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Infection by the human blood fluke, Schistosoma mansoni involves a variety of cross-species protein- protein interactions. The pathogen expresses a diverse arsenal of proteins that facilitate the breach of physical and biochemical barriers present in skin evasion of the immune system, and digestion of human plasma proteins including albumin and hemoglobin, allowing schistosomes to reside in the host for years. However, only a small number of specific interactions between S. mansoni and human proteins have been identified. We present and apply a protocol that generates testable predictions of S. mansoni-human protein interactions. In this study, we have preliminary predictions of novel interactions between schistosome and human proteins relevant to infection and the ability of the parasite to evade the immune system. We applied a computational whole-genome comparative approach to predict potential S. mansoni-human protein interactions based on similarity to known protein complexes. We first predict S. mansoni -human protein interactions based on similarity to known protein complexes. Putative interactions were then scored and assessed using several contextual filters, including the use of annotation automatically derived from literature using a simple natural language processing methodology. Next, in vitro experiments were carried out between schistosome and host proteins to validate several prospective predictions. Our method predicted 7 out of the 10 previously known cross-species interactions involved in pathogenesis between S. mansoni and its human host. Interestingly, two novel putative interactions involving Schistosoma proteins, the cercarial elastase SmCE, and the adult tegument surface protein Sm29, were also predicted and experimentally characterized. Preliminary data suggest that elafin, a host endogenous serine protease inhibitor, may be a novel substrate for SmCE. Additionally, CD59, an inhibitor of the membrane attack complex, could interact with Sm29. Furthermore, the application framework provides an integrated methodology for investigation of host-pathogen interactions and an extensive source of orthogonal data for experimental analysis. We have made the predictions available for community perusal.

Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Amongst these, schistosomiasis (bilharzia or ‘snail fever’), caused by blood flukes of the genus Schistosoma, ranks second only to malaria in terms of human impact: two hundred million people are infected and close to 800 million are at risk of infection. Drug screening against helminths poses unique challenges: the parasite cannot be cloned and is difficult to target using gene knockouts or RNAi. Consequently, both lead identification and validation involve phenotypic screening, where parasites are exposed to compounds whose effects are determined through the analysis of the ensuing phenotypic responses. The efficacy of leads thus identified derives from one or more or even unknown molecular mechanisms of action. The two most immediate and significant challenges that confront the state-of-the-art in this area are: the development of automated and quantitative phenotypic screening techniques and the mapping and quantitative characterization of the totality of phenotypic responses of the parasite. In this paper, we investigate and propose solutions for the latter problem in terms of the following: (1) mathematical formulation and algorithms that allow rigorous representation of the phenotypic response space of the parasite, (2) application of graph-theoretic and network analysis techniques for quantitative modeling and characterization of the phenotypic space, and (3) application of the aforementioned methodology to analyze the phenotypic space of S. mansoni – one of the etiological agents of schistosomiasis, induced by compounds that target its polo-like kinase 1 (PLK 1) gene – a recently validated drug target. In our approach, first, bio-image analysis algorithms are used to quantify the phenotypic responses of different drugs. Next, these responses are linearly mapped into a low- dimensional space using Principle Component Analysis (PCA). The phenotype space is modeled using neighborhood graphs which are used to represent the similarity amongst the phenotypes. These graphs are characterized and explored using network analysis algorithms. We present a number of results related to both the nature of the phenotypic space of the S. mansoni parasite as well as algorithmic issues encountered in constructing and analyzing the phenotypic-response space. In particular, the phenotype distribution of the parasite was found to have a distinct shape and topology. We have also quantitatively characterized the phenotypic space by varying critical model parameters. Finally, these maps of the phenotype space allows visualization and reasoning about complex relationships between putative drugs and their system-wide effects and can serve as a highly efficient paradigm for assimilating and unifying information from phenotypic screens both during lead identification and lead optimization.

