Discovery and molecular characterization of a Bcl-2-regulated cell death pathway in schistosomes.

Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2-regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with "BH3 mimetic" drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.

Schistosome Transgenesis: The Long Road to Success.

As research on parasitic helminths has entered the post-genomic era, research efforts have turned to deciphering the function of genes in the public databases of genome sequences. It is hoped that, by understanding the role of parasite genes in maintaining their parasitic lifestyle, critical insights can be gained to develop new intervention and control strategies. Methods to manipulate and transform parasitic worms are now developed to a point where it has become possible to gain a comprehensive understanding of the molecular mechanisms underlying host-parasite interplay, and here, we summarise and discuss the advances that have been made in schistosome transgenesis over the past 25 years. The ability to genetically manipulate schistosomes holds promise in finding new ways to control schistosomiasis, which ultimately may lead to the eradication of this debilitating disease.

Programming schistosomes - a crisper approach to transgenesis.

Ittiprasert and colleagues identified genomic safe harbour (GSH) sites in Schistosoma mansoni using computational methods and inserted a transgene into one of the sites through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-assisted homology-directed repair. This study outlines a promising strategy for functional genomics to study this parasite that causes a debilitating and neglected tropical disease.

Delays in seeking treatment for fever in children under five years of age in Nigeria: Evidence from the National Demographic Health Survey.

BACKGROUND: In countries with high child mortality rates, such as Nigeria, early intervention for common childhood illnesses (e.g., pneumonia and malaria) is essential for improving clinical outcomes. The timely reporting and treatment of fever is therefore critical in making a differential diagnosis and choosing an appropriate course of treatment. The present study aimed to investigate the prevalence and major risk factors associated with delays in seeking treatment for fever in children under five years of age in Nigeria. METHODS: This study used a total weighted sample of 7,466 children under five years of age from the 2018 National Nigerian Demographic and Health Survey. Multivariable binary logistic regression modelling was used to investigate the association between predisposing, enabling, need, health service and community level factors, and delay in treatment-seeking for fever. RESULTS: We report the delays in seeking treatment for childhood fever that was reported by mothers in the last two weeks prior to the national survey. The prevalence for delayed treatment was 62.1% (95% confidence interval [CI]: 60.1%, 64.1%). Our findings showed that there were fewer delays in seeking treatment in children aged 24-59 months (adjusted odds ratio [aOR] = 0.79, 95% CI: 0.68, 0.93), among mothers who were formally employed (aOR = 0.84; 95% CI: 0.73, 0.96), regularly attended antenatal services (aOR = 0.76, 95%CI: 0.66, 0.88), and for those who resided in wealthier households (aOR = 0.71; 95% CI: 0.56, 0.89). Children whose mothers resided in the North-West geopolitical zone of Nigeria were less likely to delay seeking treatment for fever (aOR = 0.55; 95% CI: 0.42, 0.73). However, mothers who had an unwanted pregnancy had a higher odds of delaying treatment for childhood fever (aOR = 1.58; 95% CI: 1.05, 2.39). CONCLUSION: There were significant delays in seeking treatment for childhood fever in poorer homes found in geopolitically unstable zones of Nigeria. Mothers who were poor, unemployed, and with younger children (<12 months) often delayed seeking treatment for their febrile child. Future health promotion strategies and microenterprise schemes should target both rural and urban mothers residing in poor households. Children under 12 months of age should be a priority.

Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni.

In helminth parasites, proteolytic enzymes have been implicated in facilitating host invasion, moulting, feeding, and evasion of the host immune response. These key functions render them potential targets for anti-parasite chemotherapy and immunotherapy. Schistosomes feed on host blood and the digested haemoglobin is their major source of amino acids. Haemoglobin digestion is essential for parasite development, growth, and reproduction. We recently reported the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of Schistosoma mansoni. Here, we report the design of a viral vector expressing a dsRNA hairpin to silence expression of the schistosome cathepsin B1 (SmCB1) gene. We observed 80% reduction in transcript level 72 h after virus exposure and complete silencing of enzyme activity in transduced worms. This is the first report using this technology in any helminth parasite. It will facilitate the evaluation of potential drug targets and biochemical pathways for novel interventions in schistosomes.

piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni.

The transposon piggyBac from the genome of the cabbage looper moth Trichoplusia ni has been observed in the laboratory to jump into the genomes of key model and pathogenic eukaryote organisms including mosquitoes, planarians, human and other mammalian cells, and the malaria parasite Plasmodium falciparum. Introduction of exogenous transposons into schistosomes has not been reported but transposon-mediated transgenesis of schistosomes might supersede current methods for functional genomics of this important human pathogen. In the present study we examined whether the piggyBac transposon could deliver reporter transgenes into the genome of Schistosoma mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters was introduced along with 7-methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. The activity of the helper transposase mRNA was confirmed by Southern hybridization analysis of genomic DNA from the transformed schistosomes, and hybridization signals indicated that the piggyBac transposon had integrated into numerous sites within the parasite chromosomes. piggyBac integrations were recovered by retrotransposon-anchored PCR, revealing characteristic piggyBac TTAA footprints in the vicinity of the endogenous schistosome retrotransposons Boudicca, SR1, and SR2. This is the first report of chromosomal integration of a transgene and somatic transgenesis of this important human pathogen, in this instance accomplished by mobilization of the piggyBac transposon.

Manipulating the manipulators: advances in parasitic helminth transgenesis and RNAi.

Because tropical medicine and parasitology research has moved into the postgenomic era, an enormous amount of gene sequence information for parasitic helminths is now accumulating. These sequences undoubtedly hold information that can be used for new interventions and control. However, to exploit the new resource, methods for gene manipulation and transformation of parasitic worms are needed. Until recently, gene manipulation approaches had not been seriously addressed. This situation is now changing in response to the availability of genome sequences and other advances. In this article, we review advances in the transgenesis and gene silencing of parasitic worms.

Organization and functional analysis of the Schistosoma mansoni cathepsin D-like aspartic protease gene promoter.

We have cloned a 969-bp fragment of genomic DNA that spans 821 bp of the 5' untranslated region, exon 1, a short intron, and part of exon 2 of the Schistosoma mansoni cathepsin D gene by inverse PCR. Inspection of this sequence revealed the presence of two TATA-box motifs, two inverted CCAAT-box (inverted NF-Y) motifs and sequences with homology to binding sites for the transcription factors, AP-1 and NF-Y. This sequence and deletion variants were cloned into reporter gene constructs, in order to examine the ability of these putative regulatory sequences to drive heterologous reporter gene activity. PCR products were cloned into the luciferase reporter vector pXP2. These reporter gene constructs were used to transform HeLa cells which were cultured and examined for luciferase activity. Additionally, HeLa cells transiently transfected with an EGFP reporter plasmid driven by the putative promoter from the S. mansoni cathepsin D gene were examined for EGFP transcripts and fluorescence. The 5' untranslated region of the S. mansoni cathepsin D gene, from position -772 to +40 (translation start ATG), included functional regulatory sequences capable of driving luciferase and EGFP expression, whereas shorter fragments from position -264 or -185 to +40 were insufficient to drive reporter activities.

The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements.

BACKGROUND: Of the major families of long terminal repeat (LTR) retrotransposons, the Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni. RESULTS: A proviral form of Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon. CONCLUSION: Sinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni. The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some genomes.

Studies on Acanthocheilonema viteae cystatin: genomic organization, promoter studies and expression in Caenorhabditis elegans.

Cystatins are reversible, tightly binding inhibitors of cysteine proteases. Filarial cystatins have been ascribed immunomodulatory properties and have been implicated in protective immunity. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cystatin gene locus of A. viteae. The gene is composed of 4 exons separated by 3 introns and spans approximately 2 kb of genomic DNA. The upstream genomic sequence contains transcriptional factor binding sites such as AP-1 and NF-Y, an inverted CCAAT sequence, and a TATA box. To investigate sites of cystatin expression, Caenorhabditis elegans worms were transformed by microinjection with the putative promoter region and the first exon of the A. viteae cystatin gene fused to the reporter GFP. In transgenic worms fluorescence was observed in the pharyngeal and rectal gland cells suggesting that cystatin is secreted. Additionally, A. viteae cystatin was expressed in C. elegans to explore its potential as an expression system for filarial genes.

Prospects for Vector-Based Gene Silencing to Explore Immunobiological Features of Schistosoma mansoni.

Schistosomiasis is a prevalent, socioeconomically important disease of humans caused by parasites of the genus Schistosoma (schistosomes or blood flukes). Currently, more than 200 million people worldwide are infected with schistosomes. Despite major research efforts, there is only one drug routinely used for effective treatment, and no vaccine is available to combat schistosomiasis. The purpose of the present article is to (1) provide a background on the parasites and different forms of disease; (2) describe key immunomolecular aspects of disease induced in the host; and (3) critically appraise functional genomic methods employed to explore parasite biology, parasite-host interactions and disease at the molecular level. Importantly, the article also describes the features and advantages of lentiviral delivery of artificial microRNAs to silence genes. It also discusses the first successful application of such an approach in schistosomes, in order to explore the immunobiological role of selected target proteins known to be involved in egg-induced disease. The lentiviral transduction system provides exciting prospects for future, fundamental investigations of schistosomes, and is likely to have broad applicability to other eukaryotic pathogens and infectious diseases. The ability to achieve effective and stable gene perturbation in parasites has major biotechnological implications, and might facilitate the development of radically new methods for the treatment and control of parasitic diseases.

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotransposon.

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansoni. Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRn1 and kabuki, as well as to the conserved integrase core domain, GenBank rve (). Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D.

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.

Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo.

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

Boudicca, a retrovirus-like long terminal repeat retrotransposon from the genome of the human blood fluke Schistosoma mansoni.

The genome of Schistosoma mansoni contains a proviral form of a retrovirus-like long terminal repeat (LTR) retrotransposon, designated BOUDICCA: Sequence and structural characterization of the new mobile genetic element, which was found in bacterial artificial chromosomes prepared from S. mansoni genomic DNA, revealed the presence of three putative open reading frames (ORFs) bounded by direct LTRs of 328 bp in length. ORF1 encoded a retrovirus-like major homology region and a Cys/His box motif, also present in Gag polyproteins of related retrotransposons and retroviruses. ORF2 encoded enzymatic domains and motifs characteristic of a retrovirus-like polyprotein, including aspartic protease, reverse transcriptase, RNase H, and integrase, in that order, a domain order similar to that of the gypsy/Ty3 retrotransposons. An additional ORF at the 3' end of the retrotransposon may encode an envelope protein. Phylogenetic comparison based on the reverse transcriptase domain of ORF2 confirmed that Boudicca was a gypsy-like retrotransposon and showed that it was most closely related to CsRn1 from the Oriental liver fluke Clonorchis sinensis and to kabuki from Bombyx mori. Bioinformatics approaches together with Southern hybridization analysis of genomic DNA of S. mansoni and the screening of a bacterial artificial chromosome library representing approximately 8-fold coverage of the S. mansoni genome revealed that numerous copies of Boudicca were interspersed throughout the schistosome genome. By reverse transcription-PCR, mRNA transcripts were detected in the sporocyst, cercaria, and adult developmental stages of S. mansoni, indicating that Boudicca is actively transcribed in this trematode.