Adult Schistosoma Research Articles

Efficacy and safety of single-dose artesunate plus sulfalene/pyrimethamine combined with praziquantel for the treatment of children with Schistosoma mansoni or Schistosoma haematobium in western Kenya: a randomised, open-label controlled trial

BackgroundReliance on praziquantel for the treatment and control of schistosomiasis is likely to facilitate the emergence of drug resistance. Combination therapy targeting adult and juvenile schistosome worms is urgently needed to improve praziquantel efficacy and delay the potential development of drug resistance. We assessed the efficacy and safety of single-dose praziquantel combined with single-dose artesunate plus sulfalene-pyrimethamine in the treatment of Kenyan children with schistosomiasis.MethodsThis was an open-label, randomised clinical trial involving 426 school-aged children (7–15 years old) diagnosed with Schistosoma mansoni (by Kato-Katz) or S. haematobium (by urine filtration). They were randomly assigned (1:1:1) to receive a single dose of praziquantel (40 mg/kg), a single dose of artesunate plus sulfalene-pyrimethamine (12 mg/kg artesunate) or combination therapy using a single dose of praziquantel (40 mg/kg) combined with a single dose of artesunate plus sulfalene-pyrimethamine (12 mg/kg artesunate). The primary outcome was cure and egg reduction rates at 6 weeks post-treatment in the available case population. Adverse events were assessed within 3 h after treatment.ResultsOf the 426 children enrolled, 135 received praziquantel, 150 received artesunate plus sulfalene-pyrimethamine, and 141 received combination therapy. Outcome data were available for 348 (81.7%) children. For S. mansoni-infected children (n = 335), the cure rates were 75.6%, 60.7%, and 77.8%, and the egg reduction rates were 80.1%, 85.0%, and 88.4% for praziquantel, artesunate plus sulfalene-pyrimethamine, and combination therapy, respectively. For S. haematobium-infected children (n = 145), the corresponding cure rates were 81.4%, 71.1%, and 82.2%, and the egg reduction rates were 95.6%, 97.1%, and 97.7%, respectively. Seventy-one (16.7%) children reported mild-intensity adverse events. The drugs were well tolerated and no serious adverse events were reported.ConclusionsA single oral dose of praziquantel combined with artesunate plus sulfalene-pyrimethamine cured a high proportion of children with S. haematobium but did not significantly improve the treatment efficacy for either urinary or intestinal schistosomiasis. Sequential administration of praziquantel and artesunate plus sulfalene-pyrimethamine may enhance the efficacy and safety outcomes.Graphical abstract

Therapeutic efficacy of candidate antischistosomal drugs in a murine model of schistosomiasis mansoni.

Schistosomiasis is a neglected tropical disease associated with considerable morbidity. Praziquantel (PZQ) is effective against adult schistosomes, yet, it has little effect on juvenile stages, and PZQ resistance is emerging. Adopting the drug repurposing strategy as well as assuming enhancing the efficacy and lessening the doses and side effects, the present study aimed to investigate the in vivo therapeutic efficacy of the widely used antiarrhythmic, amiodarone, and diuretic, spironolactone, and combinations of them compared to PZQ. Mice were infected by Schistosoma mansoni "S. mansoni" cercariae (Egyptian strain), then they were divided into two major groups: Early- [3weeks post-infection (wpi)] and late- [6 wpi] treated. Each group was subdivided into seven subgroups: positive control, PZQ, amiodarone, spironolactone, PZQ combined with amiodarone, PZQ combined with spironolactone, and amiodarone combined with spironolactone-treated groups. Among the early-treated groups, spironolactone had the best therapeutic impact indicated by a 69.4% reduction of total worm burden (TWB), 38.6% and 48.4% reduction of liver and intestine egg load, and a significant reduction of liver granuloma number by 49%. Whereas, among the late-treated groups, amiodarone combined with PZQ was superior to PZQ alone evidenced by 96.1% reduction of TWB with the total disappearance of female and copula in the liver and intestine, 53.1% and 84.9% reduction of liver and intestine egg load, and a significant reduction of liver granuloma number by 67.6%. Comparatively, spironolactone was superior to PZQ and amiodarone in the early treatment phase targeting immature stages, while amiodarone had a more potent effect when combined with PZQ in the late treatment phase targeting mature schistosomes.

