Search

Parasitic helminths are master manipulators of host immunity. Their strategy is complex and involves the release of excreted/secreted products, including extracellular vesicles (EVs). The protein and miRNA contents of EVs have been characterised for many parasitic helminths but, despite reports suggesting the importance of EV surface carbohydrate structures (glycans) in the interactions with target cells and thus subsequent effector functions, little is known about parasite EV glycomics. Using lectin microarrays, we identified several lectins that exhibit strong adhesion to Schistosoma mansoni EVs, suggesting the presence of multiple glycan structures on these vesicles. Interestingly, SNA-I, a lectin that recognises structures with terminal sialic acid, displayed strong affinity for S. mansoni EVs, which was completely abolished by neuraminidase treatment, suggesting sialylation in the EV sample. This finding is of interest, as sialic acids play important roles in the context of infection by aiding immune evasion, affecting target recognition, cell entry, etc., but are not thought to be synthesised by helminths. These data were validated by quantitative analysis of free sialic acid released from EVs following treatment with neuraminidase. Lectin histochemistry and fluorescence in situ hybridisation analyses on whole adult worms suggest the involvement of sub-tegumental cell bodies, as well as the digestive and excretory systems, in the release of EVs. These results support previous reports of EV biogenesis diversity in trematodes and potentially highlight new means of immune modulation and evasion employed by schistosomes.

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.

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.

Polo-like kinases are important regulators of cell cycle progression and mitosis. They constitute a family of conserved serine/threonine kinases which are highly related in their catalytic domains and contain polo boxes involved in protein-protein interactions and subcellular localization. In mammals, five Plks (Plk 1-5) encompass diverse roles in centrosome dynamics, spindle formation, intra S-phase and G2/M checkpoints and DNA damage response. Plk1 is a key positive regulator of mitosis and is overexpressed in various types of cancers. Plk4 is a divergent member of the Plk family, with essential functions in centriole duplication. Homozygous disruption of Plk1 or Plk4 in mice is lethal in embryos. Two Plk members SmPlk1 and SmSak, homologous to Plk1 and Plk4 respectively, are present in the parasitic platyhelminth Schistosoma mansoni. Structural and functional analyses of SmPlk1 have demonstrated its conserved function in the regulation of cell cycle G2/M transition in Xenopus oocytes. The anti-cancer drug BI 2536 (the most potent and selective Plk1 inhibitor) inhibits specifically the catalytic activity of SmPlk1 and induced profound alterations in schistosome gonads, indicating a role of SmPlk1 in parasite gametogenesis and its potential as a novel chemotherapeutic target against schistosomiasis. Functions of SmSak in cell cycle regulation and schistosome gonad development are currently investigatedQuinases do tipo Polo ("polo-like") são importantes reguladores da progressão do ciclo celular e da mitose. Elas constituem uma família de serina/treonina quinases que são altamente relacionadas entre si no seu domínio catalítico e contêm blocos "polo" envolvidos com interações proteína-proteína e com localização subcelular. Em mamíferos, cinco Plks (Plk 1-5) englobam diversos papéis na dinâmica do centrossomo, formação do fuso, "checkpoints" dentro da fase S e da transição G2/M, e na resposta aos danos do DNA. Plk1 é um regulador positivo chave da mitose, e é superexpresso em vários tipos de câncer. Plk4 é um membro divergente da família Plk, com funções essenciais na duplicação do centríolo. Deleção homozigótica de Plk1 ou Plk4 em camundongos é letal em embriões. Dois membros da família Plk, SmPlk1 e SmSak, homólogos a Plk1 e Plk4, respectivamente, estão presentes no parasita platelmíntico Schistosoma mansoni. Análises estruturais e funcionais de SmPlk1 demonstraram uma função conservada na regulação da transição G2/M do ciclo celular em ovócitos de Xenopus. A droga anticâncer BI2536 (o inibidor mais potente e seletivo de Plk1) inibe específicamente a atividade catalítica de SmPlk1 e induz alterações profundas nas gonadas de esquistossomos, indicando um papel de SmPlk1 na gametogênese do parasita e seu potencial como um alvo terapêutico novo contra a esquistossomose. As funções de SmSak na regulação do ciclo celular e no desenvolvimento das gônadas de esquistossomos estão sendo investigadas no momento

