h for adults, and replenished daily thereafter. ES products were stored at -80°C. Media 517 was thawed when needed, concentrated through Amicon centrifugation filters (Sigma) with a 3 518 kDa molecular weight cutoff (MWCO), buffer exchanged into phosphate buffered saline, pH 519 7.4 (PBS) and aliquoted. Protein concentration of ES products was determined using the Pierce 520 BCA™ Protein Assay kit (Thermofisher). To collect cercarial ES products, freshly-shed 521 cercariae were incubated in H2O (4000/ml) at 25°C for 3 h. H2O was filtered through Whatman 522 filter paper (11 µm) to remove cercariae and associated debris, and ES products were 523 concentrated, quantified and stored as described for adult and schistosomula ES products. 524 525

Parasite extract preparation 526
To make PBS-soluble extracts, worms were homogenized in PBS (50 µl/adult worm pair or 50 527 µl/1000 schistosomula) at 4°C using a TissueLyser II (Qiagen), homogenates were incubated 528 overnight with mixing at 4°C and the supernatants collected by centrifugation at 15,000 g for 529 1 h at 4°C. Triton X-100-soluble extracts were made from the PBS-insoluble pellets by 530 resuspension in 1% Triton X-100, 40 mM Tris-HCl, pH 7.4, mixing overnight at 4°C and the 531 supernatant collected by centrifugation at 15,000 g for 1 h at 4°C. Tegument extraction was 532 achieved using a combination of freeze/thaw/vortex [71]. In brief, parasites were slowly 533 thawed on ice, washed in TBS (10 mM Tris/HCl, 0.84% NaCl, pH 7.4) and incubated for 5 min 534 on ice in 10 mM Tris/HCl, pH 7.4 followed by vortexing (5 ´ 1 s bursts). Subsequently, the 535 tegumental extract was pelleted at 1000 g for 30 min and solubilized (3´) in 200 µl of 0.1% 536 (w/v) SDS, 1% (v/v) Triton X-100 in 40 mM Tris, pH 7.4 with pelleting at 15,000 g between 537 each wash. Protein concentration was determined using the Pierce BCA Protein Assay kit, 538 aliquoted and stored at -80°C until use. 539

Real-time qPCR 556
Real-time qPCR was used to assess developmental expression of smche genes and to determine 557 smche transcript suppression resulting from RNAi experiments. RNA from miracidia, 558 sporocysts, cercariae, adult male worms, adult female worms, and eggs were obtained from 559 BRI. Schistosomula were cultured as described above, harvested (1,000 parasites) after either 3 560 h, 24 h, 3 or 5 days, washed three times in PBS and stored at -80°C until use. Schistosomula 561 from RNAi experiments were similarly processed. Total RNA extraction was performed using 562 the Trizol (Thermofisher) reagent according to manufacturer's instructions. After air-drying, 563 RNA pellets were re-suspended in 12 µl diethylpyrocarbonate (DEPC)-treated 564 water. Concentration and purity of RNA was determined using an ND2000 Nanodrop 565 spectrophotometer (Thermofisher). Synthesis of cDNA was carried out with 1 µg of total RNA 566 using Superscript-III-Reverse Transcriptase (Invitrogen) according to the manufacturer's 567 instructions. Finally, cDNA was quantified, diluted to 50 ng/µl, aliquoted and stored at -20°C. 568 Real-time qPCR primers for each smche (supplementary Table 1) were designed using 569 Primer3 (http://frodo.wi.mit.edu/). The housekeeping gene smcox1 was selected as an internal 570 control to normalize relative smche gene expression [73]. Each qPCR (1 µl (50 ng) of cDNA, 571 5 µl of 2x SYBR green master mix (Bioline), 1µl (5 pmol/µl) each of forward and reverse 572 primers and 2 µl of nuclease-free water) was run in a Rotor-Gene Q thermal cycler (Qiagen) 573 using 40 cycles of 95°C for 10 seconds, 50-55°C for 15 seconds and 72°C for 20 seconds. 574 Stage-specific smche gene expression levels were normalized against smcox1gene expression 575 using the comparative 2 -ΔΔCT method [74]. All results represent the average of 5 independent 576 experiments with data presented as mean ± SEM. 577 578

