The esophageal gland mediates host immune evasion by the human parasite Schistosoma mansoni.

Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease affecting over 200 million people. Schistosomes develop multiple body plans while navigating their complex life cycle, which involves two different hosts: a mammalian definitive host and a molluscan intermediate host. Their survival and propagation depend upon proliferation and differentiation of stem cells necessary for parasite homeostasis and reproduction. Infective larvae released from snails carry a handful of stem cells that serve as the likely source of new tissues as the parasite adapts to life inside the mammalian host; however, the role of these stem cells during this critical life cycle stage remains unclear. Here, we characterize stem cell fates during early intramammalian development. Surprisingly, we find that the esophageal gland, an accessory organ of the digestive tract, develops before the rest of the digestive system is formed and blood feeding is initiated, suggesting a role in processes beyond nutrient uptake. To explore such a role, we examine schistosomes that lack the esophageal gland due to knockdown of a forkhead-box transcription factor, Sm-foxA, which blocks development and maintenance of the esophageal gland, without affecting the development of other somatic tissues. Intriguingly, schistosomes lacking the esophageal gland die after transplantation into naive mice, but survive in immunodeficient mice lacking B cells. We show that parasites lacking the esophageal gland are unable to lyse ingested immune cells within the esophagus before passing them into the gut. These results unveil an immune-evasion mechanism mediated by the esophageal gland, which is essential for schistosome survival and pathogenesis.

Systematically improved in vitro culture conditions reveal new insights into the reproductive biology of the human parasite Schistosoma mansoni.

Schistosomes infect over 200 million people. The prodigious egg output of these parasites is the sole driver of pathology due to infection, yet our understanding of sexual reproduction by schistosomes is limited because normal egg production is not sustained for more than a few days in vitro. Here, we describe culture conditions that support schistosome sexual development and sustained egg production in vitro. Female schistosomes rely on continuous pairing with male worms to fuel the maturation of their reproductive organs. Exploiting these new culture conditions, we explore the process of male-stimulated female maturation and demonstrate that physical contact with a male worm, and not insemination, is sufficient to induce female development and the production of viable parthenogenetic haploid embryos. We further report the characterization of a nuclear receptor (NR), which we call Vitellogenic Factor 1 (VF1), that is essential for female sexual development following pairing with a male worm. Taken together, these results provide a platform to study the fascinating sexual biology of these parasites on a molecular level, illuminating new strategies to control schistosome egg production.

A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host.

Schistosomes are parasitic flatworms that infect over 200 million people, causing the neglected tropical disease, schistosomiasis. A single drug, praziquantel, is used to treat schistosome infection. Limitations in mass drug administration programs and the emergence of schistosomiasis in nontropical areas indicate the need for new strategies to prevent infection. It has been known for several decades that rotifers colonizing the schistosome's snail intermediate host produce a water-soluble factor that paralyzes cercariae, the life cycle stage infecting humans. In spite of its potential for preventing infection, the nature of this factor has remained obscure. Here, we report the purification and chemical characterization of Schistosome Paralysis Factor (SPF), a novel tetracyclic alkaloid produced by the rotifer Rotaria rotatoria. We show that this compound paralyzes schistosome cercariae and prevents infection and does so more effectively than analogous compounds. This molecule provides new directions for understanding cercariae motility and new strategies for preventing schistosome infection.

Detection of Schistosoma mansoni long non-coding RNAs in the infected C57BL/6 mouse liver.

Long non-coding RNAs (lncRNAs) perform several types of regulatory functions and have been recently explored in the genus Schistosoma. Although sequencing and bioinformatics approaches have demonstrated the presence of hundreds of lncRNAs and microRNAs (miRNAs) in this genus, information regarding their abundance, characteristics, and potential functions linked to Schistosoma mansoni biology and parasite-host interaction is limited. Our objectives in the present study were to verify whether 15 previously identified S. mansoni lncRNAs are detectable in the host liver. In addition, we assess whether these lncRNAs are present in the S. mansoni infective form and the stages inside the definitive host. The detection of these 15 S. mansoni lncRNAs and a long terminal repeat (LTR) retrotransposon Saci 4 was performed in the eggs, cercariae, and 3.5-h schistosomula. All lncRNAs were found to be expressed in these stages; some of the lncRNAs were found in the livers of the infected C57BL/6 mice. In conclusion, S. mansoni lncRNAs were detected in host livers and quantified. Furthermore, many of the lncRNAs analyzed showed differential expression in the larval stages, indicating that they play a stage-specific regulatory role.