Polo-like kinases (Plks) are conserved regulators of mitosis. In mammals, Plk1 is over-expressed in a wide range of tumour cells and constitutes a valuable target for anti-cancer therapy. This work presents the characterisation of the Plk1 homologue (SmPlk1) of Schistosoma mansoni, a trematode responsible for schistosomiasis, one of the most important parasitic diseases, second only to malaria. The intense levels of disease transmission and the severity of pathologies are the consequences of the exceptional reproductive activity of schistosomes, in which Plks may play a decisive role. Structural and functional analyses of SmPlk1 have demonstrated its homology with other Plk1 members and its conserved function in mitotic processes. Activation of SmPlk1 was shown to be dependent on phosphorylation of its conserved threonine residue (T(182)) and the ability of active SmPlk1 to induce mitosis was demonstrated in the Xenopus oocyte model. SmPlk1 transcripts were detected abundantly in parasite stages containing a high amount of germinal cells. A potential role of SmPlk1 in mitosis and/or meiosis in schistosomes was supported by the in situ detection of SmPlk1 transcripts in female vitelline cells and oocytes as well as in male spermatocytes. Several Plk inhibitors were shown to inhibit SmPlk1 activity in Xenopus oocytes, and BI 2536 (the first-in-class prototype Plk1 inhibitor) induced in vitro dramatic alterations in schistosome gonads, which affected oogenesis and spermatogenesis. These results indicate a major role for SmPlk1 in parasite reproduction and suggest its importance as a potential new target against schistosomiasis.

SUMMARYAmong the topics of considerable interest concerning our understanding of the unusual biology of schistosomes is the sexual maturation of the female. The identification of genes coding for signal transduction proteins controlling essential steps of the pairing-dependent differentiation of the reproductive organs, vitellarium and ovary will help to substantiate our knowledge about this unique parasite. Furthermore, such signalling proteins could be potential targets to interfere with the development of this parasite to combat schistosomiasis since its pathology is caused by the eggs. This review summarises first post-genomic steps to elucidate the function of gonad-specific signalling molecules which were identified by homology-based cloning strategies, byin silicoidentification or by yeast two-hybrid interaction analyses, using a combination of novel techniques. These include thein vitroculture of adult schistosomes, their treatment with chemical inhibitors to block enzyme activity, the use of RNAi to silence gene function post-transcriptionally, and confocal laser scanning microscopy to study the morphological consequences of these experimental approaches. Finally, we propose a first model of protein networks that are active in the ovary regulating mitogenic activity and differentiation. Some of these molecules are also active in the testes of males, probably fulfilling similar roles as in the ovary.

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.

M/S n° 1, vol. 25, janvier 2009 REMERCIEMENTS Les auteurs tiennent a remercier Delphine Belanger, Marie-Eva de Villers, Paul Sidani, Tom Babcock et Rick Lin pour leurs diverses contributions a ce travail. EVS est soutenu par une bourse de clinicien-chercheur des Instituts de recherche en sante du Canada, et le laboratoire de MMM est soutenu par le National Institute of Health et le National Institute of Mental Health des Etats-Unis.

The mevalonate pathway is essential in eukaryotes and responsible for a diversity of fundamental synthetic activities. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the pathway and is targeted by the ubiquitous statin drugs to treat hypercholesterolemia. Independent reports have indicated the cidal effects of statins against the flatworm parasite, S. mansoni, and the possibility that SmHMGR is a useful drug target to develop new statin-based anti-schistosome therapies. For six commercially available statins, we demonstrate concentration- and time-dependent killing of immature (somule) and adult S. mansoni in vitro at sub-micromolar and micromolar concentrations, respectively. Cidal activity trends with statin lipophilicity whereby simvastatin and pravastatin are the most and least active, respectively. Worm death is preventable by excess mevalonate, the product of HMGR. Statin activity against somules was quantified both manually and automatically using a new, machine learning-based automated algorithm with congruent results. In addition, to chemical targeting, RNA interference (RNAi) of HMGR also kills somules in vitro and, again, lethality is blocked by excess mevalonate. Further, RNAi of HMGR of somules in vitro subsequently limits parasite survival in a mouse model of infection by up to 80%. Parasite death, either via statins or specific RNAi of HMGR, is associated with activation of apoptotic caspase activity. Together, our genetic and chemical data confirm that S. mansoni HMGR is an essential gene and the relevant target of statin drugs. We discuss our findings in context of a potential drug development program and the desired product profile for a new schistosomiasis drug.

Besides their great medical importance as causative agents of schistosomiasis, an infectious disease affecting humans and animals in tropical and subtropical regions worldwide, schistosomes exhibit distinctive biological features. Living in the blood vessels of infected hosts, these blood flukes survive permanent attacks of the immune system over many years. Furthermore, schistosomes represent the only genus of the class trematoda which live doeciously. Their most remarkable attribute, however, is the continuous pairing-contact which is both obligatory for the development of the female reproductive organs and prerequisite for egg production

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.