Cloning, expression and purification of smche gene fragments in E. coli 579
Complete ORFs for smache1, smbche1 and smache3 were synthesized by Genewiz. Attempts 580 to express full-length sequences in E. coli were unsuccessful, so primer sets incorporating NdeI 581 (forward primer) and XhoI restriction enzyme sites (reverse primer) were designed 582 (supplementary Table 1) to amplify partial, non-conserved regions of each smche, which might 583 prove more amenable to expression. Sequences (containing NdeI/ XhoI sites) for each pSmChE 584 were amplified from each full-length template by PCR and cloned into the pET41a expression 585 vector (Novagen) such that the N-terminal GST tag was removed. Protein expression was 586 induced for 24 h in E. coli BL21(DE3) by addition of 1 mM Isopropyl beta-D-1-587 thiogalactopyranoside (IPTG) using standard methods. Cultures were harvested by 588 centrifugation (8,000 g for 20 min at 4°C), re-suspended in 50 ml lysis buffer (50 mM sodium 589 phosphate, pH 8.0, 300 mM NaCl, 40 mM imidazole) and stored at -80°C. Cell pellets were 590 lysed by three freeze-thaw cycles at -80°C and 42°C followed by sonication on ice (10 ´ 5 s 591

pulses [70% amplitude] with 30 s rest periods between each pulse) with a Qsonica Sonicator. 592
Triton X-100 was added to each lysate at a final concentration of 3% and incubated for 1 h at 593 4°C with end-over-end mixing. Insoluble material (containing rSmChEs) was pelleted by 594 centrifugation at 20,000 g for 20 min at 4°C. The supernatant was discarded, and inclusion

Generation of anti-rSmChE antisera and purification of IgG 611
Three groups of five male BALB/c mice (6-week-old) were intraperitoneally immunized with 612 either rSmAChE1, rSmBChE1 or rSmAChE3 subunits (50 µg/mouse). Antigens were mixed 613 with an equal volume of Imject alum adjuvant (Thermofisher) and administered three times, 614 two weeks apart. Two weeks after the final immunization, mice were sacrificed and blood was 615 collected via cardiac puncture. Blood from all mice in each group was pooled and serum was 616 separated by centrifugation after clotting and stored at −20°C. Polyclonal antibodies were 617 purified from mouse sera using Protein A Sepharose-4B (Thermofisher) according to the 618 manufacturer's instructions. Serum from naïve mice was similarly processed. 619 620

Immunolocalization using anti-rSmChE antisera 621
Adult worm sections: Freshly perfused adult S. mansoni and S. haematobium worms were fixed 622 in 4% paraformaldehyde, embedded in paraffin and sections (7 µm thick) were cut in a cryostat. 623 Following deparaffinization in xylene and rehydration in an ethanol series, antigen retrieval 624 was performed by boiling the slides in 10 mM sodium citrate, pH 6.0, for 40 min followed by 625 a solution of 10 mM Tris, 1 mM EDTA, 0.05% Tween, pH 9.0, for 20 min. All sections were 626 then blocked with 10% heat-inactivated goat serum for 1 h RT. After washing 3 times with 627 PBST, sections were incubated with anti-SmAChE1, anti-SmBChE1, anti-SmAChE3, naïve 628 sera (negative control), S. mansoni or S. haematobium infected mouse sera (positive controls) 629 (1:50 in PBST) overnight at 4°C and then washed again (3 ´ 5 min each). were re-dissolved in 10 µl 5% formic acid and 6 µl was injected onto a 50 mm 300 µm C18 729 trap column (Agilent Technologies) followed by an initial wash step with Buffer A (5% (v/v) 730 ACN, 0.1% (v/v) formic acid) for 5 min at 30 µl/min. Peptides were eluted at a flow rate of 0.3 731 µl/min onto an analytical nano HPLC column (15 cm ´ 75 µm 300SBC18, 3.5 µm, Agilent 732 Technologies). The eluted peptides were then separated by a 55-min gradient of buffer B (90/10 733 acetonitrile/ 0.1% formic acid) 1-40% followed by a 5 min steeper gradient from 40-80%. The 734 mass spectrometer (ABSciex 5600 Triple Tof) was operated in data-dependent acquisition 735 mode, in which full scan TOF-MS data was acquired over the range of 350-1400 m/z, and over 736 the range of 80-1400 m/z for product-ion observed in the TOF-MS scan exceeding a threshold 737 of 100 counts and a charge state of +2 to +5. Analyst 1.6.1 (ABSCIEX) software was used for 738 data acquisition and analysis. 739 Three short interfering RNA duplexes (siRNAs) targeting each of the three identified smche 750 paralogs were designed (supplementary Table 2) and checked to avoid off-target silencing 751 by BLAST search using the S. mansoni genome. An irrelevant siRNA from firefly luciferase 752 (luc) was selected as a negative control [77]. All siRNAs were commercially synthesized 753 (Integrated DNA Technologies) and oligonucleoitdes were suspended to a concentration of 1 754 µg/µl in DEPC-treated water. 755 756

Infection of mice with SmChE siRNA-treated schistosomula 799
One-day-old schistosomula (10,000) were electroporated as above in 500 µl of SFB with 50 800 µg of either luc, smache1, smbche1 or smache3 siRNA or a combination of all three smche 801 siRNAs (150 µg total). Parasites were injected intramuscularly into both thighs (1,000 per 802 thigh) of male 6-8 week BALB/c mouse (5 mice per treatment group) using a 23-gauge needle. 803 A control group of mice were similarly injected with non-electroporated schistosomula. Adult 804 worms were perfused 20 days later to assess the number of worms that had matured and reached 805 the mesenteries. Experiments were performed independently in duplicate. After each 806 experiment, transcript levels of each smche from surviving worms were assessed using real-807 time qPCR. 808 809 810

Bio-scavenging of carboxylic esters by SmBChE1 811
To test the hypothesis that SmBChE1 may play a role in the bio-scavenging of AChE-inhibitory 812 molecules, we first sought to determine whether inhibition of BChE activity would potentiate 813 the AChE-inhibitory and anti-schistosome effects of organophosphates (OP)s. Schistosomula 814 extracts (20 µg) were diluted in assay buffer, then iso-OMPA was added to a final concentration 815 of either 1 or 2 mM and incubated for 20 min at RT. DDVP was then added to a final 816 concentration of 1 µM and the samples were further incubated for 20 min at RT; the final 817 reaction volume was 180 µl. ACh (final concentration 2 mM) and DTNB (final concentration 818 0.5 mM) were then added and the absorbance was monitored every 5 min at 405 nm in a 819 Polarstar Omega microplate reader (BMG Labtech). Extracts that were not treated with iso-820 OMPA with or without DDVP treatment were used as controls. Experiments were performed 821 in triplicate with data presented as the mean ± SEM. 822 The same experiments were performed on live schistosomula using either an inhibitor-or 823 RNAi-based approach. For the inhibitor-based experiment, 24 h schistosomula 824 (1,000/treatment in 1 ml SFB) were pretreated with iso-OMPA at the non-lethal concentration 825 of 100 µM and, 1 h after iso-OMPA treatment, schistosomula were treated with 1 µM DDVP 826 and cultured for 5 h at 37°C in 5% CO2. Parasites that were not treated with iso-OMPA but 827 treated with DDVP were used as controls. For the RNAi-based experiment, 24 h schistosomula 828 (1,500/100 µl SFB) were electroporated with 10 µg of either smbche1 or luc siRNA as 829 described above, added to 24 well plates containing 1 ml pre-warmed SFB and incubated 830 (37°C, 5% CO2) for 3 days before being treated with 1 µM DDVP and cultured for a further 5 831 h. For both inhibitor-and RNAi-based experiments, schistosomula viability was determined 832 using Trypan Blue staining and data is presented as the mean ± SEM of 2 biological and 3 833 technical replicates. 834 In a reverse testing of the bio-scavenging hypothesis, we sought to determine whether addition 835 of SmBChE could mitigate the effects of DDVP. Ten micrograms of fSmBChE1 was pre-836 incubated with 1 µM final concetration DDVP in AChE assay buffer (170 µl final volume) for 837 20 min at RT. Schistosomula extracts (20 µg), ACh (final concentration 2 mM) and DTNB 838 (final concentration 0.5 mM) were then added and the absorbance was monitored every 5 min 839 at 405 nm iichlorvosn a Polarstar Omega microplate reader. Reactions without fSmBChE or 840 without DDVP were used as controls. Experiments were performed in triplicate with data 841 presented as the mean ± SEM. Again, the same experiments were performed on live 842 schistosomula. After the pre-treatment of different amounts of fSmBChE (10, 5, and 2.5 µg) 843 with 1 µM DDVP in 500 µl SFB, 24 h schistosomula (1,000/treatment in 500 µl SFB) were 844 added, incubated at 37°C and 5% CO2 for 24 h and then parasite viability was measured by 845 Trypan Blue staining. Experiments where a similarly expressed and purified, but irrelevant, 846 protein (SmTSP2) was used instead of fSmBChE, and schistosomula cultured in media alone, 847 were used as controls. Data is presented as the mean ± SEM of 2 biological and 3 technical 848 replicates. 849 850

Statistical analyses 851
Data were reported as the means ± SEM. Statistical differences were assessed using 852 the student's t test. P values less than 0.05 were considered statistically significant. 853 used as a substrate) and inhibition of fSmBChE1 with iso-OMPA (BcSCh used as a substrate). 1137 Data are presented as mean ± SEM of triplicate experiments and differences between groups 1138 were measured by the student's t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.