The Prospective Use of Brazilian Marine Macroalgae in Schistosomiasis Control.

Schistosomiasis is a parasitic disease that affects more than 250 million people. The treatment is limited to praziquantel and the control of the intermediate host with the highly toxic molluscicidal niclosamide. Marine algae are a poorly explored and promising alternative that can provide lead compounds, and the use of multivariate analysis could contribute to quicker discovery. As part of our search for new natural compounds with which to control schistosomiasis, we screened 45 crude extracts obtained from 37 Brazilian seaweed species for their molluscicidal activity against Biomphalaria glabrata embryos and schistosomicidal activities against Schistosoma mansoni. Two sets of extracts were taxonomically grouped for metabolomic analysis. The extracts were analyzed by GC-MS, and the data were subjected to Pattern Hunter and Pearson correlation tests. Overall, 22 species (60%) showed activity in at least one of the two models. Multivariate analysis pointed towards 3 hits against B. glabrata veliger embryos in the Laurencia/Laurenciella set, 5 hits against B. glabrata blastula embryos, and 31 against S. mansoni in the Ochrophyta set. Preliminary annotations suggested some compounds such as triquinane alcohols, prenylated guaianes, dichotomanes, and xenianes. Despite the putative identification, this work presents potential candidates and can guide future isolation and identification.

Histone methylation changes are required for life cycle progression in the human parasite Schistosoma mansoni.

Epigenetic mechanisms and chromatin structure play an important role in development. Their impact is therefore expected to be strong in parasites with complex life cycles and multiple, strikingly different, developmental stages, i.e. developmental plasticity. Some studies have already described how the chromatin structure, through histone modifications, varies from a developmental stage to another in a few unicellular parasites. While H3K4me3 profiles remain relatively constant, H3K27 trimethylation and bivalent methylation show strong variation. Inhibitors (A366 and GSK343) of H3K27 histone methyltransferase activity in S. mansoni efficiently blocked miracidium to sporocyst transition indicating that H3K27 trimethylation is required for life cycle progression. As S. mansoni is a multicellular parasite that significantly affects both the health and economy of endemic areas, a better understanding of fluke developmental processes within the definitive host will likely highlight novel disease control strategies. Towards this goal, we also studied H4K20me1 in female cercariae and adults. In particular, we found that bivalent trimethylation of H3K4 and H3K27 at the transcription start site of genes is a landmark of the cercarial stage. In cercariae, H3K27me3 presence and strong enrichment in H4K20me1 over long regions (10-100 kb) is associated with development related genes. Here, we provide a broad overview of the chromatin structure of a metazoan parasite throughout its most important lifecycle stages. The five developmental stages studied here present distinct chromatin structures, indicating that histone methylation plays an important role during development. Hence, components of the histone methylation (and demethylation) machinery may provide suitable Schistosomiasis control targets.

Alkoxyamines Designed as Potential Drugs against Plasmodium and Schistosoma Parasites.

Malaria and schistosomiasis are major infectious causes of morbidity and mortality in the tropical and sub-tropical areas. Due to the widespread drug resistance of the parasites, the availability of new efficient and affordable drugs for these endemic pathologies is now a critical public health issue. In this study, we report the design, the synthesis and the preliminary biological evaluation of a series of alkoxyamine derivatives as potential drugs against Plasmodium and Schistosoma parasites. The compounds (RS/SR)-2F, (RR/SS)-2F, and 8F, having IC50 values in nanomolar range against drug-resistant P. falciparum strains, but also five other alkoxyamines, inducing the death of all adult worms of S. mansoni in only 1 h, can be considered as interesting chemical starting points of the series for improvement of the activity, and further structure activity, relationship studies. Moreover, investigation of the mode of action and the rate constants kd for C-ON bond homolysis of new alkoxyamines is reported, showing a possible alkyl radical mediated biological activity. A theoretical chemistry study allowed us to design new structures of alkoxyamines in order to improve the selectivity index of these drugs.

Lipid Topography in Schistosoma mansoni Cryosections, Revealed by Microembedding and High-Resolution Atmospheric-Pressure Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging.

Schistosomes are parasitic platyhelminthes that cause schistosomiasis, which is a life-threatening infectious disease for humans in the tropics and subtropics worldwide. Within the human host, female and male schistosomes develop and pair as a prerequisite for egg production. Part of the eggs get lodged in organs such as the gut, spleen, and liver, where they cause severe inflammatory processes, including liver fibrosis, which is one of the most serious pathological symptoms. High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) has been used as a powerful tool to investigate adult schistosomes at the topographic molecular level. An MSI-compatible protocol was developed, covering critical sample preparation steps and focusing on obtaining artifact-free, longitudinal cryosections. Planar, consecutive sections were prepared from ∼400 µm thick S. mansoni worm couples, comparing several microembedding approaches. High-resolution MSI at both, 10 and 5 µm lateral resolution unraveled anatomical structures and differential abundances of glycerophospholipids and saccharides in females and males. In addition, glycerophospholipids occurred differentially abundant in worm tissues of the female, such as the gut, which is essential for nutrient uptake and subsequent metabolism. Fragment ions of isobaric phospholipids were investigated by on-tissue MS2 imaging experiments, unambiguously showing isomer-specific ion signals. This study provides a solid basis for investigating schistosome parasites in chemical detail at the whole-worm level by MSI.

Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Niosomes for enhanced activity of praziquantel against Schistosoma mansoni: in vivo and in vitro evaluation.

Praziquantel (PZQ) is recommended by the WHO as the first line in treatment of schistosomiasis. Unfortunately, it exhibits low oral bioavailability which can compromise its efficacy. Nanostructures showed promising potential to overcome this problem. Accordingly, the aim of this study was to investigate the effect of niosomal encapsulation of PZQ on its activity on Schistosoma mansoni in vitro and in vivo. PZQ was encapsulated in niosomal formulation comprising span 60, cholesterol with peceol being included as absorption enhancer. The in vitro work determined the schistosomicidal activity and morphological changes after incubation with drug solution or PZQ-niosomes. The in vivo study utilized infected mice which received PZQ orally as solution or as niosomes. The activity was assessed by monitoring egg and worm count in addition to histopathological and immunohistochemical studies. The in vitro studies revealed that niosomes alone caused a 30% death of adult parasites and caused completely coiled body, destruction, and peeling of tubercles and spines, with flattening and effacement of gynecophoric canal, blebbing with niosomes vesicles attached to it. Niosomes containing PZQ at a concentration of 0.001 µg/ml increased the death from 30 to 50% with the corresponding PZQ solution causing only 10% death. The in vivo study reflected of niosome-PZQ over PZQ solution as indicated from significant reduction of adult worm count, hepatic and intestinal egg depositions, hepatic granuloma size, and numbers, with marked reduction of vascular endothelial growth factor expression. The study introduced niosomes as promising carriers for enhanced activity of PZQ.

The impact of cinnarizine and griseofulvin on juvenile and adult stages of Schistosoma mansoni.

Schistosomiasis affects millions globally. There is no vaccine, and treatment depends entirely on praziquantel (PZQ). Field isolates exhibit reduced susceptibility to PZQ, and resistance has been experimentally induced, suggesting that reliance on a single treatment is particularly dangerous. The present study investigated the value of cinnarizine and griseofulvin against Schistosoma mansoni through their in vitro effects on adult worms and oviposition as well as in vivo evaluation in early and late infection, compared to PZQ, in a preliminary experimental model. In vitro, both cinnarizine and griseofulvin showed uncoupling, sluggish worm movement and complete absence of ova at 100 µg/ml. In early infection, cinnarizine showed a significant reduction in the number of porto-mesenteric couples compared to the griseofulvin and control groups, a finding similar to PZQ. Remarkably, cinnarizine significantly exceeded PZQ and griseofulvin in reducing the total worm burden. In late infection, cinnarizine and griseofulvin showed results similar to PZQ by significantly reducing the numbers of hepatic and porto-mesenteric couples and total worm burden compared to controls. Cinnarizine performed better than griseofulvin by reducing hepatic and intestinal ovum counts, and it led to complete disappearance of the first two immature stages. The current work suggests the possibility of using cinnarizine and griseofulvin, mainly in late S. mansoni infection, especially cinnarizine, which showed similar results to PZQ and surpassed it in early infection. Further studies are required to elucidate their exact mechanisms of action and particularly their synergistic effect with PZQ.

Tissue Degeneration following Loss of Schistosoma mansoni cbp1 Is Associated with Increased Stem Cell Proliferation and Parasite Death In Vivo.

Schistosomiasis is second only to malaria in terms of the global impact among diseases caused by parasites. A striking feature of schistosomes are their ability to thrive in their hosts for decades. We have previously demonstrated that stem cells, called neoblasts, promote homeostatic tissue maintenance in adult schistosomes and suggested these cells likely contribute to parasite longevity. Whether these schistosome neoblasts have functions independent of homeostatic tissue maintenance, for example in processes such as tissue regeneration following injury, remains unexplored. Here we characterize the schistosome CBP/p300 homolog, Sm-cbp1. We found that depleting cbp1 transcript levels with RNA interference (RNAi) resulted in increased neoblast proliferation and cell death, eventually leading to organ degeneration. Based on these observations we speculated this increased rate of neoblast proliferation may be a response to mitigate tissue damage due to increased cell death. Therefore, we tested if mechanical injury was sufficient to stimulate neoblast proliferation. We found that mechanical injury induced both cell death and neoblast proliferation at wound sites, suggesting that schistosome neoblasts are capable of mounting proliferative responses to injury. Furthermore, we observed that the health of cbp1(RNAi) parasites progressively declined during the course of our in vitro experiments. To determine the fate of cbp1(RNAi) parasites in the context of a mammalian host, we coupled RNAi with an established technique to transplant schistosomes into the mesenteric veins of uninfected mice. We found transplanted cbp1(RNAi) parasites were cleared from vasculature of recipient mice and were incapable of inducing measurable pathology in their recipient hosts. Together our data suggest that injury is sufficient to induce neoblast proliferation and that cbp1 is essential for parasite survival in vivo. These studies present a new methodology to study schistosome gene function in vivo and highlight a potential role for schistosome neoblasts in promoting tissue repair following injury.

A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing.

A promising means in the search of new small molecules for the treatment of schistosomiasis (amongst other parasitic ailments) is by targeting the parasitic epigenome. In the present study, a docking based virtual screening procedure using the crystal structure of histone deacetylase 8 from Schistosoma mansoni (smHDAC8) was designed. From the developed screening protocol, we were able to identify eight novel N-(2,5-dioxopyrrolidin-3-yl)-n-alkylhydroxamate derivatives as smHDAC8 inhibitors with IC50 values ranging from 4.4-20.3 µM against smHDAC8. These newly identified inhibitors were further tested against human histone deacetylases (hsHDAC1, 6 and 8), and were found also to be exerting interesting activity against them. In silico prediction of the docking pose of the compounds was confirmed by the resolved crystal structure of one of the identified hits. This confirmed these compounds were able to chelate the catalytic zinc ion in a bidentate fashion, whilst showing an inverted binding mode of the hydroxamate group when compared to the reported smHDAC8/hydroxamates crystal structures. Therefore, they can be considered as new potential scaffold for the development of new smHDAC8 inhibitors by further investigation of their structure-activity relationship.

Schistosoma mansoni cercarial elastase (SmCE): differences in immunogenic properties of native and recombinant forms.

The Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni. In the present study, we show that in contrast to the poor immunogenicity of the enzymatically active native form of SmCE derived from a crude preparation (cercarial transformation fluid), immunization of CBA/Ca mice with two enzymatically inactive forms, namely purified native SmCE or a recombinant SmCE fused to recombinant Schistosoma japonicum glutathione S-transferase (rSmCE-SjGST), after adsorption onto aluminum hydroxide adjuvant, induced specific anti-SmCE immunoglobulin G (IgG) in all mice within 2 weeks of the second immunization. The IgG antibody response to rSmCE-SjGST was mainly of the IgG1 subclass. These results suggest that inactive forms of the antigen could be used to obtain the optimum immunogenic effects as a vaccine candidate against schistosomiasis. Mice immunized with the rSmCE-SjGST on alum had smaller mean worm burdens and lower tissue egg counts when compared with adjuvant alone- and recombinant SjGST-injected controls. The native SmCE was antigenically cross-reactive with homologous enzymes of Schistosoma haematobium and Schistosoma margrebowiei.

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs.

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galß1-4(Fucα1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcß1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galß1-3(Galß1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.

Molecular characterization of transport lectin vesicular integral membrane protein 36 kDa (VIP36) in the life cycle of Schistosoma mansoni.

VIP36 is a protein described as an L-type lectin in animals, responsible for the intracellular transport of glycoproteins within the secretory pathway, and also localized on the plasma membrane. Schistosoma mansoni has a complex system of vesicles and protein transport machinery to the cell surface. The excreted/secreted products of the larvae and eggs are known to be exposed to the host immune system. Hence, characterizing the role and action of SmVIP36 in the S. mansoni life cycle is important for a better understanding of the parasite-host relationship. To this purpose, we firstly performed in silico analysis. Analysis of SmVIP36 in silico revealed that it contains a lectin leg-like domain with a jellyroll fold as seen by its putative 3D tertiary structure. Additionally, it was also observed that its CRD contains calcium ion-binding amino acids, suggesting that the binding of SmVIP36 to glycoproteins is calcium-dependent. Finally, we observed that the SmVIP36 predicted amino acid sequence relative to its orthologs was conserved. However, phylogenetic analysis revealed that SmVIP36 follows species evolution, forming a further cluster with its definitive host Homo sapiens. Moreover, q-PCR analysis in the S. mansoni life cycle points to a significant increase in gene expression in the eggs, schistosomulae, and female adult stages. Similarly, protein expression increased in eggs, cercariae, schistosomulae, and adult worm stages. These results suggest that SmVIP36 might participate in the complex secretory activity within the egg envelope and tegument proteins, both important for the stages of the parasite that interact with the host.

Cross-species prophylactic efficacy of Sm-p80-based vaccine and intracellular localization of Sm-p80/Sm-p80 ortholog proteins during development in Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium.

Schistosomiasis remains a major global health problem. Despite large-scale schistosomiasis control efforts, clear limitations such as possible emergence of drug resistance and reinfection rates highlight the need for an effective schistosomiasis vaccine. Schistosoma mansoni large subunit of calpain (Sm-p80)-based vaccine formulations have shown remarkable efficacy in protecting against S. mansoni challenge infections in mice and baboons. In this study, we evaluated the cross-species protective efficacy of Sm-p80 vaccine against S. japonicum and S. haematobium challenge infections in rodent models. We also elucidated the expression of Sm-p80 and Sm-p80 ortholog proteins in different developmental stages of S. mansoni, S. haematobium, and S. japonicum. Immunization with Sm-p80 vaccine reduced worm burden by 46.75% against S. japonicum challenge infection in mice. DNA prime/protein boost (1 + 1 dose administered on a single day) resulted in 26.95% reduction in worm burden in S. haematobium-hamster infection/challenge model. A balanced Th1 (IFN-γ, TNF-α, IL-2, and IL-12) and Th2 (IL-4, IgG1) type of responses were observed following vaccination in both S. japonicum and S. haematobium challenge trials and these are associated with the prophylactic efficacy of Sm-p80 vaccine. Immunohistochemistry demonstrated that Sm-p80/Sm-p80 ortholog proteins are expressed in different life cycle stages of the three major human species of schistosomes studied. The data presented in this study reinforce the potential of Sm-p80-based vaccine for both hepatic/intestinal and urogenital schistosomiasis occurring in different geographical areas of the world. Differential expression of Sm-p80/Sm-p80 protein orthologs in different life cycle makes this vaccine potentially useful in targeting different levels of infection, disease, and transmission.

Discovery of Antischistosomal Drug Leads Based on Tetraazamacrocyclic Derivatives and Their Metal Complexes.

Praziquantel (PZQ) is the only drug available for the treatment of schistosomiasis, and since its large-scale use might be associated with the onset of resistance, new antischistosomal drugs should be developed. A series of 26 synthetic tetraazamacrocyclic derivatives and their metal complexes were synthesized, characterized, and screened for antischistosomal activity by application of a phased screening program. The compounds were first screened against newly transformed schistosomula (NTS) of harvested Schistosoma mansoni cercariae, then against adult worms, and finally, in vivo using the mouse model of S. mansoni infection. At a concentration of 33 µM, incubation with a total of 12 compounds resulted in the mortality of NTS at the 62% to 100% level. Five of these showing 100% inhibition of viability of NTS at 10 µM were selected for further screening for determination of the 50 inhibitory concentrations (IC50s) against both NTS and adult worms. Against NTS, all 5 compounds showed IC50s comparable to the IC50 of the standard drug, PZQ (0.87 to 9.65 µM for the 5 compounds versus 2.20 µM for PZQ). Three of these, which are the bisquinoline derivative of cyclen and its Fe(2+) and Mn(2+) complexes, showed micromolar IC50s (1.62 µM, 1.34 µM, and 4.12 µM, respectively, versus 0.10 µM for PZQ) against adult worms. In vivo, the worm burden reductions were 12.3%, 88.4%, and 74.5%, respectively, at a single oral dose of 400 mg/kg of body weight. The Fe(2+) complex exhibited activity in vivo comparable to that of PZQ, pointing to the discovery of a novel drug lead for schistosomiasis.

Epigenetic changes modulate schistosome egg formation and are a novel target for reducing transmission of schistosomiasis.

Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.

Ly6C(high) monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1(GFP/+) mice. CX3CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX3CR1-GFP⁺ Ly6C(low) monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX3CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX3CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C(high) and Ly6C(low) monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C(high) monocytes, but that these cells may transition through a Ly6C(low) state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C(high) monocytes via help from CD4⁺ T cells.