Genetic characterization of schistosome species from cattle in Côte d’Ivoire

BackgroundSchistosomiasis is a water-based parasitic disease that affects humans, livestock and wild animals. While considerable resources are dedicated to the surveillance, disease mapping, control and elimination of human schistosomiasis, this is not the case for livestock schistosomiasis. Indeed, there are important data and knowledge gaps concerning the species present, population genetic diversity, infection prevalence, morbidity and economic impact. This study aimed to identify circulating schistosome species in cattle across Côte d’Ivoire and to investigate their population diversity and structuring.MethodsOverall, 400 adult schistosomes were collected from slaughtered cattle at six sites across Côte d’Ivoire. Additionally, 114 miracidia were collected from live cattle at one site: Ferkessédougou, in the northern part of Côte d’Ivoire. DNA from all specimens was extracted and the cox1 and ITS1/2 regions amplified and analysed to confirm species. The genetic diversity and structuring of the schistosome populations were investigated using 12 microsatellite markers.ResultsAll adult schistosomes and miracidia presented Schistosoma bovis mitochondrial cox1 profile. Nuclear ITS1/2 data were obtained from 101 adult schistosomes and four miracidia, all of which presented an S. bovis profile. Genetic diversity indices revealed a deficiency of heterozygotes and signals of inbreeding across all sites, while structure analyses displayed little geographic structuring and differentiation. Cattle in Côte d’Ivoire thus appear to be mono-species infected with S. bovis. Hybrids of Schistosoma haematobium × S. bovis have not been identified in this study. Cattle schistosomes appear to be panmictic across the country.ConclusionsOur results contribute to a deeper understanding of schistosome populations in Ivorian cattle and emphasize a One Health approach of joint human and animal surveillance and prevention and control programmes for schistosomiasis.Graphical

SCHISTOACT: a protocol for an open-label, five-arm, non-inferiority, individually randomized controlled trial of the efficacy and safety of praziquantel plus artemisinin-based combinations in the treatment of Schistosoma mansoni infection

BackgroundSchistosomiasis control relies on praziquantel for preventive chemotherapy. Alternative drugs are needed for the treatment and control of schistosomiasis. Praziquantel is effective against adult schistosome worms but ineffective against larval stages of the parasite and cannot prevent re-infection or interrupt the transmission of infection. Continued reliance on praziquantel for wide-scale schistosomiasis control will likely accelerate the emergence of drug resistance. Artemisinin derivatives are effective against the juvenile stages but ineffective against adult worms. The SCHISTOACT study aimed to evaluate the efficacy and safety of praziquantel plus one of four artemisinin-based combinations in treating Schistosoma mansoni infection in Kenya.MethodsThe SCHISTOACT study is an open-label, head-to-head, five-arm, proof-of-concept, non-inferiority, individually randomized controlled trial with a follow-up of 12 weeks. A total of 540 primary school-aged children from the Mwea area, Kirinyaga County in central Kenya, diagnosed with S. mansoni infection (by Kato-Katz method) are randomly allocated (1:1:1:1:1) to a single dose of praziquantel plus a 3-day course of artesunate-sulfalene/pyrimethamine, or artesunate-amodiaquine, or artesunate plus mefloquine, or dihydroartemisinin-piperaquine, or praziquantel control arm. The primary endpoints are efficacy (cure rate, assessed by microscopy) and safety (adverse events) of each study arm 6 weeks after treatment. Secondary endpoints include cumulative cure rate, egg reduction rate, and re-infection 12 weeks after treatment. The non-inferiority margin is set at − 10 for the risk difference in cure rates between praziquantel and the combined treatment.DiscussionThis study assesses a strategy for repurposing artemisinin-based combination therapies (ACTs) for treating schistosomiasis. It adopts a head-to-head comparison of four different ACTs to test a non-inferiority hypothesis and to strengthen local capacity to conduct clinical trials for interventions against neglected tropical diseases.Trial registrationPan-African Clinical Trials Registry PACTR202001919442161. Retrospectively registered on 6 January 2020

Antiparasitic properties of 4-nerolidylcatechol from Pothomorphe umbellata (L.) Miq. (Piperaceae) in vitro and in mice models with either prepatent or patent Schistosoma mansoni infections

Ethnopharmacological relevanceRoots of Pothomorphe umbellata (L.) Miq. are used in traditional medicine of Africa and South America for the treatment of malaria and helminthiasis. However, neither P. umbellata nor its isolated compounds have been evaluated against Schistosoma species. Aims of this studyTo investigate the antischistosomal effects of P. umbellata root extracts and the isolated compound 4-nerolidylcatechol (4-NC) against Schistosoma mansoni ex vivo and in murine models of schistosomiasis. Materials and methodsThe crude hydroalcoholic (PuE) and hexane (PuH) extracts of P. umbellata roots were prepared and initially submitted to an ex vivo phenotypic screening against adult S. mansoni. PuH was analyzed by HPLC-DAD, characterized by UHPLC-HRMS/MS, and submitted to chromatographic fractionation, leading to the isolation of 4-NC. The anthelmintic properties of 4-NC were assayed ex vivo against adult schistosomes and in murine models of schistosomiasis for both patent and prepatent S. mansoni infections. Praziquantel (PZQ) was used as a reference compound. ResultsPuE (EC50: 18.7 μg/mL) and PuH (EC50: 9.2 μg/mL) kill adult schistosomes ex vivo. The UHPLC–HRMS/MS analysis of PuH, the most active extract, revealed the presence of 4-NC, peltatol A, and peltatol B or C. After isolation from PuH, 4-NC presented remarkable in vitro schistosomicidal activity with EC50 of 2.9 μM (0.91 μg/mL) and a selectivity index higher than 68 against Vero mammalian cells, without affecting viability of nematode Caenorhabditis elegans. In patent S. mansoni infection, the oral treatment with 4-NC decreased worm burden and egg production in 52.1% and 52.3%, respectively, also reducing splenomegaly and hepatomegaly. 4-NC, unlike PZQ, showed in vivo efficacy against juvenile S. mansoni, decreasing worm burden in 52.4%. ConclusionsThis study demonstrates that P. umbellata roots possess antischistosomal activity, giving support for the medicinal use of this plant against parasites. 4-NC was identified from P. umbellata roots as one of the effective in vitro and in vivo antischistosomal compound and as a potential lead for the development of novel anthelmintics.

Identification and characterisation of the tegument-expressed aldehyde dehydrogenase SmALDH_312 of Schistosoma mansoni, a target of disulfiram.

Schistosomiasis is an infectious disease caused by blood flukes of the genus Schistosoma and affects approximately 200 million people worldwide. Since Praziquantel (PZQ) is the only drug for schistosomiasis, alternatives are needed. By a biochemical approach, we identified a tegumentally expressed aldehyde dehydrogenase (ALDH) of S. mansoni, SmALDH_312. Molecular analyses of adult parasites showed Smaldh_312 transcripts in both genders and different tissues. Physiological and cell-biological experiments exhibited detrimental effects of the drug disulfiram (DSF), a known ALDH inhibitor, on larval and adult schistosomes in vitro. DSF also reduced stem-cell proliferation and caused severe tegument damage in treated worms. In silico-modelling of SmALDH_312 and docking analyses predicted DSF binding, which we finally confirmed by enzyme assays with recombinant SmALDH_312. Furthermore, we identified compounds of the Medicine for Malaria Venture (MMV) pathogen box inhibiting SmALDH_312 activity. Our findings represent a promising starting point for further development towards new drugs for schistosomiasis.

Using ChEMBL to Complement Schistosome Drug Discovery.

Schistosomiasis is one of the most important neglected tropical diseases. Until an effective vaccine is registered for use, the cornerstone of schistosomiasis control remains chemotherapy with praziquantel. The sustainability of this strategy is at substantial risk due to the possibility of praziquantel insensitive/resistant schistosomes developing. Considerable time and effort could be saved in the schistosome drug discovery pipeline if available functional genomics, bioinformatics, cheminformatics and phenotypic resources are systematically leveraged. Our approach, described here, outlines how schistosome-specific resources/methodologies, coupled to the open-access drug discovery database ChEMBL, can be cooperatively used to accelerate early-stage, schistosome drug discovery efforts. Our process identified seven compounds (fimepinostat, trichostatin A, NVP-BEP800, luminespib, epoxomicin, CGP60474 and staurosporine) with ex vivo anti-schistosomula potencies in the sub-micromolar range. Three of those compounds (epoxomicin, CGP60474 and staurosporine) also demonstrated potent and fast-acting ex vivo effects on adult schistosomes and completely inhibited egg production. ChEMBL toxicity data were also leveraged to provide further support for progressing CGP60474 (as well as luminespib and TAE684) as a novel anti-schistosomal compound. As very few compounds are currently at the advanced stages of the anti-schistosomal pipeline, our approaches highlight a strategy by which new chemical matter can be identified and quickly progressed through preclinical development.

Life stage-specific glycosylation of extracellular vesicles from Schistosoma mansoni schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL.

Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells via C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host.

In Vitro and In Vivo Antischistosomal Activity Profiling and Pharmacokinetics of Ozonide Carboxylic Acids.

Praziquantel, the only drug in clinical use for the treatment and control of schistosomiasis, is inactive against developing infections. Ozonides are synthetic peroxide derivatives inspired by the naturally occurring artemisinin and show particularly promising activity against juvenile schistosomes. We conducted an in-depth characterization of the in vitro and in vivo antischistosomal activity and pharmacokinetics of lead ozonide carboxylic acid OZ418 and four of its active analogs. In vitro, the ozonides featured rapid and consistent activity against schistosomula and adult schistosomes at double-digit micromolar EC50 values. Potency did not vary considerably between Schistosoma spp. The zwitterionic OZ740 and OZ772 were more active in vivo compared to their non-amphoteric carboxylic acids OZ418 and OZ748, despite their much lower systemic plasma exposure (AUC). The most active compound in vivo was ethyl ester OZ780, which was rapidly transformed to its parent zwitterion OZ740 and achieved ED50 values of 35 ± 2.4 and 29 ± 2.4 mg/kg against adult and juvenile Schistosoma mansoni, respectively. Ozonide carboxylic acids represent promising candidates for further optimization and development due to their good efficacy against both life stages together with their broad activity range against all relevant parasite species.

Phenotypic Profiling of Macrocyclic Lactones on Parasitic Schistosoma Flatworms.

Macrocyclic lactones are front-line therapies for parasitic roundworm infections; however, there are no comprehensive studies on the activity of this drug class against parasitic flatworms. Ivermectin is well known to be inactive against flatworms. However, the structure-activity relationship of macrocyclic lactones may vary across phyla, and it is entirely possible other members of this drug class do in fact show antiparasitic activity on flatworms. For example, there are several reports hinting at the anti-schistosomal activity of doramectin and moxidectin. To explore this class further, we developed an automated imaging assay combined with measurement of lactate levels from worm media. This assay was applied to the screening of 21 macrocyclic lactones (avermectins, milbemycins, and others such as spinosyns) against adult schistosomes. These in vitro assays identified several macrocyclic lactones (emamectin, milbemycin oxime, and the moxidectin metabolite 23-ketonemadectin) that caused contractile paralysis and lack of lactate production. Several of these were also active against miracidia, which infect the snail intermediate host. Hits prioritized from these in vitro assays were administered to mice harboring patent schistosome infections. However, no reduction in worm burden was observed. Nevertheless, these data show the utility of a multiplexed in vitro screening platform to quantitatively assess drug action and exclude inactive compounds from a chemical series before proceeding to in vivo studies. While the prototypical macrocyclic lactone ivermectin displays minimal activity against adult Schistosoma mansoni, this family of compounds does contain schistocidal compounds which may serve as a starting point for development of new anti-flatworm chemotherapies.

Comparative proteomics analysis of Schistosoma japonicum developed in different Oncomelania snails as intermediate hosts.

Schistosomiasis is a tropical parasitic disease that seriously endangers humans and animals. In this study, two Oncomelania snails, Oncomelania hupensis (O. hupensis) and Oncomelania weishan (O. weishan), were infected with Schistosoma japonicum (S. japonicum) cercariae during the early period, and ICR mice were subsequently infected with two kinds of miracidia that developed in male and female adult worms. In this study, isobaric tags for relative and absolute quantification (iTRAQ) were used to identify four channels: 113, 115, 117, and 119.A total of 2364 adult schistosome proteins were identified, and 1901 proteins were quantitative. Our results revealed 68 differentially expressed proteins (DEPs) in female adult worms, including 24 upregulated proteins and 44 downregulated proteins, and 55 DEPs in male adult worms, including 25 upregulated proteins and 30 downregulated proteins. LC-MS/MS and bioinformatics analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function and catabolism pathways. In summary, this proteomics analysis of adult schistosomes that hatched in two intermediate hosts helps to improve our understanding of the growth and developmental mechanisms of S. japonicum.

Single-cell RNA-seq analyses show that long non-coding RNAs are conspicuously expressed in Schistosoma mansoni gamete and tegument progenitor cell populations.

Schistosoma mansoni is a flatworm that causes schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. New therapeutic targets are needed with only one drug available for treatment and no vaccine. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential. In other organisms, they have been shown as involved with reproduction, stem cell maintenance and drug resistance, and they tend to exhibit tissue-specific expression patterns. S. mansoni expresses thousands of lncRNA genes; however, the cell type expression patterns of lncRNAs in the parasite remain uncharacterized. Here, we have re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) data obtained from adult S. mansoni to identify the lncRNAs signature of adult schistosome cell types. A total of 8023 lncRNAs (79% of all lncRNAs) were detected. Analyses of the lncRNAs expression profiles in the cells using statistically stringent criteria were performed to identify 74 lncRNA gene markers of cell clusters. Male gamete and tegument progenitor lineages clusters contained most of the cluster-specific lncRNA markers. We also identified lncRNA markers of specific neural clusters. Whole-mount in situ hybridization (WISH) and double fluorescence in situ hybridization were used to validate the cluster-specific expression of 13 out of 16 selected lncRNA genes (81%) in the male and female adult parasite tissues; for one of these 16 gene loci, probes for two different lncRNA isoforms were used, which showed differential isoform expression in testis and ovary. An atlas of the expression profiles across the cell clusters of all lncRNAs detected in our analysis is available as a public website resource (http://verjolab.usp.br:8081). The results presented here give strong support to a tissue-specific expression and to a regulated expression program of lncRNAs in S. mansoni. This will be the basis for further exploration of lncRNA genes as potential therapeutic targets.

Status of Intestinal Schistosomiasis in Buffaloes and Cattle in Rajshahi, Bangladesh

Schistosoma infection causes serious health impediment in livestock with poor productive and reproductive performances. This study was carried out to know the status of Schistosoma infection in buffaloes and cattle in Rajshahi, Bangladesh. The adult schistosomes were collected from mesenteries of buffaloes and cattle of Rajshahi. Out of 240 mesenteries (120 from each species) examined, 113 (47.08%) were found infected. The infection rate in buffaloes and cattle were 54.16% and 40%, respectively. Female buffalo and cattle (57.69% and 46%) were more prone to the infection than male (51.47% and 36.71%) although the differences were not statistically significant. Significant variations (p<0.05) were observed in infection rate in different season. The highest infection rate was observed in rainy season (85% and 65%) followed by summer (52.5% and 40%) and winter (25% and 15%) in both the animals. Intensity of the infection greatly varied. Mostly, half of the infected animal (55% buffalo and 46% cattle) showed moderate infection intensity (51 to 100 parasites/animal). High degree of infection (>100 parasites/animal) was found in 25% and 33% infected buffalo and cattle, respectively. Two species of intestinal schistosomes (S. indicum and S. spindale) were identified microscopically by observing egg of female parasites. S. indicum was present in all the infected mesenteries whereas only 29% buffaloes and 35% cattle samples contained S. spindale. The study determined the current scenario of intestinal schistosome infection in buffaloes and cattle of Rajshahi. The findings might be helpful to develop effective strategic plan to overcome the harmful effects of schistosomes in livestock of Bangladesh.
 Vol. 9, No. 2, August 2022: 201-211

In Vivo Evaluation of an Antibody-Functionalized Lipoidal Nanosystem for Schistosomiasis Intervention.

This study employed nanotechnological techniques to design and develop a praziquantel nanoliposomal (NLP) system and surface-functionalized the NLP with anti-calpain antibody (anti-calpain-NLP) for targeted praziquantel (PZQ) delivery in the treatment of schistosomiasis. Anti-calpain-NLPs were prepared and validated for their physicochemical parameters, in vitro and in vivo toxicity, drug entrapment efficiency (DEE), drug loading capacity (DLC), drug release, and parasitological cure rate. The particle sizes for the formulated nanoliposomes ranged from 88.3 to 92.7 nm (PDI = 0.17–0.35), and zeta potential ranged from −20.2 to −31.9 mV. The DLC and DEE ranged from 9.03 to 14.16 and 92.07 to 94.63, respectively. The functionalization of the nanoliposome surface was stable, uniform, and spherical. Fourier-transform infrared (FTIR), thermal behavior and X-ray powder diffraction (XRPD) analysis confirmed that the anti-calpain antibody and PZQ were attached to the surface and the nanoliposomes inner core, respectively. The drug sustained release was shown to be 93.2 and 91.1% within 24 h for NLP and anti-calpain-NLP, respectively. In the in vitro analysis study, the nanoliposome concentrations range of 30 to 120 μg/mL employed revealed acceptable levels of cell viability, with no significant cytotoxic effects on RAW 264.7 murine macrophage as well as 3T3 human fibroblast cells. Biochemical markers and histopathological analysis showed that the formulated nanoliposomes present no or minimal oxidative stress and confer hepatoprotective effects on the animals. The cure rate of the anti-calpain-NLP and PZQ was assessed by parasitological analysis, and it was discovered that treatment with 250 mg/kg anti-calpain-NLP demonstrated greater activity on the total worm burden, and ova count for both the juvenile and adult schistosomes in the intestine and liver of infected mice. The findings so obtained supported the ability of oral anti-calpain-NLP to target young and adult schistosomes in the liver and porto-mesenteric locations, resulting in improved effectiveness of PZQ.

Differential Excretory/Secretory Proteome of the Adult Female and Male Stages of the Human Blood Fluke, Schistosoma mansoni

Intricate molecular communication between schistosome flatworms and their mammalian host, as well as between paired male and female schistosomes has shaped the secreted proteome of these flatworms. Whereas the schistosome egg is responsible for the disease manifestations of chronic schistosomiasis, the long lived, adult female and male stages also release different mediators including glycans, lipids, proteins and small molecules, known as excretory/secretory products (ESPs), that facilitate their survival. Given their importance, deeper analysis focused on analyzing the ESPs from adult schistosomes would likely be informative, beyond current understanding of the complement of ESP proteins. Here, taking advantage of highly accurate and sensitive mass spectrometers, the excretory/secretory proteome from culturedSchistosoma mansonimale or female adult worms was identified, quantified, compared and contrasted using a label-free proteomic approach. Approximately 1,000 proteins were identified, from which almost 800 could be quantified. Considering the proteins uniquely identified and proteins with a significantly regulated expression pattern in male or female flukes, a total of 370 and 140 proteins were uniquely or more abundantly secreted by males and females, respectively. Using functional analysis networks showing the gene ontology terms and KEGG pathways with the highest significance, we observed that male schistosomes secrete proteins related to carbohydrate metabolism and cytoskeletal organization more abundantly than females, while female worms secreted more hydrolases and proteins involved in cellular homeostasis than males. This analysis doubles the number of reported excreted/secreted proteins fromS. mansoni, contributing to deeper understanding of the host-parasite interaction and parasitism. Furthermore, these findings expand potential vaccine and diagnostic candidates for this neglected tropical disease pathogen, and thereby also provide leads for novel intervention to control this disease and its transmission.

Schistosome Sulfotransferases: Mode of Action, Expression and Localization.

Oxamniquine (OXA) is a prodrug activated by a sulfotransferase (SULT) that was only active against Schistosoma mansoni. We have reengineered OXA to be effective against S. haematobium and S. japonicum. Three derivatives stand out, CIDD-0066790, CIDD-0072229, and CIDD-0149830 as they kill all three major human schistosome species. However, questions remain. Is the OXA mode of action conserved in derivatives? RNA-interference experiments demonstrate that knockdown of the SmSULT, ShSULT, and SjSULT results in resistance to CIDD-0066790. Confirming that the OXA-derivative mode of action is conserved. Next is the level of expression of the schistosome SULTs in each species, as well as changes in SULT expression throughout development in S. mansoni. Using multiple tools, our data show that SmSULT has higher expression compared to ShSULT and SjSULT. Third, is the localization of SULT in the adult, multicellular eucaryotic schistosome species. We utilized fluorescence in situ hybridization and uptake of radiolabeled OXA to determine that multiple cell types throughout the adult schistosome worm express SULT. Thus, we hypothesize the ability of many cells to express the sulfotransferase accounts for the ability of the OXA derivatives to kill adult worms. Our studies demonstrate that the OXA derivatives are able to kill all three human schistosome species and thus will be a useful complement to PZQ.

Sex-Specific Modulation of the Host Transcriptome in the Spleen of Schistosoma mansoni-Infected Mice.

BackgroundSchistosomiasis is a severe parasitic disease that is primarily driven by the host’s immune response to schistosome eggs trapped in tissue and by the granulomatous inflammatory and fibrotic reaction they cause. Despite significant progress in understanding the complex immunological processes involved in the relationship between schistosomes and their host, neither an effective vaccine against the infection nor anti-fibrotic drugs currently exists, making the search for new targets for schistosome drugs and vaccine candidates even more important. In order to identify new molecular targets for defense against or elimination of the parasite, we investigate herein the interplay between the host and male or female schistosomes, clearly separating this from the action of the parasite eggs.MethodsFor this purpose, we infected 6–8-week-old female NMRI mice with 100 male (M), female (F), or both (MF) S. mansoni cercariae and performed a comparative transcriptomic and flow cytometric analysis of their spleens.ResultsPrincipal component analysis of a total of 22,207 transcripts showed a clear clustering of the experimental groups. We identified a total of 1,293 genes in group M, 512 genes in group F, and 4,062 genes in group MF that were differentially expressed compared to naive controls. The highest percentage of regulated genes (2,972; 65.9%) was found in group MF alone, but there was a large overlap between groups M and MF (798; 17.7%) and a small overlap between groups F and MF (91; 2.0%). Only 4.5% of genes (201) were revealed to be regulated in all experimental groups (M/F/MF). In addition, we were able to show that both worm sexes trigger immune responses in an egg-independent manner (non-polarized Th1 and Th2 response), with female worms exerting less regulatory influence than males.ConclusionOur data show that adult schistosomes trigger sex-specific, egg-independent immune responses. The lists of genes regulated by adult female or male worms presented here may be useful in deciphering host–parasite interactions to identify targets for schistosome elimination.

Adult schistosomes have an epithelial bacterial population distinct from the surrounding mammalian host blood.

BackgroundSchistosomiasis is a neglected tropical parasitic and chronic disease affecting hundreds of millions of people. Adult schistosomes reside in the blood stream of the definitive mammalian host. These helminth parasites possess two epithelial surfaces, the tegument and the gastrodermis, both of which interact with the host during immune evasion and in nutrient uptake.MethodsFemale ARC Swiss mice (4–6 weeks old) were infected percutaneously with Schistosoma japonicum cercariae freshly shed from Oncomelania hupensis quadrasi snails (Philippines strain). Fluorescent in situ hybridisation (FISH) was performed by using fresh adult S. japonicum perfused from those infected mice. Adult S. japonicum worms were processed to isolate the tegument from the carcass containing the gastrodermis; blood and bile were collected individually from infected and uninfected mice. Total DNA extracted from all those samples were used for microbiome profiling.ResultsFISH and microbiome profiling showed the presence of bacterial populations on two epithelial surfaces of adult worms, suggesting they were distinct not only from the host blood but also from each other. Whereas microbial diversity was reduced overall in the parasite epithelial tissues when compared with that of host blood, specific bacterial taxa, including Anoxybacillus and Escherichia, were elevated on the tegument. Minimal differences were evident in the microbiome of host blood during an active infection, compared with that of control uninfected blood. However, sampling of bile from infected animals identified some differences compared with controls, including elevated levels of Limnohabitans, Clostridium and Curvibacter.ConclusionsUsing FISH and microbial profiling, we were able to demonstrate, for the first time, that bacteria are presented on the epithelial surfaces of adult schistosomes. These schistosome surface-associated bacteria, which are distinct from the host blood microenvironment, should be considered as a new and important component of the host-schistosome interaction. The importance of individual bacterial species in relation to schistosome parasitism needs further elucidation.

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Distinct Schistosoma mansoni-Specific Immunoglobulin Subclasses Are Induced by Different Schistosoma mansoni Stages-A Tool to Decipher Schistosoma mansoni Infection Stages.

Despite the existence of an effective medication against schistosomiasis, the disease remains a major health problem in affected areas, especially for those lacking appropriate sanitary facilities. Moreover, treatment cannot prevent re-infection since it is only effective on adult schistosome worms. Previous retrospective studies in the Sudan have discovered unique immuno-epidemiological profiles in uninfected individuals and those positive for Schistosoma mansoni via polymerase chain reaction (PCR) but egg-negative and those with eggs in their stool. Expanding on these data, serum samples from these individuals were further investigated for the presence of cercarial (SmCTF)-specific antibodies, which would indicate immune responses at the early stages of infection. Indeed, SmCTF IgG1, 2, 3 and 4 levels were significantly elevated in SmPCR+ individuals when compared to egg+ patients. Following multivariable regression analysis, including SmCTF-specific Igs, Schistosoma egg antigen (SEA)-specific and Schistosoma worm antigen (SWA)-specific immunoglobulins revealed a specific immunoglobulin (Ig) profile of individuals presenting different states of infection, which may be a useful future tool in order to identify egg− individuals and thereby prevent unnecessary treatments.