Large-scale RNAi screening uncovers therapeutic targets in the parasite Schistosoma mansoni

Abstract

Schistosome parasites kill 250,000 people every year. Treatment of schistosomiasis relies on the drug praziquantel. Unfortunately, a scarcity of molecular tools has hindered the discovery of new drug targets. Here, we describe a large-scale RNA interference screen in adult Schistosoma mansoni examining the function of 2,216 genes. We discovered 250 genes with phenotypes affecting neuromuscular function, tissue integrity, stem cell maintenance, and parasite survival. Leveraging these data, we prioritize compounds with activity against the parasites and uncover a pair of protein kinases (TAO and STK25) that cooperate to maintain muscle-specific mRNA transcription. Loss of either of these kinases results in paralysis and worm death in a mammalian host. These studies may help expedite therapeutic development and invigorate studies of these neglected parasites.

One Sentence Summary:

An RNAi screen examines the function of 20% of S. mansoni genes uncovering new phenotypes and suggesting new therapeutic targets.

Studies of gene function in intra-mammalian schistosome parasites have been limited to relatively small numbers of genes(1). Therefore, we developed a large-scale RNAi screening platform on adult schistosomes that prioritized a list of 2,320 of the worm’s ~10,000 protein coding genes (Fig. S1A, Table S1). We generated dsRNAs, and treated adult male and female pairs with dsRNA over the course of a 30-day experiment (Fig. 1A). After filtering genes that either did not amplify by PCR, or failed to generate sufficient concentrations of dsRNA, a total of 2,216 genes were screened (Table S1).

Fig. 1. Summary of RNAi phenotypes.

(A) RNAi treatment regime. Parasites were monitored for visible abnormalities and at D29 EdU was added to media to label proliferative cells. (B) Categories of RNAi phenotypes observed. kin-17 (Smp_023250), cog1 (Smp_132980), p97 (Smp_018240), c44 (Smp_136260), prpf4b (Smp_068960), gtf2f1 (Smp_088460), stk25 (Smp_096640). Scale bar: 100 μm

Schistosomes live in the veins surrounding the host intestines, and attach to the vascular endothelium to avoid being swept away in the blood and trapped in host organs. Under in vitro culture conditions healthy parasites attach to the substrate with a combination of their oral and ventral suckers (Movie S1). We thus reasoned that substrate attachment would be a useful quantitative metric to define RNAi treatments that affect parasite vitality and predict in vivo survival. Therefore, we monitored parasites for 30 days to identify substrate attachment and other visible defects.

Schistosomes possess adult somatic stem cells (neoblasts) that rejuvenate parasite tissues, including the intestine and tegument (skin)(2, 3). The parasites also contain large numbers of proliferative germline stem cells (GSCs) in their reproductive organs(2) which are essential for producing eggs; the central driver of parasite-induced pathology in vivo(4). Therefore, we monitored the maintenance of neoblasts and GSCs by labeling parasites with the thymidine analog EdU prior to the conclusion of the experiment (Fig. 1A). Due to the variable rate at which the reproductive organs of female worms degenerate during in vitro culture(5), stem cell proliferation was only monitored in male worms. For genes with RNAi phenotypes uncovered during our screen, we confirmed gene identity by sequencing, and mitigated potential off-target effects by designing an additional dsRNA targeting a non-overlapping gene region or by examining sequence identity of hits with other S. mansoni genes (Fig. S1). These studies identified 195 genes with fully-penetrant attachment phenotypes of which 121 possessed phenotypes in addition to attachment, including: tissue and intestinal edema (36), head (26) and/or tegument (78) degeneration, muscular hypercontraction (6), and death (36) (Fig. 1B, Table S2). RNAi of an additional 66 genes resulted in stem cell maintenance defects but caused no other visible phenotypes (e.g., attachment) suggesting an essential role for stem cell maintenance (Fig. S2, Table S3). We cannot rule out the possibility of false negatives among the genes with no phenotype and encourage greater scrutiny of such genes by alternate knockdown approaches or analysis of different phenotypic readouts.

Of the 66 genes required for stem cell survival, RNAi of over 90% (60/66) led to defects in the maintenance of both proliferative cells in the male testes and neoblasts (Fig. S2). However, for a minority of genes this maintenance defect appeared to be specific to either proliferative cells in the testes or the neoblasts (Fig. S2). Gene Ontology enrichment analysis identified genes important for protein translation, including gene products involved in ribosomal structure, tRNA aminoacylation, and rRNA processing as putative regulators of proliferative cell maintenance (Fig. S3A). Although this could reflect an enhanced sensitivity of actively proliferating cells to alterations in protein translation, studies have highlighted “non-housekeeping” roles for translational regulators in controlling stem cell function(6).