International audience; prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections. Nuclear receptors (NRs) are transcription factors involved in key physiological processes such as growth and development, aging and reproduction, as well as metabolism and detoxification of endo-and xenobiotics 1-4. They typically bind small lipophilic molecules, resulting in their activation, which allows the receptor to recruit co-regulators of expression of specific genes 5. Because of their central role in critical metabolic processes, including development, NRs are relevant targets for drug discovery 2. It is estimated that 10-20% of the pharmaceutical market targets these receptors in humans 5. NRs are conserved among metazoans including helminths 6. While humans harbor 49 NRs in their genome, Caenorhabditis elegans has 284 NRs 7 and some are essential for development and reproduction but also survival by enabling this free-living worm to adapt to variable environmental conditions. DAF-12 was described as a NR in C. elegans, due to the discovery of its ligands, dafachronic acids (Δ4-and Δ7-DA) 8-10. In this nematode, DAF-12 acts as a molecular switch in the signaling pathway that regulates dauer formation, or alternative progression to reproductive development and the adult stage, by sensing the environmental quality and bacterial nutrient availability 9,11. When environmental conditions are favorable, and bacteria are available, C. elegans stimulates the production of DA steroid hormones, which activate DAF-12 7,10,12. CelDAF-12 then induces transcription of development genes that promote growth and reproduction 13-15. In contrast, in unfavorable conditions, DAs are not synthesized and the DAF-12 apo-receptor interacts with a co-repressor, preventing target gene expression and resulting in arrest of development at dauer diapause larvae, a non-feeding, non-reproducing, stress-resistant, open

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments.

Background Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer’s disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). Methodology/Principal findings A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite’s genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. Conclusions/Significance The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A’s contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets., Author summary Just one drug, praziquantel (PZQ), is available to treat schistosomiasis, a flatworm disease that infects over 240 million people, mainly in Africa. With the expanding distribution of PZQ, and the associated threat of drug resistance, new drugs and drug targets are needed. We screened Schistosoma mansoni worms with over 1,000 benzoxaborole chemical molecules from Anacor Pharmaceuticals to identify a subset of human cyclic nucleotide phosphodiesterase 4 (huPDE4) inhibitors that cause parasite hypermotility and degeneration. We identified four PDE4 genes in the genome of the parasite and recombinantly expressed one of them (SmPDE4A) in bacteria. This enzyme was then used to uncover a relationship between the degree of enzyme inhibition, and the generation of parasite hypermotility and degeneration. To understand whether the SmPDE4A enzyme is the target of the benzoxaboroles in the parasite, we incorporated the coding DNA for SmPDE4A into the model nematode Caenorhabditis elegans that lacked its own functional PDE4 and, as a consequence, was hypermotile. These ‘transgenic’ worms displayed normal motility which could be increased by applying the most potent huPDE4 benzoxaborole inhibitors. In summary, the chemical, biological and genetic approaches taken identify SmPDE4A as a potential drug target for treating schistosomiasis. The potential value of C. elegans as a tool to test and optimize therapeutic chemistries for a flatworm disease is also highlighted.

Background Schistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets. Methodology/Principal Findings We show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1–2 μM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays. Conclusions/Significance The reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals., Author Summary Just one drug is available to treat schistosomiasis, a parasitic disease that affects hundreds of millions of people in developing countries. In the search for new drugs and drug targets, therefore, we have been interested in the schistosome version of human polo-like kinase (huPLK)1, an enzyme with critical functions in cell division. We used RNA interference to knock down messenger RNA for the SmPLK1 –the Schistosoma mansoni parasite’s version of huPLK1. This interference caused disruptive changes in the morphology of the immature ‘somule’ stage of the parasite, indicating that SmPLK1 is an important protein for survival. We then purchased, or acquired from GlaxoSmithKline (GSK), various small chemical inhibitors of huPLK1 and tested these against both the somules and adult parasites in culture. Many of these inhibitors caused severe changes in the parasite and, for somules, the differences could be computationally mapped and distinguished from unexposed parasites. For the GSK inhibitors, we observed ‘somule-adult bioactivity clustering,’ that is, chemicals active against the adults were also active against somules. This suggests that certain chemical attributes in the inhibitors are being favoured. Interestingly, many of the GSK inhibitors most active against the parasite are also known to both potently inhibit huPLK1 and kill cancer cells. Overall, our data suggest that SmPLK1 is a possible drug target and that the GSK chemistries could form the basis for developing a new drug to treat schistosomiasis.