Of the 195 genes essential for parasite attachment, a large fraction share sequence similarity with other organisms, including other medically-relevant schistosome species (Table S4). Additionally, most genes with an attachment phenotype (172/195 genes; 88%) possess a close homolog from C. elegans or D. melanogaster that likewise has a loss-of-function phenotype (Table S5). Gene Ontology analyses of genes with attachment phenotypes further revealed that the dominant group of enriched genes were those encoding components necessary for protein turnover via the ubiquitin-proteasome system (UPS) (Fig. S3B). Proteolysis is important for larval and adult viability in vitro(7, 8) and our data identified that key components from virtually every arm of the UPS were required for adult parasite vitality during in vitro culture (Fig. S4A). Indeed, RNAi of nearly all of UPS components resulted in extensive tissue degeneration and in some cases adult parasite death (Fig. S4B–C).

To determine if any genes associated with attachment phenotypes encoded proteins targeted by existing pharmacological agents, we searched the literature and the ChEMBL database(9) (Table S6). This analysis uncovered 205 compounds potentially targeting 49 S. mansoni proteins. We selected 14 of these compounds (Table S7) and examined the activities of these compounds on worms cultured in vitro either by automated worm movement tracking or parasite attachment. More than half of the compounds tested (8/14) on worms at 10 μM reduced parasite movement >75% and half of the compounds (7/14) caused fully-penetrant substrate attachment defects by D7 post-treatment (Fig. 2A–B, Movie S2). Similarly, 7 of these compounds affected the movement of post-infective larvae (schistosomula), suggesting activity against multiple life-cycle stages (Table S8).

Fig. 2. Compounds prioritized from RNAi studies have effects on schistosomes in vitro.

(A) Compounds (red text) predicted to target schistosome proteins (blue text) essential for parasite attachment at 10 μM for their effects on worm motility. n = 12 worm pairs, n = 3 biological replicates; data are mean +/− SD. Dashed line, threshold for 75% reduction in motility (B) Heatmap showing time course measuring the fraction of male worms attached to the substrate following treatment with compounds for 72 hours as in A. n = 3 biological replicates (C) Effects of CB-5083 or NMS-873 (10 μM, 72 hrs) treatment on male parasites. (D) Percent recovery p97(RNAi) or control(RNAi) male parasites surgically transplanted into mice. n = 8 transplants for each group. ****, p < 0.0001, t-test; data are mean +/− 95% CI. (E) Hematoxylin and Eosin staining of livers from mice receiving control or p97(RNAi) parasites. Egg-induced granulomas (yellow arrows). (top right) counts of eggs/cm liver section are shown, counts from sections from n = 3 livers. Scale bars: C, E, 100 μm.

Among the compounds that emerged from these studies was simvastatin, an HMG-CoA reductase inhibitor, with known effects on parasites both in vitro and in vivo(10). The proteasome inhibitor ixazomib affected both schistosome movement (Fig. 2A) and attachment (Fig. 2B), mirroring a recent report using the proteasome inhibitor bortezomib (7). However, the most potent effects on adult parasites were observed with CB-5083 and NMS-873 that inhibit the UPS component p97 by either competing with ATP (11) or binding to an allosteric site(12), respectively (Fig. S5A). Similar to the death observed following long-term p97 RNAi treatment (Fig. 1B), both p97 inhibitors led to death in vitro (Movie S3). Despite their distinct biochemical mechanisms of action, we noted similar deformations in the structure of the parasite tegument following treatment with either CB-5083 or NMS-873, suggesting that these compounds have similar pharmacological effects on the parasite (Fig. 2C).

We then assessed if NMS-873 and CB-5083 affected UPS function by measuring the accumulation of ubiquitinated proteins using an antibody that recognizes K48 polyubiquitinated proteins marked for proteasome-mediated destruction(13). We observed the accumulation of polyubiquitinated proteins following RNAi of p97 and after treatment of schistosomes with either CB-5083 or NMS-873 (Fig. S5B). Consistent with CB-5083 or NMS-873 acting via p97 inhibition to blunt protein degradation, we found that p97 RNAi-treatment together with low concentrations of either drug led to additive increases in polyubiquitinated protein accumulation (Fig. S5C). We also observed accumulation of polyubiquitinated proteins following either RNAi of proteasome subunit beta type-2 or treatment with ixazomib (Fig. S5D). These effects on the degradation of ubiquitinated proteins appeared to be specific to inhibition of UPS function, rather than a non-specific effect due to reduced worm vitality (Fig. S5D).

We then depleted UPS components using RNAi and surgically transplanted these worms into the mesenteric veins of recipient mice (Fig. S6A) and measured parasite egg deposition in host tissues and parasite survival. Following hepatic portal perfusion, we recovered about 55% of transplanted control RNAi-treated worms (Figs. 2D, S6B) and these parasites established patent infections, depositing large number of eggs into the livers of recipient mice (Figs. 2E, S6C–D). In contrast, we failed to recover parasites following hepatic portal perfusion from mice receiving p97 (Fig. 2D) or proteasome subunit beta type-2 (Fig. S6B). As a consequence, the livers in these mice were devoid of eggs and we observed no signs of egg-induced granulomas (Figs. 2E, S6C–D). We also observed RNAi-treated parasites at various stages of infiltration by host immune cells (Fig. S6E–F). This suggests that these parasites are unable to remain in the portal vasculature and are cleared via the liver by the immune system. Taken together, these data highlight the essentially and druggability of the schistosome UPS.

Another group of potentially druggable targets to emerge from our RNAi screen were protein kinases, 19 of which led to defects in either parasite attachment or stem cell maintenance. RNAi of two STE20 serine-threonine kinases: tao and stk25, which are homologs of the human TAO1/2/3 and STK25/YSK1 protein kinases, respectively, led to rapid detachment from the substrate (Fig. S7A) and a concomitant posterior paralysis and hypercontraction of the body, such that the parasites took on a distinctive banana-shaped morphology (Figs. 3A, S7B, Movie S4). Aside from RNAi of stk25 and tao, this banana-shaped phenotype of worms was unique, only observed following RNAi of a CCM3/PDCD10 homolog (Smp_031950), a known heterodimerization partner with the mammalian STK25 kinase(14). We failed to observe death of either stk25- or tao- depleted parasites during in vitro culture. However, following surgical transplantation, we noted a reduction in the recovery of tao or stk25 RNAi-treated parasites from recipient mice and these recipient mice displayed few signs of egg-induced granuloma formation (Figs. 3B–C, S7C–D). Thus, both tao and stk25 appear to be essential for schistosome survival in vivo.

Fig. 3. The protein kinases SmSTK25 and SmTAO are essential to maintain muscular function.

(A) RNAi of stk25 or tao causes parasite hypercontraction. (B-C) Percent recovery stk25(RNAi), tao(RNAi), or control(RNAi) male parasites surgically transplanted into mice. n ≥ 11 transplants for each group. ****, p < 0.0001, t-test; data are mean +/− 95% CI. (D) Western blot to detect phospho-T173 (p-SmSTK25) or total SmSTK25 following an in vitro kinase reaction +/− ATP. kinase dead SmTAO(kdSmTAO). Representative of n = 2 experiments. (E) Scatter plot showing the relationship between the differentially expressed genes (p adj < 0.001, Benjamini-Hochberg corrected Wald test) following stk25 or tao RNAi-treatment (r = 0.9, p < 0.0001, Pearson correlation). (F) Heatmap showing expression of tissue-specific transcripts following RNAi of tao or stk25. (G). in situ hybridization for tropomyosin 1 (tpm1, Smp_340760) and a myosin light chain (myl2, Smp_132670) following RNAi at D13. Scale bars: A, 500 μm; G, 100 μm.

Given the unique and specific nature of the stk25 and tao associated “banana” phenotype we reasoned that these kinases may be acting in concert to mediate signaling processes in the worm. The Drosophila STK25 ortholog (GCK3) is a substrate of TAO and these proteins function in a signaling cascade essential for tracheal development(15). Likewise we observed that recombinant S. mansoni STK25 (SmSTK25) could serve as a substrate for the S. mansoni TAO (SmTAO) in an in vitro kinase assay (Fig. S8A–B). The human STK25 is activated by phosphorylation of a conserved threonine residue within its activation loop(16). By mass spectrometry we observed that this conserved threonine within the predicted SmSTK25 activation loop (T¹⁷³) was phosphorylated following incubation of recombinant SmTAO with catalytically inactive SmSTK25 (Fig. S8C–D). Using an antibody that recognizes activation loop phosphorylation in STK25 orthologs(16), western blotting revealed SmSTK25 T¹⁷³ autophosphorylation following an in vitro kinase reaction; this signal was abrogated in controls lacking ATP and when the SmSTK25 catalytic K⁴⁸ residue was mutated to R (Figs. 3D, S8E). Consistent with our mass spectrometry results, we detected robust phosphorylation of T¹⁷³ when recombinant SmTAO was incubated with kinase dead SmSTK25 (Fig. 3D), suggesting that SmTAO can phosphorylate a key residue for the activation of the mammalian STK25.

We reasoned that the schistosome TAO and STK25 might be acting in a signaling module to mediate critical processes in the parasite. To define these processes, we performed transcriptional profiling on RNAi-treated parasites just prior to observing detachment and hypercontraction (Day 6 and Day 9 for tao and stk25 RNAi treatments, respectively) (Fig. S9A–B). We found that expression of differentially regulated genes following RNAi of either tao or stk25 were correlated (Fig. 3E) and that more than half of these differentially regulated genes were common in both datasets (Fig. S9C, Table S9). Importantly, RNAi of either tao or stk25 was specific, not affecting expression of the other kinase gene of this pair (Fig. 3E–F).

Examining the tissue-specific expression of differentially-regulated genes on an adult schistosome single cell expression atlas using cells from schistosome somatic tissues(17), we found that roughly 40% (51/129) of the most down-regulated genes following tao and stk25 RNAi were specific markers of parasite muscle cells (Fig. S10A–C, Table S9). Indeed, nearly half of all mRNAs specifically-enriched in muscle cells (60/135) from this single cell atlas, including key muscle contractile proteins, were down-regulated following RNAi of both tao and stk25 (Figs. 3F, S10B, D, Table S10). Importantly, these transcriptional effects appeared to be largely specific to parasite muscles (Figs. 3F, S10B, Table S10).

In principle, loss of muscle-specific mRNAs could be due to either loss of muscle cells or down-regulation of muscle-specific mRNAs. We thus labeled F-actin in schistosome muscle fibers with phalloidin and performed in situ hybridization to detect muscle-specific mRNAs. RNAi-treated parasites exhibited a reduction in the expression of mRNAs encoding the contractile proteins Tropomyosin 1 and a Myosin Light Chain by in situ hybridization (Fig. 3G), but no qualitative defects in phalloidin staining of body wall muscles (Fig. S11). Thus, it appears that these kinases are required to maintain the transcription of many muscle-specific mRNAs in intact muscle cells. Interestingly, the heads of tao and stk25 RNAi parasites retained their capacity for movement (Movie S4) and partially maintained the expression of muscle-specific mRNAs (Fig. 3G). Thus, there appears to be a relationship between the maintenance of muscle-specific mRNA expression and locomotion.

Taken in their entirety, our data suggest that STK25 and TAO kinases cooperate in the schistosome to mediate signaling essential for sustaining the transcription of muscle-specific mRNAs. As a consequence, loss of either SmSTK25 or SmTAO activity results in muscular function defects compromising parasite survival in vivo. As whole-body knockouts of mouse STK25 are homozygous viable displaying no obvious deleterious phenotypes(18), SmSTK25 represents an attractive target for therapeutic intervention. Similar cross-examination of genes associated with other phenotypic classes (e.g., tissue edema) may provide insights into the specificity of the various phenotypes observed in this work.

Technological advances have paved the way for large-scale analyses of gene function in protozoan parasites(19–21), but, have been lacking in helminth parasites. Our systematic analysis of gene function in schistosomes allowed us to effectively prioritize targets and potential specific inhibitors with activities on worms. Future efforts should not only further explore the potency and selectivity of compounds our studies have uncovered (Fig. 2A–B, Table S6), but larger libraries of compounds with known molecular targets (22). However, to mitigate potential issues with host toxicity it is also worth exploring whether parasite-selective inhibitors of validated target proteins can be uncovered. Not only does this study enhance our understanding of schistosome biology, it provides a new lens to prioritize genes of interest in other medically- and agriculturally-important parasitic flatworms. Collectively, we anticipate this study will expedite the discovery of new anti-helminthics.