Background Among more than 20,000 species of hermaphroditic trematodes, Schistosomatidae are unusual since they have evolved gonochorism. In schistosomes, sex is determined by a female heterogametic system, but phenotypic sexual dimorphism appears only after infection of the vertebrate definitive host. The completion of gonad maturation occurs even later, after pairing. To date, the molecular mechanisms that trigger the sexual differentiation in these species remain unknown, and in vivo studies on the developing schistosomulum stages are lacking. To study the molecular basis of sex determination and sexual differentiation in schistosomes, we investigated the whole transcriptome of the human parasite Schistosoma mansoni in a stage- and sex-comparative manner. Methodology/ Principal Findings We performed a RNA-seq on males and females for five developmental stages: cercariae larvae, three in vivo schistosomulum stages and adults. We detected 7,168 genes differentially expressed between sexes in at least one of the developmental stages, and 4,065 of them were functionally annotated. Transcriptome data were completed with H3K27me3 histone modification analysis using ChIP-Seq before (in cercariae) and after (in adults) the phenotypic sexual dimorphism appearance. In this paper we present (i) candidate determinants of the sexual differentiation, (ii) sex-biased players of the interaction with the vertebrate host, and (iii) different dynamic of the H3K27me3 histone mark between sexes as an illustration of sex-biased epigenetic landscapes. Conclusions/ Significance Our work presents evidence that sexual differentiation in S. mansoni is accompanied by distinct male and female transcriptional landscapes of known players of the host-parasite crosstalk, genetic determinants and epigenetic regulators. Our results suggest that such combination could lead to the optimized sexual dimorphism of this parasitic species. As S. mansoni is pathogenic for humans, this study represents a promising source of therapeutic targets, providing not only data on the parasite development in interaction with its vertebrate host, but also new insights on its reproductive function., Author Summary Parasitic flatworms include more than 20,000 species that are classically hermaphrodites. Among them, the roughly hundred species of Schistosomatidae are intriguing because they are gonochoric. Schistosomes are responsible of the second most important parasitic disease worldwide, and eggs are the main cause of the inflammatory symptoms. Thus, studying the sexual reproduction mechanisms of schistosomes is of particular interest for drug development. Schistosome’s sex is genetically determined by the presence of sex chromosomes: ZZ in males or ZW in females. There is, however, no phenotypic dimorphism in the larval stages: sexual dimorphism appears only in the vertebrate host. In order to understand the molecular mechanisms underlying phenotypic sexual dimorphism, we performed a transcriptome analysis (RNA-Seq) in five different stages of the parasite lifecycle as well as a chromatin status analysis (ChIP-Seq) in the non-differentiated stage cercariae and in the adult differentiated stage, for males and females separately. Our work presents evidence that sexual differentiation in S. mansoni is accompanied by distinct male and female transcriptional landscapes of known players of the host-parasite crosstalk, developmental pathways and epigenetic regulators. Our sex-comparative approach provides therefore new potential therapeutic targets to affect development and sexual reproduction of parasite.

Graphical abstract, Highlights • The Abl tyrosine-kinase inhibitor Imatinib is toxic to S. mansoni in vitro but not in vivo in rodents. • Blood components like serum albumin and alpha-1 acid glycoprotein (AGP) negated Imatinib’s toxicity in vitro. • Erythromycin partially restored the toxicity of Imatinib in vitro. • High levels of AGP upon infection make rodents poor models for examining some small molecule inhibitors., In the search for new drugs and drug targets to treat the flatworm disease schistosomiasis, protein kinases (PKs) have come under particular scrutiny because of their essential roles in developmental and physiological processes in schistosome parasites. In this context the application of the anti-cancer Abl tyrosine kinase (TK) inhibitor Imatinib (Gleevec/Glivec; STI-571) to adult Schistosoma mansoni in vitro has indicated negative effects on diverse physiological processes including survival. Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA) and alpha-1 acid glycoprotein (AGP or orosomucoid) negated Imatinib’s deleterious effects on adult S. mansoni and schistosomula (post-infective larvae) in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6–8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments.